Peptides, peptide complexes, and their uses that possess anti-diabetic activity

Abstract

The present invention relates to a peptide conjugate having anti-diabetic activity and uses thereof. The peptide conjugate of the present invention exhibits blood glucose level lowering efficacy by promoting glucose absorption into cells, suppressing insulin resistance signals, promoting insulin sensitivity signals, and suppressing the death of pancreatic β cells, which are insulin-producing cells. The present invention also relates to a peptide having anti-diabetic and anti-obesity activities and uses thereof. The peptide of the present invention has activities of suppressing insulin resistance signals, promoting insulin sensitivity signals, and promoting fat breakdown in adipocytes.

Description

[Technical Field]

[0001] This invention relates to peptide complexes having anti-diabetic activity and their applications. Furthermore, this invention relates to peptides having anti-obesity and anti-diabetic activity and their applications. [Background technology]

[0002] Diabetes mellitus is a type of metabolic disorder characterized by insufficient insulin secretion or impaired insulin function, resulting in hyperglycemia (high blood glucose levels). This hyperglycemia causes various symptoms and signs, and glucose is excreted in the urine. Recently, the incidence of diabetes mellitus has been increasing dramatically due to the rise in obesity rates, particularly abdominal obesity. Diabetes mellitus is broadly divided into type 1 diabetes mellitus, which is insulin-dependent, and type 2 diabetes mellitus, which is non-insulin-dependent. Type 2 diabetes mellitus is characterized by hyperglycemia, insulin resistance, and relative insulin secretion impairment.

[0003] When food is ingested, glucose from the food is absorbed in the digestive tract, stimulating insulin secretion in the β cells of the pancreas and intestines. The secreted insulin promotes glucose absorption into muscles. Insulin is also partially involved in glucose absorption in the liver, but mainly suppresses glucose production in the liver. Insulin lowers blood glucose concentration by suppressing glucose production in the liver and promoting glucose absorption into peripheral tissues, including muscles. Insulin resistance refers to a condition in which the blood glucose response to insulin is lower than normal under a given insulin concentration. Insulin regulates blood glucose by promoting glucose uptake into muscles or suppressing glucose production in the liver, and insulin resistance means a state in which this insulin action is reduced even when there is no insulin deficiency. In the process of glucose absorption into peripheral tissue cells, insulin receptors on the cell membrane become involved, and insulin resistance occurs when the number of insulin receptors decreases or when intracellular defects occur after receptor binding. In type 2 diabetes, while insulin receptor defects are sometimes found, intracellular defects after receptor function, specifically impaired phosphorylation / dephosphorylation regulated by insulin, are known to play a more significant role. Among these mechanisms, impaired PI3K (Phosphoinositide 3-kinase) signaling is known to reduce the translocation of the glucose transporter GLUT-4 (Glucose transporter type 4) to the cell membrane.

[0004] Currently, methods for regulating blood glucose include lifestyle modifications (dietary therapy, exercise therapy) and drug therapy. However, dietary therapy and exercise therapy are difficult to strictly manage and implement, and their effectiveness is limited. Therefore, most diabetic patients rely on lifestyle modifications along with drug-based blood glucose regulation, such as insulin, insulin secretagogues, insulin sensitivity enhancers, and hypoglycemic agents.

[0005] Obesity is a condition in which excess energy is stored as body fat when energy intake from food is not balanced with energy expenditure, resulting in an excessive amount of adipose tissue in the body. According to the World Health Organization (WHO), more than one billion adults worldwide are overweight, and of those, at least three million are clinically obese, with a significant increase in the United States and Europe. Being overweight and obese raises blood pressure and cholesterol levels, causing various diseases such as heart disease, diabetes, and arthritis, and increases the incidence of various lifestyle-related diseases. Furthermore, being overweight and obese are contributing factors that increase the incidence of various lifestyle-related diseases such as arteriosclerosis, hypertension, hyperlipidemia, and heart disease not only in adults but also in children and adolescents.

[0006] Currently, the most widely prescribed obesity treatments approved by the US FDA include a group of drugs that act on the central nervous system to suppress appetite, and orlistat (Xenical), an inhibitor of lipase, a digestive enzyme secreted from the pancreas. Many central nervous system-acting drugs, such as sibutramine, have had their approval revoked due to cardiovascular and psychiatric side effects, and orlistat has the limitation that its effects vary depending on fat intake, along with a variety of other side effects. On the other hand, among endocrine peptide-targeted drugs, liraglutide, a glucagon-like peptide-1 (GLP-1) receptor activator, is approved and in use, but concerns about its risk of thyroid cancer have emerged. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2016-175362 [Patent Document 2] WO2018-074682 [Overview of the project] [Problems that the invention aims to solve]

[0008] The inventors have made research efforts to find an active substance that is more effective and safer for treating diabetes or obesity, in terms of suppressing insulin resistance and promoting glucose absorption by increasing insulin sensitivity, protecting pancreatic and intestinal β-cells from free fatty acids, and promoting lipolysis in adipocytes. As a result, they have experimentally confirmed that a complex of two peptides and a peptide having a novel amino acid sequence satisfy the above requirements, leading to the completion of the present invention.

[0009] Therefore, the object of the present invention is to provide a peptide complex having anti-diabetic activity.

[0010] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes containing the aforementioned peptide complex as an active ingredient.

[0011] Another object of the present invention is to provide a functional food composition for regulating blood glucose levels, which contains the aforementioned peptide complex having the activity described above as an active ingredient.

[0012] Another object of the present invention is to provide a novel peptide having anti-diabetic activity and anti-obesity activity.

[0013] Another object of the present invention is to provide pharmaceutical compositions and functional food compositions for the prevention, treatment, or improvement of diabetes, comprising a novel peptide having the aforementioned activity as an active ingredient.

[0014] Another object of the present invention is to provide pharmaceutical compositions and functional food compositions for the prevention, treatment, or improvement of obesity, comprising the aforementioned active peptides as active ingredients. [Means for solving the problem]

[0015] In order to achieve the aforementioned objective, One aspect of the present invention provides a peptide complex comprising: (i) a peptide comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 2.

[0016] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising the peptide complex as an active ingredient.

[0017] Still another aspect of the present invention provides a functional food composition for preventing or improving diabetes, comprising the peptide complex as an active ingredient.

[0018] Another aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 2.

[0019] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising the peptide as an active ingredient.

[0020] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating obesity, comprising the peptide as an active ingredient.

[0021] Still another aspect of the present invention provides a functional food composition for regulating blood glucose levels, comprising the peptide as an active ingredient.

[0022] Still another aspect of the present invention provides a functional food composition for preventing or improving obesity, comprising the peptide as an active ingredient.

[0023] The present invention will be described in detail below.

[0024] 1. Peptides, Peptide Complexes and Their Activities According to one aspect of the present invention, there is provided a peptide complex comprising: (i) a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1; and (ii) a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 2.

[0025] Another aspect of the present invention provides a peptide comprising the amino acid sequence disclosed in Sequence ID No. 2.

[0026] As used herein, the term "peptide" refers to a linear molecule formed by the bonding of amino acid residues to one another via peptide bonds.

[0027] The peptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention may be used without modification. However, amino acid variants or fragments having different sequences due to deletion, insertion, substitution, or combination thereof of amino acid residues may also be used, provided that the original activity of the peptide, such as its anti-diabetic activity, is not affected.

[0028] The peptide of the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within the limits that do not alter its activity.

