Peptide and peptide complex having antidiabetic activity and use thereof

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] The present invention relates to a peptide conjugate having anti-diabetic activity and uses thereof. The present invention also relates to a peptide having anti-obesity and anti-diabetic activity and uses thereof. [Background technology]

[0002] Diabetes is a type of metabolic disease characterized by insufficient insulin secretion or normal function, and is characterized by hyperglycemia, which is an increase in the concentration of glucose in the blood, which causes various symptoms and signs, and glucose is excreted in the urine. Recently, the incidence of diabetes has been increasing explosively due to the increase in obesity rates, especially abdominal obesity. Diabetes is broadly divided into type 1 diabetes, which is insulin-dependent diabetes, and type 2 diabetes, which is non-insulin-dependent diabetes. Type 2 diabetes is characterized by hyperglycemia, insulin resistance, and relative insulin secretion disorders.

[0003] When food is ingested, glucose from the food is absorbed in the digestive tract, stimulating insulin secretion in the beta cells of the pancreas, and the secreted insulin promotes glucose absorption into muscles. Insulin is also involved in glucose absorption in the liver, but mainly inhibits glucose production in the liver. Insulin reduces blood glucose levels by inhibiting glucose production in the liver and promoting glucose absorption into peripheral tissues, including muscles. Insulin resistance is a state in which blood glucose response to insulin is lower than normal at a given insulin concentration. Insulin regulates blood glucose by promoting glucose uptake into muscles or inhibiting glucose production in the liver, and insulin resistance refers to a state in which such insulin action is reduced even when there is no insulin deficiency. Insulin receptors on the cell membrane become involved in the process of absorbing glucose into cells of peripheral tissues, and insulin resistance occurs when the number of insulin receptors decreases or intracellular defects occur after receptor binding. In type 2 diabetes, although defects in the insulin receptor are sometimes found, it is known that the main cause is intracellular defects after the receptor, i.e., impaired phosphorylation / dephosphorylation regulated by insulin. Among these mechanisms, it is known that impaired PI3K (Phosphoinositide 3-kinase) signaling reduces the movement of the glucose transporter GLUT-4 (Glucose transporter type 4) to the cell membrane.

[0004] Currently, lifestyle modification (dietary therapy, exercise therapy) and drug therapy are used to regulate blood sugar. However, strict management and implementation of dietary therapy and exercise therapy are difficult, and their effectiveness is limited. Therefore, most diabetic patients rely on lifestyle modification and drug therapy such as insulin, insulin secretagogues, insulin sensitizers, and hypoglycemic agents to regulate blood sugar.

[0005] Obesity refers to a state in which excess energy is stored as body fat when there is an imbalance between the energy intake from food and the energy consumed, resulting in the excess of adipose tissue in the body. According to the World Health Organisation (WHO), over one billion adults worldwide are overweight, of which at least three million are clinically obese, with a significant increase in the United States and Europe. Overweight and obesity increase blood pressure and cholesterol levels, causing various diseases such as heart disease, diabetes and arthritis, and increasing the incidence of various adult diseases. Moreover, overweight and obesity are one of the factors that increase the incidence of various adult diseases such as arteriosclerosis, high blood pressure, hyperlipidemia and heart disease not only in adults but also in children and adolescents.

[0006] Representative obesity treatment drugs currently approved by the US FDA and widely prescribed include drugs that act on the central nervous system to act as appetite suppressants, and orlistat (Xenical), an inhibitor of the digestive enzyme lipase secreted from the pancreas. Many central nervous system drugs, including sibutramine, have been revoked due to cardiovascular and psychiatric side effects, and orlistat has the limitation that its effects vary depending on the amount of fat intake, along with various side effects. Meanwhile, liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has been approved and is being used as an endocrine peptide targeting drug, but there is a growing concern about the risk of thyroid cancer. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2016-175362 [Patent Document 2] WO2018-074682 Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have conducted research and development efforts to find an active substance with improved efficacy and safety in the treatment of diabetes or obesity from the standpoint of suppressing insulin resistance and promoting glucose absorption by increasing insulin sensitivity, protecting pancreatic beta cells from free fatty acids, and promoting lipolysis in adipocytes. As a result, the present inventors have experimentally confirmed that a complex of two types of peptides and a peptide having a novel amino acid sequence satisfy the above-mentioned requirements, thereby completing the present invention.

[0009] It is therefore an object of the present invention to provide a peptide conjugate having antidiabetic activity.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, which contains as an active ingredient the peptide complex having the above-mentioned activity.

[0011] It is yet another object of the present invention to provide a functional food composition for regulating blood sugar levels, which contains as an active ingredient a peptide complex having the above-mentioned activity.

[0012] It is still another object of the present invention to provide a novel peptide having antidiabetic and antiobesity activities.

[0013] It is still another object of the present invention to provide a pharmaceutical composition and a functional food composition for preventing, treating, or ameliorating diabetes, which contain the novel peptide having the above-mentioned activity as an active ingredient.

[0014] It is still another object of the present invention to provide a pharmaceutical composition and a functional food composition for preventing, treating, or improving obesity, which contain a peptide having the above-mentioned activity as an active ingredient. [Means for solving the problem]

[0015] In order to achieve the above objective, One aspect of the 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, which comprises the peptide complex as an active ingredient.

[0017] Yet another aspect of the present invention provides a functional food composition for preventing or ameliorating diabetes, which contains 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] In still another aspect, the present invention provides a pharmaceutical composition for preventing or treating obesity, comprising the peptide as an active ingredient.

[0021] In yet another aspect, the present invention provides a functional food composition for regulating blood sugar levels, which contains the peptide as an active ingredient.

[0022] Yet another aspect of the present invention provides a functional food composition for preventing or ameliorating 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] According to another aspect of the present invention there is provided a peptide comprising the amino acid sequence disclosed in SEQ ID NO:2.

[0026] As used herein, the term "peptide" refers to a linear molecule formed by amino acid residues joined together by peptide bonds.

[0027] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO:1 or the amino acid sequence of SEQ ID NO:2 may be used without modification, but amino acid mutants or fragments having different sequences due to deletion, insertion, substitution, or a combination thereof of amino acid residues may also be used within a range that does not affect the original activity of the peptide, such as anti-diabetic activity.

[0028] The peptides of the present invention may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, and the like, within the limits that do not alter their activity.

[0029] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a peptide comprising the amino acid sequence of SEQ ID NO: 2, and a variant or active fragment thereof. The substantially identical amino acid sequence means an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more, respectively, with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. The peptide may further include a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically produced to increase half-life or peptide stability.

[0030] The peptide of the present invention may be one in which a partial site of the amino acid sequence is selected and modified at the N-terminus and / or C-terminus in order to increase its activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" includes not only in vivo stability that protects the peptide of the present invention from attack by in vivo protease, but also storage stability (e.g., room temperature storage stability).

[0031] The N-terminal modification may be 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 a polyethylene glycol (PEG) attached to the N-terminus of the peptide. The C-terminal modification may be a protecting group selected from the group consisting of a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), or the like attached to the C-terminus of the peptide, but is not limited thereto.

[0032] The peptides of the present invention can be prepared by various methods well known in the art, for example, by chemical synthesis methods well 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 (U.S. Pat. No. 5,516,891).

[0033] The peptide conjugates of the present invention include a peptide comprising the amino acid sequence of SEQ ID NO:1 and a peptide comprising the amino acid sequence of SEQ ID NO:2.

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

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

[0036] The peptide conjugate of the present invention has antidiabetic activity.

[0037] The peptide conjugate of the present invention has an activity of promoting glucose absorption into cells, which may be fat cells (adipocytes), muscle cells (myocytes), or liver cells (hepatocytes).

[0038] The peptide complex of the present invention has an activity of promoting 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 activity of suppressing insulin resistance signaling.