[0029] The peptides of the present invention include peptides containing an amino acid sequence substantially identical to that of a peptide containing the amino acid sequence of SEQ ID NO: 1 or a peptide containing the amino acid sequence of SEQ ID NO: 2, as well as their variants or active fragments. A substantially identical amino acid sequence means an amino acid sequence having 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. The peptides may further include targeted sequences, tags, labeled residues, or amino acid sequences produced for specific purposes such as increasing half-life or peptide stability.

[0030] The peptides of the present invention may have their N-terminus and / or C-terminus modified by selecting a portion of the amino acid sequence to increase their activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptides of the present invention, for example, by increasing their half-life when administered in vivo. The term "stability" includes not only in vivo stability that protects the peptides of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0031] The modification of the N-terminus may involve attaching a protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) to the N-terminus of the peptide. The modification of the C-terminus may involve attaching a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), etc., to the C-terminus of the peptide, but is not limited to these.

[0032] The peptides of the present invention can be produced by a variety of widely known methods in the art to which the present invention pertains. For example, the peptides of the present invention can be produced by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis techniques (US Patent No. 5,516,891).

[0033] The peptide complex of the present invention comprises a peptide containing the amino acid sequence of SEQ ID NO: 1 and a peptide containing the amino acid sequence of SEQ ID NO: 2.

[0034] The peptide complex of the present invention may mean a mixture of a peptide containing the amino acid sequence of SEQ ID NO: 1 and a peptide containing the amino acid sequence of SEQ ID NO: 2.

[0035] In the peptide complex of the present invention, the ratio of the peptide containing the amino acid sequence of SEQ ID NO: 1 and the peptide containing the amino acid sequence of SEQ ID NO: 2 is not limited to a specific range. For example, the ratio may be selected and used within an appropriate range of weight ratio from 1:0.1 to 100.

[0036] The peptide complex of the present invention has anti-diabetic activity.

[0037] The peptide complex of the present invention has the activity to promote glucose absorption into cells. The cells may be adipocytes, myocytes, or hepatocytes.

[0038] The peptide complex of the present invention has the activity to promote the expression of one or more genes selected from the group consisting of Leptin, Adiponectin, IRS-1 (Insulin receptor substrate 1), GLUT4 (Glucose transporter type 4), PGC-1α (Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha), ACOX-1 (Acyl-CoA Oxidase 1), PPAR-α (Peroxisome proliferator-activated receptor-alpha), and CPT-1α (Carnitine palmitoyltransferase 1 alpha) in cells.

[0039] The peptide complex of the present invention has the activity to suppress insulin resistance signals.

[0040] The peptide complex of the present invention has the activity to suppress the phosphorylation of Ser302 of IRS (Insulin receptor kinase) or the phosphorylation of JNK (c-JunN-terminal kinase).

[0041] The peptide complex of the present invention has the activity to suppress the expression of the TNF-α gene, the mTOR (mammalian Target of Rapamycin) gene, or the p70S6K gene under an insulin resistance-inducing environment.

[0042] The peptide complex of the present invention has activity that promotes insulin sensitivity signals.

[0043] The peptide complex of the present invention has the activity to increase the phosphorylation of IRS (Insulin Receptor Substrate) Tyr632, or to promote the activation of PI3K (Phosphoinositide 3-kinase), ATK, or AMPK (AMP-activated protein kinase).

[0044] The peptide complex of the present invention has the activity to suppress the generation of reactive oxygen species (ROS) induced by free fatty acids, the expression of the TNF-α gene, the expression of the TNF-α protein, or the expression of the IL-1β protein.

[0045] The peptide complex of the present invention has the activity to suppress the death of pancreatic and intestinal β-cells induced by free fatty acids.

[0046] Because the peptide complex of the present invention described above possesses the activity described above, it can exhibit excellent efficacy in the treatment of diabetes.

[0047] The peptide comprising the amino acid sequence of Sequence ID No. 2 of the present invention has anti-diabetic activity.

[0048] The peptide comprising the amino acid sequence of SEQ ID NO: 2 of the present invention has the activity to suppress insulin resistance signals or promote insulin sensitivity signals.

[0049] Specifically, the peptide containing the amino acid sequence of Sequence ID No. 2 of the present invention can promote insulin sensitivity signals through its phosphorylation-promoting activity of Tyr632 of the IRS (Insulin receptor substrate), its activation-promoting activity of phospho-AKT, or its phosphorylation-promoting activity of AMPK. Furthermore, the peptide of the present invention can suppress insulin resistance signals under an insulin resistance-inducing environment through its phosphorylation-inhibiting activity of Sre302 of the IRS.

[0050] The peptide comprising the amino acid sequence of Sequence ID No. 2 of the present invention has anti-obesity activity.

[0051] The peptide comprising the amino acid sequence of Sequence ID No. 2 of the present invention has the activity to promote the breakdown of fat in adipocytes.

[0052] Specifically, the peptide containing the amino acid sequence of Sequence ID No. 2 of the present invention can increase the expression of lipolytic enzyme proteins such as ATGL (Adipose triglyceride lipase), pHSL (phosphorylated hormone-sensitive lipase), or PLIN (Perilipin, lipid droplet-associated protein) in adipocytes.

[0053] The peptide containing the amino acid sequence of Sequence ID No. 2 of the present invention, as described above, exhibits the activity described above, and can therefore exert excellent efficacy in the treatment, prevention, or improvement of diabetes and obesity.

[0054] 2. Compositions for the prevention, treatment, or improvement of diabetes and obesity Pharmaceutical composition Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes, comprising as an active ingredient a peptide complex comprising (i) a peptide comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 2.

[0055] As described above, the peptide complex of the present invention has glucose absorption-promoting activity, insulin resistance-inhibiting activity, insulin sensitivity-promoting activity, and pancreatic and intestinal β-cell protective activity, and therefore has excellent therapeutic or preventive activity for diabetes.

[0056] In the present invention, the diabetes may be type 1 diabetes or type 2 diabetes, and more specifically, it may be type 2 diabetes.

[0057] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide complex can promote glucose absorption.

[0058] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide complex can suppress insulin resistance signals or promote insulin sensitivity signals.

[0059] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide complex can suppress the phosphorylation of Ser302 of IRS (Insulin receptor kinase) or the phosphorylation of JNK (c-JunN-terminal kinase).

[0060] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide complex can suppress the expression of the TNF-α gene, the mTOR (mammalian Target Of Rapamycin) gene, or the p70S6K gene under an insulin resistance-inducing environment.

[0061] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide complex can increase the phosphorylation of IRS (Insulin Receptor Substrate) Tyr632, or promote the activation of PI3K (Phosphoinositide 3-kinase), ATK, or AMPK (AMP-activated protein kinase).

[0062] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide complex can promote the expression of one or more genes selected from the group consisting of Leptin, Adiponectin, IRS-1 (Insulin receptor substrate 1), GLUT4 (Glucose transporter type 4), PGC-1α, ACOX-1, PPAR-α, and CPT-1α.

[0063] In the pharmaceutical composition for the prevention or treatment of diabetes, the peptide complex can suppress the generation of reactive oxygen species (ROS) induced by free fatty acids, the expression of the TNF-α gene, the expression of the TNF-α protein, or the expression of the IL-1β protein, or suppress the death of pancreatic and intestinal β cells induced by free fatty acids.

[0064] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes, comprising a peptide containing the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

[0065] As described above, the peptide of the present invention has insulin resistance signal suppression activity and insulin sensitivity promoting activity, and therefore has excellent activity for treating or preventing diabetes.