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

[0041] The peptide complex of the present invention has activity of suppressing 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 conjugates of the present invention have the activity of promoting insulin-sensitizing signals.

[0043] The peptide complex of the present invention has the activity of increasing phosphorylation of Tyr632 of the insulin receptor substrate (IRS), or promoting the activation of PI3K (phosphoinositide 3-kinase), ATK, or AMPK (AMP-activated protein kinase).

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

[0045] The peptide complex of the present invention has the activity of suppressing the death of pancreatic β cells induced by free fatty acids.

[0046] Since the above-mentioned peptide complex of the present invention has the above-mentioned activity, it can exert a very excellent effect in the treatment of diabetes.

[0047] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 2 has antidiabetic activity.

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

[0049] Specifically, a peptide comprising the amino acid sequence of SEQ ID NO:2 of the present invention can promote insulin sensitivity signals through the phosphorylation-promoting activity of Tyr632 of IRS (insulin receptor substrate), the activation-promoting activity of phospho-AKT, or the phosphorylation-promoting activity of AMPK, and the peptide of the present invention can suppress insulin resistance signals through the phosphorylation-inhibiting activity of Sre302 of IRS under an insulin resistance-inducing environment.

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

[0051] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO:2 has the activity of promoting fat decomposition in adipocytes.

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

[0053] The peptide comprising the amino acid sequence of SEQ ID NO: 2 of the present invention has the above-mentioned activity, and therefore can exert excellent efficacy in treating, preventing or ameliorating diabetes and obesity.

[0054] 2. Composition for preventing, treating, or ameliorating diabetes and obesity Pharmaceutical Compositions According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient a peptide complex comprising: (i) a peptide having the amino acid sequence of SEQ ID NO:1; and (ii) a peptide having 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 suppressing activity, insulin sensitivity promoting activity, and pancreatic β-cell protecting activity, and therefore has excellent activity for treating or preventing diabetes.

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

[0057] In the pharmaceutical composition for preventing or treating diabetes, the peptide complex can promote glucose absorption.

[0058] In the pharmaceutical composition for preventing or treating diabetes, the peptide complex can suppress insulin resistance signaling or promote insulin sensitivity signaling.

[0059] In the pharmaceutical composition for preventing or treating diabetes, the peptide complex can inhibit phosphorylation of Ser302 of IRS (Insulin receptor kinase) or phosphorylation of JNK (c-Jun N-terminal kinase).

[0060] In the pharmaceutical composition for preventing or treating 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 pharmaceutical composition for preventing or treating diabetes, the peptide complex can increase phosphorylation of Tyr632 of IRS (Insulin Receptor Substrate), or promote activation of PI3K (Phosphoinositide 3-kinase), activation of ATK, or activation of AMPK (AMP-activated protein kinase).

[0062] In the pharmaceutical composition for preventing or treating 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 preventing or treating diabetes, the peptide conjugate can suppress the production of reactive oxygen species (ROS), TNF-α gene expression, TNF-α protein expression, or IL-1β protein expression induced by free fatty acids, or suppress the death of pancreatic β cells induced by free fatty acids.

[0064] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating diabetes, which comprises a peptide comprising 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 suppressing activity and insulin sensitivity promoting activity, and therefore has excellent therapeutic or preventive activity for diabetes.

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

[0067] In the pharmaceutical composition for preventing or treating diabetes, the peptide can suppress insulin resistance signaling or promote insulin sensitivity signaling.

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

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

[0070] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating obesity, which comprises a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0071] In the pharmaceutical composition for preventing or treating obesity, the peptide can promote the decomposition of fat in adipocytes.

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

[0073] The pharmaceutical compositions of the present invention may comprise a therapeutically effective amount of the peptide conjugate or peptide and a pharma- ceutically acceptable carrier.

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

[0075] The pharma- ceutically acceptable carriers include those commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil, but are not limited thereto.

[0076] The pharmaceutical composition of the present invention may further contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like, but are not limited thereto.

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

[0078] The pharmaceutical compositions of the present invention may be administered by any suitable route for treating diabetes or obesity, for example, orally or parenterally, and in the case of parenteral administration, may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, etc.

[0079] The dosage of the pharmaceutical composition may be, but is not limited to, 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, and may be variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0080] The pharmaceutical composition of the present invention may be prepared in a unit dose form or in a multi-dose container by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person having ordinary skill in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets or capsules, and may further include a dispersant or stabilizer.

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

[0082] In the functional food composition of the present invention, the blood sugar level regulation may be the regulation of blood sugar 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, specifically type 2 diabetes.

[0084] In the functional food composition of the present invention, the regulation of blood sugar levels may be lowering of blood sugar levels.

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

[0086] According to another aspect of the present invention, there is provided a functional food composition for regulating blood sugar levels, comprising a peptide comprising the amino acid sequence of SEQ ID NO:2 as an active ingredient.

[0087] In the functional food composition of the present invention, the blood sugar level regulation may be the regulation of blood sugar 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, specifically type 2 diabetes.

[0089] In the functional food composition of the present invention, the regulation of blood glucose level may be lowering of blood glucose level.

[0090] In the functional food composition for regulating blood sugar level, the peptide can suppress insulin resistance signaling or promote insulin sensitivity signaling.

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

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

[0093] According to another aspect of the present invention, there is provided a functional food composition for preventing or ameliorating obesity, which comprises a peptide comprising the amino acid sequence of SEQ ID NO:2 as an active ingredient.

[0094] In the functional food composition for preventing or ameliorating obesity, the peptide can promote the decomposition of fat in adipocytes.

[0095] In the functional food composition for preventing or improving obesity, the peptide can increase the expression of lipolytic enzyme proteins ATGL (Adipose triglyceride lipase), pHSL (phosphorylated hormone-sensitive lipase), or PLIN (Perilipin, lipid droplet-associated protein) in adipocytes.

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

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

[0098] The food composition of the present invention may contain not only the peptide complex or peptide as the active ingredient but also ingredients that are usually 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, fructose, disaccharides, such as maltose, sucrose, oligosaccharides, and polysaccharides, such as normal sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors, thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, and the like), and synthetic flavors (saccharin, aspartame, and the like) may be used as flavoring agents. The proportion of the carbohydrates may generally be 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 above-mentioned ingredients, the functional food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and thickeners (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. In addition, it may contain fruit pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks.

[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 extract, jujube extract, licorice extract, etc.

[0101] In another aspect of the present invention, there is provided a method for treating diabetes, comprising administering to a diabetic patient 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.

[0102] In yet another aspect of the present invention, there is provided a method for regulating blood glucose, comprising administering a therapeutically effective amount of a peptide complex comprising the aforementioned (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 in need of blood glucose regulation.

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

[0104] In yet another aspect of the present invention, there is provided a method for regulating blood glucose, comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:2 to a subject in need of blood glucose regulation.

[0105] In another aspect of the present invention, there is provided a method for treating, ameliorating, or preventing obesity, comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:2 to a subject in need of treatment for obesity. Effect of the Invention

[0106] The peptide complex or peptide of the present invention can exhibit the effect of lowering blood glucose levels by promoting glucose absorption into cells, suppressing insulin resistance signals, promoting insulin sensitivity signals, and inhibiting the death of pancreatic β cells, which are insulin-producing cells. The peptide of the present invention also exhibits the effect of promoting fat decomposition in adipocytes. Therefore, the peptide complex or peptide of the present invention can be used for the treatment, prevention, and amelioration of diabetes or obesity, and can be usefully used for lowering blood glucose in diabetic patients or high-risk patients in the pre-diabetic stage, and can be used for the treatment, prevention, and amelioration of obesity.