[0066] In the present invention, the diabetes may be type 1 diabetes or type 2 diabetes, and more specifically, it may be type 2 diabetes.

[0067] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide can suppress insulin resistance signals or promote insulin sensitivity signals.

[0068] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide can promote the phosphorylation of Tyr632 of IRS (Insulin receptor substrate), promote the activation of phospho-AKT, or promote the phosphorylation of AMPK.

[0069] In the aforementioned pharmaceutical composition for the prevention or treatment of diabetes, the peptide can suppress the phosphorylation of Sre302 of the IRS under an insulin resistance-inducing environment.

[0070] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of obesity, comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0071] In the aforementioned pharmaceutical composition for the prevention or treatment of obesity, the peptide can promote the breakdown of fat in adipocytes.

[0072] In the aforementioned pharmaceutical composition for the prevention or treatment of obesity, the peptide can increase the expression of lipolytic enzyme proteins such as ATGL (Adipose triglyceride lipase), pHSL (phosphorylated hormone-sensitive lipase), or PLIN (Perilipin, lipid droplet-associated protein) in adipocytes.

[0073] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of the peptide complex or peptide and a pharmaceutically acceptable carrier.

[0074] The term "therapeutic effective amount" means an amount sufficient to achieve the activity or efficacy of the peptide complex, which is the active ingredient of the pharmaceutical composition of the present invention, for example, an amount sufficient to achieve the efficacy of treating or preventing diabetes or obesity.

[0075] The pharmaceutically acceptable carriers mentioned above include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil, which are commonly used in formulation.

[0076] The pharmaceutical composition of the present invention may further contain, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspension agents, preservatives, and the like, in addition to the above-mentioned components.

[0077] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0078] The pharmaceutical composition of the present invention may be administered by any suitable route for treating diabetes or obesity, for example, by or without a port. In the case of or without a port, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, local administration, or transdermal administration.

[0079] The dosage of the pharmaceutical composition may be 0.0001 ug to 100 mg, 0.001 ug to 100 mg, 0.01 ug to 100 mg, 0.1 ug to 100 mg, or 1.0 ug to 1000 mg per day, but is not limited thereto, and can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0080] The pharmaceutical compositions of the present invention may be manufactured in unit volume form or encapsulated in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. 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, tablet or capsule, and may further contain a dispersant or stabilizer.

[0081] Food composition According to yet another aspect of the present invention, a functional food composition for regulating blood glucose levels is provided, comprising as an active ingredient a peptide complex comprising (i) a peptide comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 2.

[0082] In the functional food composition of the present invention, the adjustment of blood glucose levels may be the adjustment of blood glucose levels in diabetic patients or high-risk patients in the pre-diabetic stage.

[0083] In the functional food composition of the present invention, the diabetes may be type 1 diabetes or type 2 diabetes, and more specifically, it may be type 2 diabetes.

[0084] In the functional food composition of the present invention, the regulation of blood glucose levels may be a decrease in blood glucose.

[0085] In the functional food composition of the present invention, the peptide complex may be included in an appropriate amount selected within the range of 0.0001% to 10% by weight relative to the total weight of the composition.

[0086] Another aspect of the present invention provides a functional food composition for regulating blood glucose levels, comprising a peptide containing the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

[0087] In the functional food composition of the present invention, the adjustment of blood glucose levels may be the adjustment of blood glucose levels in diabetic patients or high-risk patients in the pre-diabetic stage.

[0088] In the functional food composition of the present invention, the diabetes may be type 1 diabetes or type 2 diabetes, and more specifically, it may be type 2 diabetes.

[0089] In the functional food composition of the present invention, the regulation of blood glucose levels may be a decrease in blood glucose levels.

[0090] In the functional food composition for regulating blood glucose levels, the peptide can suppress insulin resistance signals or promote insulin sensitivity signals.

[0091] In the functional food composition for regulating blood glucose levels, the peptide can promote the phosphorylation of Tyr632 of IRS (Insulin receptor substrate), promote the activation of phospho-AKT, or promote the phosphorylation of AMPK.

[0092] In the functional food composition for regulating blood glucose levels, the peptide can suppress the phosphorylation of Sre302 of the IRS under an insulin resistance-inducing environment.

[0093] Another aspect of the present invention provides a functional food composition for preventing or improving obesity, comprising a peptide containing the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

[0094] In the functional food composition for preventing or improving obesity, the peptide can promote the breakdown of fat in fat cells.

[0095] In the functional food composition for preventing or improving obesity, the peptide can increase the expression of lipolytic enzyme proteins such as ATGL (Adipose triglyceride lipase), pHSL (phosphorylated Hormone-Sensitive Lipase), or PLIN (Perilipin, lipiddroplet-associated protein) in adipocytes.

[0096] In the functional food composition of the present invention, the peptide may be included in an appropriate amount selected within the range of 0.0001% to 10% by weight relative to the total weight of the composition.

[0097] In one embodiment, the functional food composition of the present invention may contain a food-grade effective amount of the peptide complex or peptide and a food-grade acceptable carrier.

[0098] The food composition of the present invention may contain, as the active ingredient, a peptide complex, or not only peptides but also ingredients that are normally added during food production, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of the carbohydrates mentioned above may be monosaccharides, such as glucose and fructose; disaccharides, such as maltose, sucrose, and oligosaccharides; and polysaccharides, such as dextrin and cyclodextrin, which are common sugars, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavorings, natural flavorings, thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.) may be used. The proportion of the carbohydrates may be generally about 1 to 20 g, preferably about 5 to 12 g, per 100 g of the food composition of the present invention, but is not limited thereto.

[0099] In addition to the components mentioned above, the functional food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (for cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. It may also contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0100] For example, when the functional food composition of the present invention is manufactured as a drink, in addition to the peptide complex which is the active ingredient of the present invention, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, and the like.

[0101] In another aspect of the present invention, the present invention provides a method for treating diabetes, comprising the step of administering a therapeutically effective amount of a peptide complex comprising (i) a peptide comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 2 to a diabetic patient.

[0102] In yet another aspect of the present invention, the present invention provides a method for controlling blood glucose, comprising the step of administering a therapeutically effective amount of a peptide complex comprising (i) a peptide comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 2 to a subject requiring blood glucose control.

[0103] In another aspect of the present invention, a method for treating, preventing, or improving diabetes is provided, comprising the step of administering a therapeutically effective amount of a peptide containing the amino acid sequence of SEQ ID NO: 2 described above to a diabetic patient.

[0104] In yet another aspect of the present invention, a method for controlling blood glucose is provided, which includes the step of administering a therapeutically effective amount of a peptide containing the amino acid sequence of SEQ ID NO: 2 described above to a subject requiring blood glucose control.

[0105] In another aspect of the present invention, the present invention provides a method for treating, improving, or preventing obesity, comprising the step of administering a therapeutically effective amount of a peptide containing the amino acid sequence of Sequence ID No. 2 described above to a subject in need of treatment for obesity. [Effects of the Invention]

[0106] The peptide complex or peptide of the present invention can lower blood glucose levels by promoting glucose absorption into cells, suppressing insulin resistance signals, promoting insulin sensitivity signals, and inhibiting the death of pancreatic and gallbladder β-cells, which are insulin-producing cells. Furthermore, the peptide of the present invention promotes the breakdown of fat in adipocytes. Therefore, the peptide complex or peptide of the present invention can be used for the treatment, prevention, and improvement of diabetes or obesity, can be usefully used to lower blood glucose levels in diabetic patients or high-risk patients in the prediabetic stage, and can be used for the treatment, prevention, and improvement of obesity.

[0107] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawing]

[0108] [Figure 1A] These experimental results demonstrate that the peptide complex of the present invention promotes glucose absorption into adipocytes. [Figure 1B] These experimental results demonstrate that the peptide complex of the present invention promotes glucose absorption into adipocytes. [Figure 2A] These experimental results demonstrate that the peptide complex of the present invention promotes glucose absorption by myoblasts. [Figure 2B] These experimental results demonstrate that the peptide complex of the present invention promotes glucose absorption by myoblasts. [Figure 3A] These experimental results demonstrate that the peptide complex of the present invention has the efficacy of promoting the activity of Phospho-AMPK and Phospho-ACC, which are insulin sensitivity-promoting factors. [Figure 3B] These experimental results demonstrate that the peptide complex of the present invention has the efficacy of promoting the activity of Phospho-AMPK and Phospho-ACC, which are insulin sensitivity-promoting factors. [Figure 4A] These experimental results demonstrate that the peptide complex of the present invention has the efficacy to suppress the phosphorylation of IRSdml Serin302 and JNK, which are insulin resistance signaling factors. [Figure 4B] These experimental results demonstrate that the peptide complex of the present invention has the efficacy to suppress the phosphorylation of IRSdml Serin302 and JNK, which are insulin resistance signaling factors. [Figure 5A] These experimental results demonstrate that the peptide complex of the present invention reduces the increased expression of the insulin resistance-inducing cytokine TNF-α and the insulin resistance-promoting signaling factors mTOR and p70S6K genes, which are induced by rhTNF-α treatment. [Figure 5B] These experimental results demonstrate that the peptide complex of the present invention reduces the increased expression of the insulin resistance-inducing cytokine TNF-α and the insulin resistance-promoting signaling factors mTOR and p70S6K genes, which are induced by rhTNF-α treatment. [Figure 6A] Experimental results show that when the peptide complex of the present invention is treated with adipocytes in which insulin resistance has been induced by rhTNF-α treatment, the phosphorylation of IRSTyrosin632, an insulin sensitivity-promoting factor, increases, and the activation of PI3K, ATK, and AMPK is also promoted. [Figure 6B] Experimental results show that when the peptide complex of the present invention is treated with adipocytes in which insulin resistance has been induced by rhTNF-α treatment, the phosphorylation of IRSTyrosin632, an insulin sensitivity-promoting factor, increases, and the activation of PI3K, ATK, and AMPK is also promoted. [Figure 7A]Experimental results show that in adipocytes, the expression of glucose absorption-related genes Leptin, Adiponectin, IRS-1, and GLUT4, which were reduced by rhTNF-α treatment, increased upon treatment with the peptide complex of the present invention. [Figure 7B] Experimental results show that in adipocytes, the expression of glucose absorption-related genes Leptin, Adiponectin, IRS-1, and GLUT4, which were reduced by rhTNF-α treatment, increased upon treatment with the peptide complex of the present invention. [Figure 8A] These experimental results demonstrate that palmitic acid-induced reactive oxygen species (ROS) are reduced by the peptide complex of the present invention. [Figure 8B] These experimental results demonstrate that palmitic acid-induced reactive oxygen species (ROS) are reduced by the peptide complex of the present invention. [Figure 9A] This experimental result demonstrates that the gene expression of TNF-α, an inflammatory protein induced by palmitic acid, is significantly reduced by treatment with the peptide complex of the present invention. [Figure 9B] This experimental result demonstrates that the gene expression of TNF-α, an inflammatory protein induced by palmitic acid, is significantly reduced by treatment with the peptide complex of the present invention. [Figure 10A] These experimental results demonstrate that the protein expression levels of TNF-α and IL-1β, which were increased by palmitic acid treatment in INS-1 (Rat pancreatic beta cell) cells, were significantly reduced again by treatment with the peptide complex of the present invention. [Figure 10B] These experimental results demonstrate that the protein expression levels of TNF-α and IL-1β, which were increased by palmitic acid treatment in INS-1 (Rat pancreatic beta cell) cells, were significantly reduced again by treatment with the peptide complex of the present invention. [Figure 11] These experimental results demonstrate that the increase in INS-1 cell death induced by palmitic acid treatment is reduced again and the cell viability increases upon treatment with the peptide complex of the present invention. [Figure 12A] These experimental results demonstrate that the expression of FFA beta oxidation-related genes, such as PGC-1α, ACOX-1, PPAR-α, or CPT-1α, is promoted by the peptide complex of the present invention in HepG2 cells. [Figure 12B] These experimental results demonstrate that the expression of FFA beta oxidation-related genes, such as PGC-1α, ACOX-1, PPAR-α, or CPT-1α, is promoted by the peptide complex of the present invention in HepG2 cells. [Figure 13A] The peptide of the present invention exhibits activity that increases the expression of lipolytic enzyme proteins ATGL (Adipose triglyceride lipase), pHSL (phosphorylated hormone-sensitive lipase), and PLIN (Perilipin, lipid droplet-associated protein) in adipocytes. [Figure 13B] The peptide of the present invention exhibits activity that increases the expression of lipolytic enzyme proteins ATGL (Adipose triglyceride lipase), pHSL (phosphorylated hormone-sensitive lipase), and PLIN (Perilipin, lipid droplet-associated protein) in adipocytes. [Figure 14] When the peptide of the present invention is used to treat adipocytes, the amount of glycerol released, which is a product of lipolysis, increases in a concentration-dependent manner with respect to the treated peptide. [Figure 15A] When adipocytes are treated with the peptide of the present invention, the phosphorylation of IRS (Insulin Receptor Substrate) Tyr632, an insulin sensitivity-promoting factor, and the activation of phosphorylation-AKT are promoted, and the phosphorylation of the signaling protein AMPK is increased. On the other hand, the phosphorylation of IRS (Insulin Receptor Substrate) Ser302 is reduced under an insulin resistance-inducing environment. [Figure 15B] When adipocytes are treated with the peptide of the present invention, the phosphorylation of IRS (Insulin Receptor Substrate) Tyr632, an insulin sensitivity-promoting factor, and the activation of phosphorylation-AKT are promoted, leading to an increase in the phosphorylation of the signaling protein AMPK. On the other hand, the phosphorylation of IRS (Insulin Receptor Substrate) Ser302 is reduced under an insulin resistance-inducing environment. [Modes for carrying out the invention]

[0109] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0110] Examples Manufacturing Example 1: Production of Peptides and Peptide Complexes Peptides having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, as listed in Table 1 below, were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA). These synthesized peptides were then separated into pure molecules using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLCBEH300C18 (2.1mm x 100mm, 1.7um, Waters Co, USA).

[0111] [Table 1]

[0112] Equal amounts of the peptides produced under Sequence ID No. 1 and No. 2 were mixed to produce a peptide complex, and its efficacy was evaluated. The efficacy of the peptide produced under Sequence ID No. 2 was also evaluated.

[0113] Experimental Example 1: Promoting glucose absorption by adipocytes. We conducted experiments using adipocytes to investigate whether the peptide complex produced in Production Example 1 promotes glucose uptake into cells.