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

[0108] [Figure 1A] 1 shows experimental results showing that the peptide complex of the present invention promotes glucose absorption into fat cells. [Figure 1B] 1 shows experimental results showing that the peptide complex of the present invention promotes glucose absorption into fat cells. [Figure 2A] 1 shows experimental results showing that the peptide complex of the present invention promotes glucose absorption into myoblasts. [Figure 2B] 1 shows experimental results showing that the peptide complex of the present invention promotes glucose absorption into myoblasts. [Figure 3A] 1 shows experimental results indicating that the peptide complex of the present invention has the effect of promoting the activity of phospho-AMPK and phospho-ACC, which are insulin sensitivity promoting factors. [Figure 3B] 1 shows experimental results indicating that the peptide complex of the present invention has the effect of promoting the activity of phospho-AMPK and phospho-ACC, which are insulin sensitivity promoting factors. [Figure 4A] 1 shows experimental results indicating that the peptide complex of the present invention has the effect of suppressing phosphorylation of Serine 302 of IRSdml and phosphorylation of JNK, which are insulin resistance signaling factors. [Figure 4B] 1 shows experimental results indicating that the peptide complex of the present invention has the effect of suppressing phosphorylation of Serine 302 of IRSdml and phosphorylation of JNK, which are insulin resistance signaling factors. [Figure 5A] These are experimental results showing that the peptide complex of the present invention again reduces the increased expression of the insulin resistance-inducing cytokine TNF-α and the insulin resistance-promoting signaling factors mTOR and p70S6K genes induced by rhTNF-α treatment. [Figure 5B] These are experimental results showing that the peptide complex of the present invention again reduces the increased expression of the insulin resistance-inducing cytokine TNF-α and the insulin resistance-promoting signaling factors mTOR and p70S6K genes induced by rhTNF-α treatment. [Figure 6A] These experimental results show that when adipocytes in which insulin resistance has been induced by treatment with rhTNF-α are treated with the peptide complex of the present invention, the phosphorylation of IRSTyrosin632, an insulin sensitivity promoting factor, increases, and the activation of PI3K, ATK, and AMPK is also promoted. [Figure 6B] These experimental results show that when adipocytes in which insulin resistance has been induced by treatment with rhTNF-α are treated with the peptide complex of the present invention, the phosphorylation of IRSTyrosin632, an insulin sensitivity promoting factor, increases, and the activation of PI3K, ATK, and AMPK is also promoted. [Figure 7A]1 shows experimental results indicating that the expression of glucose absorption-related genes leptin, adiponectin, IRS-1, and GLUT4, which was decreased by rhTNF-α treatment in adipocytes, was increased by treatment with the peptide complex of the present invention. [Figure 7B] 1 shows experimental results indicating that the expression of glucose absorption-related genes leptin, adiponectin, IRS-1, and GLUT4, which was decreased by rhTNF-α treatment in adipocytes, was increased by treatment with the peptide complex of the present invention. [Figure 8A] 1 shows experimental results showing that palmitic acid-induced reactive oxygen species (ROS) are reduced by the peptide complex of the present invention. [Figure 8B] 1 shows experimental results showing that palmitic acid-induced reactive oxygen species (ROS) are reduced by the peptide complex of the present invention. [Figure 9A] 1 shows experimental results indicating that 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] 1 shows experimental results indicating that 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 are experimental results showing that the expression levels of TNF-α and IL-1β proteins, which were increased by palmitic acid treatment in INS-1 (Rat pancreatic beta cell) cells, were significantly decreased again by treatment with the peptide complex of the present invention. [Figure 10B] These are experimental results showing that the expression levels of TNF-α and IL-1β proteins, which were increased by palmitic acid treatment in INS-1 (Rat pancreatic beta cell) cells, were significantly decreased again by treatment with the peptide complex of the present invention. [Figure 11] These are experimental results showing that the increase in INS-1 cell death induced by palmitic acid treatment is again reduced by treatment with the peptide complex of the present invention, resulting in an increase in cell viability. [Figure 12A] 1 shows experimental results indicating that the expression of PGC-1α, ACOX-1, PPAR-α, or CPT-1α gene, which is an FFA beta oxidation-related gene, is promoted by the peptide complex of the present invention in HepG2 cells. [Figure 12B] 1 shows experimental results indicating that the expression of PGC-1α, ACOX-1, PPAR-α, or CPT-1α gene, which is an FFA beta oxidation-related gene, is promoted by the peptide complex of the present invention in HepG2 cells. [Figure 13A] The peptides of the present invention exhibit the activity of increasing 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. [Figure 13B] The peptides of the present invention exhibit the activity of increasing 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. [Figure 14] When adipocytes are treated with the peptide of the present invention, the amount of released glycerol, a product of lipolysis, increases in a peptide concentration-dependent manner. [Figure 15A] When adipocytes are treated with the peptide of the present invention, phosphorylation of the insulin receptor substrate (IRS) Tyr632, which is an insulin sensitivity promoter, and activation of phosphorylation-AKT are promoted, and phosphorylation of the signal transduction protein AMPK is increased. On the other hand, phosphorylation of the insulin receptor substrate (IRS) Ser302 is decreased under an insulin resistance-inducing environment. [Figure 15B] When adipocytes are treated with the peptide of the present invention, phosphorylation of the insulin receptor substrate (IRS) Tyr632, which is an insulin sensitivity promoter, and activation of phosphorylation-AKT are promoted, and phosphorylation of the signal transduction protein AMPK is increased. On the other hand, phosphorylation of the insulin receptor substrate (IRS) Ser302 is decreased under an insulin resistance-inducing environment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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] Working Example Production Example 1: Production of peptides and peptide complexes The peptides having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 shown in Table 1 were synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified and separated using C18 reversed-phase high performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300C18 (2.1 mm x 100 mm, 1.7 um, Waters Co, USA).