[0114] When 3T3-L1 predipocytes reach 80% confluence, 5 x 10 units are placed in a 96-well plate. 3 Cells were seeded in wells and used for the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / mL insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were cultured for 16 hours in glucose-free medium, and then starvation was induced by culturing for 40 minutes in 100 uL KRPH buffer containing 2% BSA. Samples were prepared by treating with rhTNF-α (2 nM), insulin (10 ug / ml), and peptide complex (2, 20 ug / ml) and culturing for 1 hour, followed by treatment with 80 uM 2-NBDG and culturing for 1 hour. Glucose absorption was measured using the "Glucose uptake assay kit" (Abcam, Cambridge, UK), and glucose absorbed by cells was imaged using a fluorescence microscope (ECLIPSE 80i, Nikon, Japan).

[0115] The experimental results, as shown in Figures 1A and 1B, confirmed that the intracellular absorption of glucose, which was suppressed by TNF-α, increased upon treatment with the peptide complex. Furthermore, in Experimental Example 3 described later, it was confirmed that this effect occurs via the AMPK (AMP-activated protein kinase) and ACC (Acetyl-CoA carboxylase) signaling pathway. Through these experimental results, it was confirmed that the peptide complex significantly increased the suppression of intracellular glucose absorption induced by inflammation.

[0116] Experimental Example 2: Promoting glucose absorption in muscle cells (myocytes) We experimented with whether the peptide complex produced in Production Example 1 promotes glucose absorption into cells using myoblasts.

[0117] C2C12 myoblasts were placed in 5x10 cells in a 96-well plate. 3 Cells were seeded in wells and cultured for 2 days in 10% FBS medium containing DMEM. After changing the DMEM medium with 2% horse serum, C2C12 myoblasts were cultured for 6 days to induce differentiation into myotubes. Cultured myotubes were treated with rhTNF-α (2nM), insulin (10ug / ml), and peptide complex (2, 20ug / ml) and cultured for 1 hour, then treated with 80uM 2-NBDG and cultured for 1 hour to prepare samples. Glucose uptake assay kit (Abcam, Cambridge, UK) was measured, and glucose absorbed by cells was imaged via fluorescence microscopy (ECLIPSE 80i, Nikon, Japan). The experimental results, as shown in Figures 2A and 2B, confirmed that glucose absorption into myoblasts, which was suppressed by rhTNF-α treatment, was increased by the peptide complex of the present invention.

[0118] Experimental Example 3: Signaling pathway of peptide complexes in glucose absorption enhancement We confirmed whether the peptide complex of Production Example 1 has the effect of promoting the activity of insulin sensitivity enhancers through protein expression analysis (western blot analysis).

[0119] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 96-well plate. 3Cells were seeded in wells and used for the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / mL insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to serum-free medium and cultured for 4 hours to induce starvation. The peptide complexes from Preparation Example 1 (0.2, 2, 20 ug / ml) were then treated and cultured for 30 minutes, followed by lysis buffer to dissolve the proteins. The proteins obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes were quantified using a BCA kit. The proteins were then subjected to SDD-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and electrotransferred onto a membrane. After blocking the protein-coated membrane with 5% skim milk, the primary antibody was reacted overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour, washed again with PBS-T, and then visualized using Gel Doc (Bio-Rad, Hercules, CA, USA) via Western detection reagent (Elpis Biotech, Daejeon, Korea). The antibodies used in the experiment were: anti-Phospho-AMPK antibody (Cell signaling technology (CST), USA), anti-Phospho-ACC antibody (Cell signaling technology (CST), USA), and anti-α-tubulin antibody (Santa Cruz Biotechnology). As shown in Figures 3A and 3B, the experimental results confirmed that the peptide complex of the present invention has the efficacy to promote the activity of the insulin sensitivity-enhancing factors Phospho-AMPK and Phospho-ACC.

[0120] Experimental Example 4: Suppression of insulin resistance signals by peptide complexes We confirmed whether the peptide complex produced in Production Example 1 has the effect of suppressing insulin resistance signals via protein expression analysis (western blot analysis).

[0121] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 6-well plate. 5Cells were seeded in wells and used for the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / mL insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to serum-free medium and cultured for 4 hours to induce starvation. After treating the cells with rhTNF-α (2 nM), insulin (1 ug / ml), and peptide complexes (0.2, 2, 20 ug / ml) and culturing for 30 minutes, the cells from each treatment group were lysed with lysis buffer, and the obtained proteins were quantified using a BCA kit after centrifugation at 4°C and 12,000 rpm for 30 minutes. Proteins were electrotransferred onto a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membranes with attached proteins were blocked by treatment with 5% skim milk, and then the primary antibody was reacted overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour, washed again with PBS-T, and then visualized via Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA). The antibodies used in the experiment were: anti-Phospho-IRS (Ser302) antibody (Cell signaling technology (CST), USA), anti-Phospho-JNK antibody (Santa Cruz Biotechnology, USA), and anti-α-tubulin antibody (Santa Cruz Biotechnology, USA).As shown in Figures 4A and 4B, the experimental results confirm that the peptide complex of the present invention suppresses the phosphorylation of Serin302 and JNK (c-Jun N-terminal kinase), which are insulin resistance signaling factors induced by TNF-α treatment.

[0122] Experimental Example 5: Suppression of insulin resistance-inducing gene expression by peptide complexes We experimented to see if the peptide complex produced in Production Example 1 suppressed the expression of insulin resistance-inducing genes. After inducing insulin resistance in adipocytes by treating them with rhTNF-α, we treated them with the peptide complex and then measured the expression levels of the mTOR and p70S6K genes.

[0123] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 6-well plate. 5Cells were seeded in wells and used for the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / mL insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to serum-free medium and cultured for 4 hours to induce starvation. A control group with no treatment and a control group treated with rhTNF-α (2 nM), insulin (1 ug / ml), and peptide complexes (0.2, 2, 20 ug / ml) were used, and RNA was extracted after 24 hours and RT-PCR was performed. RNA was extracted from 3T3-L1 cells using the easy-BLUE™ Total RNA Extraction Kit (Qiagen, Germany). The extracted RNA was converted to cDNA using an RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). After preparing the reaction mixture using a PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers targeting TNF-α, mTOR (mammalian Target of Rapamycin), p70S6K, or the GAPDH gene, PCR was performed using a PCR machine (Eppendorf, Germany). Subsequently, the mRNA expression pattern was determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiment are shown in Table 2.

[0124] [Table 2]

[0125] The experimental results, as shown in Figures 5A and 5B, showed that the expression of the TNF-α gene, an insulin resistance-inducing inflammatory cytokine, and the mTOR and p70S6K genes, which are insulin resistance-promoting signaling factors, increased upon rhTNF-α treatment. However, it was confirmed that the expression of these genes decreased again upon treatment with the peptide complex.

[0126] Experimental Example 6: Enhancing insulin sensitivity signaling by peptide complexes We experimented to see if the peptide complex produced in Production Example 1 promotes insulin sensitivity signals.