[0111] [Table 1]

[0112] The peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2 were mixed in equal amounts to prepare a peptide complex, and the efficacy of the peptide of SEQ ID NO: 2 was evaluated.

[0113] Experimental example 1: Promoting glucose absorption into adipocytes An experiment was carried out using adipocytes to determine whether the peptide complex prepared in Preparation Example 1 promotes the uptake of glucose into cells.

[0114] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 3 Cells were seeded at 1 / well and used for the experiment. Two days after reaching confluence, the cells were switched to differentiation induction medium to induce differentiation into adipocytes, and two days after induction, the cells were cultured in medium containing 10% FBS and 10ug / mL insulin for two days, and then the medium was switched to 10% FBS every two days until differentiation into adipocytes. When differentiation was complete, the medium was switched to glucose free medium and cultured for 16 hours, and then cultured in 100uL KRPH buffer containing 2% BSA for 40 minutes to induce starvation. The cells were treated with rhTNF-α (2nM), insulin (10ug / ml), and peptide complex (2, 20ug / ml) and cultured for 1 hour, and then treated with 80uM 2-NBDG and cultured for 1 hour to prepare samples. Glucose uptake was measured using a “Glucose uptake assay kit” (Abcam, Cambridge, UK), and glucose absorbed into the cells was photographed via a fluorescence microscope (ECLIPSE 80i, Nikon, Japan).

[0115] As shown in Figures 1A and 1B, the results of the experiment confirmed that the intracellular glucose uptake suppressed by TNF-α was increased by the peptide complex treatment. In addition, it was confirmed that this effect occurs via the AMPK (AMP-activated protein kinase) and ACC (Acetyl-CoA carboxylase) signaling pathway in Experimental Example 3 described below. Through these experimental results, it was confirmed that the peptide complex significantly increases the inhibition of intracellular glucose uptake induced by inflammation.

[0116] Experimental example 2: Promotion of glucose absorption into myocytes An experiment was carried out using myoblasts to determine whether the peptide complex prepared in Preparation Example 1 promotes the absorption of glucose into cells.

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

[0118] Experimental Example 3: Signal transduction pathway of peptide complex in promoting glucose absorption Whether the peptide conjugate of Preparation 1 has the effect of promoting the activity of insulin sensitizer was confirmed by protein expression analysis (western blot analysis).

[0119] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were plated in a 96-well plate. 3The cells were seeded at 1 cell / well and used in the experiment. Two days after reaching confluence, the differentiation medium was changed to induce differentiation into adipocytes, and two days after the induction, the cells were cultured in a medium containing 10% FBS and 10ug / mL insulin for two days, and then changed to a medium containing 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the cells were cultured for 4 hours in a serum-free medium to induce starvation. The cells were treated with the peptide complex (0.2, 2, 20ug / mL) of Preparation Example 1 and cultured for 30 minutes, then dissolved in a dissolution buffer, and centrifuged at 4℃ and 12,000 rpm for 30 minutes. The proteins obtained were quantified using a BCA kit. Proteins were subjected to SDD-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and electrotransferred to a membrane. The membrane with the attached protein was treated with 5% skim milk for blocking, 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, and after washing again with PBS-T, the protein was visualized using Gel Doc (Bio-Rad, Hercules, CA, USA) and Western detection reagent (Elpis Biotech, Daejeon, Korea). The antibodies used in the experiment were as follows: 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 a result of the experiment, as shown in Figures 3A and 3B, it was confirmed that the peptide complex of the present invention has the effect of promoting the activity of phospho-AMPK and phospho-ACC, which are insulin sensitivity promoting factors.

[0120] Experimental Example 4: Insulin resistance signal suppression by peptide complex Whether the peptide complex prepared in Preparation Example 1 has an effect of suppressing insulin resistance signaling was confirmed through protein expression analysis (western blot analysis).

[0121] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were cultured in a 6-well plate. 5Cells were seeded at 1 cell / well and used in the experiment. Two days after reaching confluence, differentiation into adipocytes was induced by switching to differentiation induction medium. Two days after induction, the cells were cultured in medium containing 10% FBS and 10ug / mL insulin for two days, and then the medium was changed to 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the cells were cultured in serum-free medium for 4 hours to induce starvation. After treating with rhTNF-α (2nM), insulin (1ug / ml) and peptide complex (0.2, 2, 20ug / ml) and culturing for 30 minutes, the cells of each treatment group were dissolved by adding lysis buffer, centrifuged at 4℃ at 12,000 rpm for 30 minutes, and the protein obtained was quantified using a BCA kit. Proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membrane with the attached proteins was blocked by treating it with 5% skim milk, and then reacted with the primary antibody overnight at 4℃. After washing with PBS-T, the membrane was reacted with the secondary antibody for 1 hour at room temperature, and after washing again with PBS-T, the proteins were visualized using Gel Doc (Bio-Rad, Hercules, CA, USA) and western detection reagent (Elpis Biotech, Daejeon, Korea). The antibodies used in the experiment were as follows: 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 a result of the experiment, as shown in Figures 4A and 4B, it was confirmed that the peptide conjugate of the present invention has the effect of suppressing the phosphorylation of Serine 302 of IRS (Insulin receptor kinase) and the phosphorylation of JNK (c-Jun N-terminal kinase), which are insulin resistance signaling factors induced by TNF-α treatment.

[0122] Experimental Example 5: Inhibition of insulin resistance-inducing gene expression by peptide complexes An experiment was conducted to see whether the peptide complex prepared in Preparation Example 1 suppresses the expression of genes that induce insulin resistance. Adipocytes were treated with rhTNF-α to induce insulin resistance, and then the expression levels of mTOR and p70S6K genes were measured after treatment with the peptide complex.