[0127] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 6-well plate. 5Cells were seeded in wells and used for the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / mL insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to serum-free medium and cultured for 4 hours to induce starvation. After treating the cells with rhTNF-α (2 nM), insulin (1 ug / ml), and peptide complexes (0.2, 2, 20 ug / ml) and culturing for 30 minutes, the cells from each treatment group were lysed with lysis buffer, and the obtained proteins were quantified using a BCA kit after centrifugation at 4°C and 12,000 rpm for 30 minutes. Proteins were electrotransferred onto a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membranes with attached proteins were blocked by treating them with 5% skim milk, and then the primary antibody was reacted overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour, washed again with PBS-T, and then visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) with Gel Doc (Bio-Rad, Hercules, CA, USA). The antibodies used in the experiment were as follows: anti-Phospho-IRS(Tyr632) antibody (Cell signaling technology (CST), USA), anti-Phospho-PI3K antibody (Cell signaling technology (CST), USA), anti-Phospho-AKT antibody (Cell signaling technology (CST), USA), anti-Phospho-AMPK antibody (Cell signaling technology (CST), USA), and anti-α-tubulin antibody (Santa Cruz Biotechnology, USA).The experimental results, as shown in Figures 6A and 6B, confirmed that in adipocytes in which insulin resistance was induced by rhTNF-α treatment, treatment with the peptide complex increased the phosphorylation of IRS (Insulin Receptor Substrate) Tyrosin 632, an insulin sensitivity-promoting factor, and thereby also promoted the activation of PI3K (Phosphoinositide 3-kinase), ATK, and AMPK (AMP-activated protein kinase).

[0128] Experimental Example 7: Promotion of glucose absorption-related gene expression We confirmed whether the peptide complex produced in Production Example 1 promotes the expression of glucose absorption-related genes by analyzing the expression of Leptin, Adiponectin, IRS-1 (Insulin Receptor Substrate 1), and GLUT4 (Glucose transporter type 4) genes.

[0129] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 6-well plate. 5Cells were seeded in wells and used for the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / mL insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to serum-free medium and cultured for 4 hours to induce starvation. A control group with no treatment and a control group treated with rhTNF-α (2 nM), insulin (1 ug / ml), and peptide complexes (0.2, 2, 20 ug / ml) were used, and RNA was extracted after 24 hours and RT-PCR was performed. RNA was extracted from 3T3-L1 cells using the easy-BLUE™ Total RNA Extraction Kit (Qiagen, Germany). The extracted RNA was converted to cDNA using an RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). After preparing the reaction mixture using a PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for each gene, PCR was performed using a PCR machine (Eppendorf, Germany). Subsequently, the mRNA expression pattern was determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiment are shown in Table 3.

[0130] [Table 3]

[0131] The experimental results, as shown in Figures 7A and 7B, confirmed that the expression of glucose absorption-related genes Leptin, Adiponectin, IRS-1, and GLUT4, which were decreased in adipocytes by rhTNF-α treatment, increased with peptide complex treatment.

[0132] Experimental Example 8: Suppression of ROS generation induced by palmitate. We confirmed, via intracellular ROS detection analysis (FACS), whether the peptide complex produced in Production Example 1 suppresses the production of reactive oxygen species (ROS) induced by palmitic acid, a type of free fatty acid.

[0133] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 6-well plate. 5 Cells were seeded in wells and used in the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / mL insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to serum-free medium and cultured for 4 hours to induce starvation. A control group with no treatment and cells treated with rhTNF-α (2 nM), insulin (1 ug / ml), and peptide complex (2 ug / ml) were cultured for 24 hours, and oxidative activity was measured by the degree of fluorescence using FACS (Becton Dickinson & Company (BD), USA) 30 minutes after treatment with DCF-DH. As shown in the graphs in Figures 8A and 8B, the measurement results confirmed that palmitic acid-induced reactive oxygen species (ROS) were reduced by the peptide complex.

[0134] Experimental Example 9: Suppression of TNF-α gene expression induced by palmitic acid. We confirmed, via gene expression analysis (RT-PCR), whether the peptide complex produced in Production Example 1 suppresses inflammation induced by palmitic acid, a type of free fatty acid.

[0135] INS-1 cells were starved using serum-free media RPMI 1640 (Gibco, New York, USA) and pretreated with 25 μM palmitic acid for 2 hours. These cells were then treated with peptide complexes (0.2, 2, 20 μg / ml). After 24 hours, RNA was extracted using the easy-BLUE™ Total RNA Extraction Kit (Qiagen, Germany). The extracted RNA was converted to cDNA using an RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). A reaction mixture was prepared using the PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for the TNF-α and GAPDH genes. PCR was then performed using a PCR machine (Eppendorf, Germany). Subsequently, mRNA expression patterns were determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are shown in Table 4.

[0136] [Table 4]

[0137] The experimental results, as shown in Figures 9A and 9B, confirmed that the gene expression of TNF-α, an inflammatory protein induced by palmitic acid, was significantly reduced by peptide complex treatment.

[0138] Experimental Example 10: Suppression of inflammatory cytokine expression induced by palmitic acid We confirmed, via protein expression analysis (western blot analysis), whether the peptide complex produced in Production Example 1 suppresses the expression of inflammatory cytokines induced by palmitic acid, a type of free fatty acid.

[0139] INS-1 cells (Rat pancreatic beta cells) were starved in serum-free medium RPMI 1640 (Gibco, New York, USA) and pretreated with 25 μM palmitic acid for 2 hours. Peptide complexes (0.2, 2, 20 μg / ml) were then added. After 24 hours, the cells were washed once with PBS. A lysis buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 1% NP-40, and a protease inhibitor (Millipore, Darmstadt, Germany) was added, and the cells were dissolved on ice for 30 minutes. The proteins obtained were then centrifuged at 4°C and 13,000 rpm for 10 minutes and quantified using a BCA kit (Thermo Fisher Scientific, Waltham, USA). Equal amounts of protein were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred onto a polyvinylidene difluoride membrane. To block nonspecific antibody binding, the protein-bound membranes were treated with 5% skim milk (BD, New Jersey, USA) and blocked for 1 hour. The primary antibody was then reacted overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1-2 hours, washed again with PBS-T, and the protein was visualized on X-ray film using ECL (enhanced chemiluminescence) (Thermo Fisher Scientific, Waltham, USA). The antibodies used in the experiment were as follows: anti-TNF-α antibody (Cell signaling technology (CST), USA), anti-IL-1β antibody (Cell signaling technology (CST), USA), and anti-Actin antibody (Santa Cruz Biotechnology, USA).The experimental results, as shown in Figures 10A and 10B, confirmed that the expression levels of TNF-α and IL-1β proteins, which increased in INS-1 (Rat pancreatic beta cell) cells after palmitic acid treatment, significantly decreased again after peptide complex treatment.

[0140] Experimental Example 11: Suppression of palmitic acid-induced pancreatic β-cell death We confirmed, via cell cytotoxicity assay, whether the peptide complex produced in Production Example 1 suppresses the death of pancreatic beta cells induced by palmitic acid, a type of free fatty acid.

[0141] INS-1 cells (Rat pancreatic beta cells) were starved in serum-free medium RPMI 1640 (Gibco, New York, USA) and pretreated with 25 μM palmitic acid for 2 hours. They were then treated with peptide complexes (0.2, 2, 20 μg / ml). After 24 hours, CCK-8 solution (Dojindo, Kumamoto, Japan) was added to 1 / 10 of the culture fluid volume. Absorbance was measured at 30-minute or 1-hour intervals, and the experiment was terminated when the average absorbance of the control group reached 1.0. The absorbance of the reaction product was measured at a wavelength of 450 nm using a microplate reader. As shown in Figure 11, the experimental results confirmed that the increase in INS-1 cell death induced by palmitic acid treatment decreased again after treatment with the peptide complex, and cell viability increased.

[0142] Experimental Example 12: Promotion of glucose absorption-related gene expression Regarding whether the peptide complex produced in Production Example 1 promotes the expression of genes related to glucose absorption, PGC-1α (Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha), ACOX-1 (Acyl-CoA Oxidase 1), PPAR-α (Peroxisome proliferator-activated receptor-alpha), or CPT-1α (Carnitine palmitoyltransferase 1 alpha), it was confirmed via gene expression analysis (RT-PCR).

[0143] When HepG2 liver cancer cells reached 80% confluence, they were seeded at 2x10 5 cells / well in a 12-well plate and used for the experiment. The next day, serum-free media was alternated to induce a 4-hour starvation state. After 4 hours, a control group that was not treated and groups treated with the peptide complex (0.2, 2, 20 ug / ml) were set up, and RNA was extracted after 24 hours and RT-PCR was performed. RNA was extracted using the easy-BLUE™ Total RNA extraction kit (Qiagen, Germany). The extracted RNA was converted to cDNA using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). After preparing a reaction mixture with PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for the PGC-1α, ACOX-1, PPAR-α, or CPT-1α genes, PCR was performed using a PCR machine (Eppendorf, Germany). Subsequently, the mRNA expression pattern was determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiment are shown in Table 5 below.

[0144]

Table 5

[0145] As shown in Figures 12A and 12B, the experimental results confirmed that the expression of FFA beta oxidation-related genes, such as PGC-1α, ACOX-1, PPAR-α, or CPT-1α, is promoted in HepG2 cells by peptide complexes.

[0146] Experimental Example 13: Promotion of lipolytic enzyme protein expression in adipocytes We experimentally investigated whether the peptide with the amino acid sequence of Sequence ID No. 2, produced in Production Example 1, promotes the expression of lipolytic enzyme proteins in adipocytes via protein expression analysis (western blot analysis).

[0147] When 3T3-L1 predipocytes reach 80% confluence, 5 x 10 units are placed in a 6-well plate. 5Cells were seeded in wells and used in the experiment. Two days after reaching confluence, differentiation into adipocytes was induced by switching to differentiation induction medium [DMEM medium containing 10% FBS, 1 ug / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 uM dexamethasone (Sigma, USA)]. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / ml insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to srum-free medium and cultured for 4 hours to induce starvation. Cells were divided into three groups: an untreated group (negative control group), a group treated with the peptide from Production Example 1 (2, 20 ug / ml), and a group treated with TNFα (20 nM) (positive control group). Cells were treated with the specified concentrations of each substance and cultured for 1 hour. Then, lysis buffer was added to the cells of each treatment group to lyse them. After lysis, the cells were centrifuged at 4°C and 12,000 rpm for 30 minutes, and the obtained proteins were quantified using a BCA kit. The proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred onto a membrane. The membranes with attached proteins were blocked by treating them with 5% skim milk, and then the primary antibody was reacted overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour, washed again with PBS-T, and then visualized via Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).The antibodies used in the experiment were as follows: anti-ATGL antibody (Cell signaling technology (CST), USA), anti-pHSL antibody (Cell signaling technology (CST), USA), anti-PLIN antibody (Cell signaling technology (CST), USA), and anti-α-tubulin antibody (Santa Cruz Biotechnology, USA). As shown in Figures 13A and 13b, the experimental results confirmed that treatment with the peptide of the present invention increased the expression of the lipolytic enzyme proteins ATGL (Adipose triglyceride lipase), pHSL (phosphorylated Hormone-Sensitive Lipase), and PLIN (Perilipin, lipid droplet-associated protein) in adipocytes.

[0148] Experimental Example 14: Promoting lipolysis in fat cells To determine whether the peptide with the amino acid sequence of Sequence ID No. 2, produced in Production Example 1, promotes lipolysis in adipocytes, we analyzed the amount of glycerol released, a product of lipolysis.

[0149] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 6-well plate. 5Cells were seeded in wells and used in the experiment. Two days after reaching confluence, differentiation into adipocytes was induced by switching to differentiation induction medium [DMEM medium containing 10% FBS, 1 ug / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 uM dexamethasone (Sigma, USA)]. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / ml insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to srum-free medium and cultured for 4 hours to induce starvation. Cells were divided into three groups: an untreated group (negative control group), a group treated with the peptide from Production Example 1 (2, 20 ug / ml), and a group treated with TNF-α (20 nM) (positive control group). Each group was treated with the specified concentration of the substance and cultured for 48 hours to obtain the supernatant. The supernatant was then subjected to a glycerol release assay using the "Glycerol Colormetric assay kit" (Cayman Chemical, USA) to compare the amount of glycerol released. As shown in Figure 14, the glycerol release assay confirmed that when treated with the peptide of the present invention, the amount of glycerol released from adipocytes, a product of lipolysis, increased in a concentration-dependent manner with the treated peptide.

[0150] Experimental Example 15: Suppression of insulin resistance signals and enhancement of insulin sensitivity signals We experimentally investigated whether the peptide with the amino acid sequence of Sequence ID No. 2, produced in Production Example 1, suppresses insulin resistance and promotes insulin sensitivity signaling in adipocytes by analyzing protein expression.

[0151] When 3T3-L1 adipocytes reach 80% confluence, 5 x 10⁶ cells are placed in a 6-well plate. 5Cells were seeded in wells and used in the experiment. Two days after reaching confluence, differentiation into adipocytes was induced by switching to differentiation induction medium [DMEM medium containing 10% FBS, 1 ug / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 uM dexamethasone (Sigma, USA)]. Two days after induction, the cells were cultured for two days in a medium containing 10% FBS and 10 ug / ml insulin, and then switched to a medium containing 10% FBS every two days until differentiation into adipocytes was complete. Once differentiation was complete, the cells were switched to srum-free medium and cultured for 4 hours to induce starvation. Then, rhTNF-α (2 nM), insulin (1 ug / ml), and the peptide from Production Example 1 (2, 20 ug / ml) were treated and cultured for 1 hour. After 30 minutes of incubation, cells from each treatment group were lysed with lysis buffer, and the obtained proteins were centrifuged at 12,000 rpm at 4°C for 30 minutes. The proteins were then quantified using a BCA kit. The proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred onto a membrane. The membranes with attached proteins were blocked by treating them with 5% skim milk, and then reacted with the primary antibody overnight at 4°C. After washing with PBS-T, the secondary antibody was reacted at room temperature for 1 hour, washed again with PBS-T, and then visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) with Gel Doc (Bio-Rad, Hercules, CA, USA).The antibodies used in the experiment were as follows: anti-Phospho-IRS(Ser302) antibody (Cell signaling technology (CST), USA), anti-Phospho-IRS(Tyr632) antibody (Cell signaling technology (CST), USA), anti-Phospho-AKT antibody (Cell signaling technology (CST), USA), anti-Phospho-AMPK antibody (Cell signaling technology (CST), USA), and anti-α-tubulin antibody (Santa Cruz Biotechnology, USA). The experimental results, as shown in Figures 15A and 15B, confirmed that treatment with the peptide of the present invention promoted the phosphorylation of IRS (Insulin Receptor Substrate) Tyr632 and the activation of phosphorylation-AKT, which are insulin sensitivity-enhancing factors. Furthermore, an increase in the phosphorylation of AMPK, an insulin sensitivity-enhancing signaling protein, was confirmed. On the other hand, we confirmed that reducing the phosphorylation of IRS (Insulin Receptor Substrate) Ser302 under an insulin resistance-inducing environment suppresses insulin resistance.

[0152] Manufacturing Example 2: Manufacturing of Pharmaceutical Compositions 2-1. Manufacturing of powdered medicines 2g of the peptide complex or peptide of the present invention 1g lactose The aforementioned components were mixed and filled into a sealed bag to produce a powder.

[0153] 2-2. Manufacturing of Tablets 100 mg of the peptide complex or peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg After mixing the aforementioned components, tablets were manufactured by compressing them using a conventional tablet manufacturing method.

[0154] 2-3. Manufacturing of Capsules 100 mg of the peptide complex or peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg After mixing the aforementioned components, the mixture was filled into gelatin capsules using a conventional capsule manufacturing method to produce capsules.

[0155] 2-4. Ring Manufacturing 1 g of the peptide complex or peptide of the present invention Lactose 1.5g 1g glycerin 0.5g xylitol After mixing the aforementioned components, the product was manufactured using a conventional method to produce 4 g per ring.

[0156] 2-5. Granule production 150 mg of the peptide complex or peptide of the present invention Soybean extract 50mg Glucose 200mg Starch 600mg After mixing the aforementioned components, 100 mg of 30% ethanol was added and dried at 60°C to form granules, which were then filled into bags.

[0157] Manufacturing Example 3: Manufacturing of Functional Food Compositions 3-1. Manufacturing of health foods The peptide complex of the present invention or 500 µg of the peptide of the present invention Appropriate amount of vitamin mixture Vitamin A acetate 70mg Vitamin E 1.0 mg Vitamin D 0.13mg Vitamin B2 0.15mg Vitamin B6 0.5mg Vitamin B12 0.2mg Vitamin C 10mg Biotin 10mg Nicotinamide 1.7mg Folic acid 50mg Calcium pantothenate 0.5mg Appropriate amount of inorganic mixture Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Monopotassium phosphate 15 mg Dicalcium phosphate 55mg Potassium citrate 90mg Calcium carbonate 100mg Magnesium chloride 24.8 mg The composition ratio of the vitamin and mineral mixture is a mixture of components relatively suitable for health foods as shown in the preferred examples, but the mixing ratio can be arbitrarily modified. After mixing the components using a conventional health food manufacturing method, granules can be produced and used in the manufacture of health food compositions using conventional methods.

[0158] 3-2. Manufacturing of health drinks 500ug of the peptide complex or peptide of the present invention Citric acid 1000mg Oligosaccharide 100g Plum concentrate 2g Taurine 1g Add purified water to make a total of 900ml After mixing the ingredients using a conventional method for manufacturing health beverages, the mixture was stirred and heated at 85°C for approximately one hour. The resulting solution was then filtered, collected in a sterilized container, sealed and sterilized, and then refrigerated before being used in the production of health beverage compositions. The aforementioned composition ratio is a mixture of ingredients relatively suitable for beverages, as preferred in the examples, but the blending ratio can be arbitrarily modified according to regional and ethnic preferences such as demand levels, demand countries, and intended use.

[0159] While the above has provided illustrative examples of typical embodiments of this application, the scope of this application is not limited to such specific embodiments, and any person with ordinary skill in the art can appropriately modify the claims of this application.

Claims

1. A peptide mixture comprising (i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, and (ii) a peptide consisting of the amino acid sequence of SEQ ID NO:

2.

2. The peptide mixture according to claim 1, wherein the peptide mixture has anti-diabetic activity.

3. A pharmaceutical composition for the prevention or treatment of diabetes, comprising the peptide mixture described in claim 1 as an active ingredient.

4. The peptide mixture promotes glucose absorption, as described in claim 3, for the pharmaceutical composition for the prevention or treatment of diabetes.

5. The peptide mixture suppresses insulin resistance signals or promotes insulin sensitivity signals, the pharmaceutical composition for the prevention or treatment of diabetes according to claim 3.

6. The peptide mixture inhibits the phosphorylation of Ser302 of IRS (Insulin Receptor Substrate) or the phosphorylation of JNK (c-Jun N-terminal kinase), as described in claim 3, for the prevention or treatment of diabetes.

7. The peptide mixture suppresses the expression of the TNF-α gene, the mTOR (mammalian Target Of Rapamycin) gene, or the p70S6K gene under an insulin resistance-inducing environment, as described in claim 3, for the prevention or treatment of diabetes.

8. The peptide mixture increases the phosphorylation of IRS (Insulin Receptor Substrate) Tyr632 or promotes the activation of PI3K (Phosphoinoside 3-kinase), AKT, or AMPK (AMP-activated protein kinase), according to claim 3, a pharmaceutical composition for the prevention or treatment of diabetes.

9. The peptide mixture promotes the expression of one or more genes selected from the group consisting of Leptin, Adiponectin, IRS-1 (Insulin Receptor Substrate 1), GLUT4 (Glucose transporter type 4), PGC-1α, ACOX-1, PPAR-α, and CPT-1α, as described in claim 3, for the prevention or treatment of diabetes.

10. The peptide mixture suppresses the generation of reactive oxygen species (ROS) induced by free fatty acids, the expression of the TNF-α gene, the expression of the TNF-α protein, or the expression of the IL-1β protein, or suppresses the death of pancreatic β cells induced by free fatty acids, as described in claim 3, for the prevention or treatment of diabetes.

11. A functional food composition for regulating blood glucose levels, comprising the peptide mixture described in claim 1 as an active ingredient.

12. The peptide mixture promotes glucose absorption, as described in claim 11, for the functional food composition for regulating blood glucose levels.

13. The peptide mixture suppresses insulin resistance signals or promotes insulin sensitivity signals, as described in claim 11, for functional food composition for regulating blood glucose levels.

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

2.

15. The peptide according to claim 14, wherein the peptide has anti-diabetic activity or anti-obesity activity.

16. A pharmaceutical composition for the prevention or treatment of diabetes, comprising the peptide described in claim 14 as an active ingredient.

17. The peptide suppresses insulin resistance signals or promotes insulin sensitivity signals, the pharmaceutical composition for the prevention or treatment of diabetes according to claim 16.

18. The peptide has the activity to promote the phosphorylation of Tyr632 of IRS (Insulin Receptor Substrate), the activation of phosphor-AKT, or the phosphorylation of AMPK. Alternatively, the pharmaceutical composition for the prevention or treatment of diabetes according to claim 16, which has phosphorylation inhibitory activity of Ser302 of IRS under an insulin resistance-inducing environment.

19. A pharmaceutical composition for the prevention or treatment of obesity, comprising the peptide described in claim 14 as an active ingredient.

20. The peptide promotes the breakdown of fat in adipocytes, as described in claim 19, for the pharmaceutical composition for the prevention or treatment of obesity.

21. The pharmaceutically active composition for the prevention or treatment of obesity according to claim 19, wherein the peptide increases the expression of lipolytic enzyme proteins ATGL (Adipose triglyceride lipase), pHSL (phosphorylated hormone-sensitive lipase), or PLIN (Perilipin, lipid droplet-associated protein) in adipocytes.

22. A functional food composition for regulating blood glucose levels, comprising the peptide described in claim 14 as an active ingredient.

23. The peptide suppresses insulin resistance signals or promotes insulin sensitivity signals, as described in claim 22, for the functional food composition for regulating blood glucose levels.

24. A functional food composition for preventing or improving obesity, comprising the peptide described in claim 14 as an active ingredient.

25. The peptide promotes the breakdown of fat in fat cells, as described in claim 24, for the functional food composition for preventing or improving obesity.

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