[0123] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were cultured in a 6-well plate. 5Cells were seeded at 1 cell / well and used for the experiment. Two days after reaching confluence, differentiation into adipocytes was induced by switching to differentiation induction medium. Two days after induction, cells were cultured in medium containing 10% FBS and 10ug / mL insulin for two days, and then switched to medium containing 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, cells were cultured in serum-free medium for 4 hours to induce starvation. The control group was treated with rhTNF-α (2nM), insulin (1ug / ml), and peptide complex (0.2, 2, 20ug / ml), and RNA was extracted after 24 hours and RT-PCR was performed. RNA was extracted from 3T3-L1 cells using easy-BLUETM Total RNA extraction kit (Qiagen, Germany). The extracted RNA was converted to cDNA using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). A reaction mixture was prepared using PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for TNF-α, mTOR (mammalian Target Of Rapamycin), p70S6K, or GAPDH genes, and then PCR was performed using a PCR machine (Eppendorf, Germany). The mRNA expression pattern was then determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are shown in Table 2 below.

[0124] [Table 2]

[0125] As a result of the experiment, as shown in Figures 5A and 5B, the expression of the TNF-α gene, an inflammatory cytokine that induces insulin resistance, and the mTOR and p70S6K genes, which are insulin resistance-promoting signaling factors, increased when treated with rhTNF-α, but it was confirmed that the expression of the above genes decreased again when treated with the peptide complex.

[0126] Experimental Example 6: Peptide conjugates promote insulin-sensitivity signaling An experiment was carried out to determine whether the peptide complex prepared in Preparation Example 1 promotes insulin-sensitivity signaling.

[0127] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were cultured in a 6-well plate. 5Cells were seeded at 100 cells / well and used in the experiment. Two days after reaching confluence, the differentiation medium was changed to induce differentiation into adipocytes, and two days after the induction, the cells were cultured in a medium containing 10% FBS and 10ug / mL insulin for two days, and then the medium was changed to 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the medium was changed to serum-free medium and cultured for 4 hours to induce starvation. After treating with rhTNF-α (2nM), insulin (1ug / ml) and peptide complex (0.2, 2, 20ug / ml) and culturing for 30 minutes, the cells of each treatment group were dissolved by adding lysis buffer, and centrifuged at 4℃ at 12,000 rpm for 30 minutes, and the protein obtained was quantified using a BCA kit. Proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membrane with the attached proteins was blocked by treating it with 5% skim milk and reacted with the primary antibody at 4℃ overnight. After washing with PBS-T, the membrane was reacted with the secondary antibody at room temperature for 1 hour, washed again with PBS-T, and visualized with Gel Doc (Bio-Rad, Hercules, CA, USA) and western detection reagent (Elpis Biotech, Daejeon, Korea). The antibodies used in the experiments 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).As shown in Figures 6A and 6B, the experimental results showed 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) Tyrosin632, an insulin sensitivity-promoting factor, thereby promoting 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 Whether the peptide complex produced in Production Example 1 promotes the expression of glucose absorption-related genes was confirmed by analyzing the expression of leptin, adiponectin, IRS-1 (insulin receptor substrate 1), and GLUT4 (glucose transporter type 4) genes.

[0129] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were cultured in a 6-well plate. 5Cells were seeded at 1000 cells / well and used in the experiment. Two days after reaching confluence, the differentiation medium was changed to induce differentiation into adipocytes, and two days after the induction, the cells were cultured in a medium containing 10% FBS and 10ug / mL insulin for two days, and then the medium was changed to 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the cells were changed to serum-free medium and cultured for 4 hours to induce starvation. The control group was treated with rhTNF-α (2nM), insulin (1ug / ml), and peptide complex (0.2, 2, 20ug / ml), and RNA was extracted after 24 hours and RT-PCR was performed. RNA was extracted from 3T3-L1 cells using easy-BLUETM 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 each gene, PCR was performed using a PCR machine (Eppendorf, Germany). Then, the mRNA expression pattern was determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are shown in Table 3 below.

[0130] [Table 3]

[0131] As a result of the experiment, as shown in Figures 7A and 7B, it was confirmed that the expression of glucose absorption-related genes leptin, adiponectin, IRS-1, and GLUT4, which were decreased by rhTNF-α treatment in adipocytes, was increased by treatment with the peptide complex.

[0132] Experimental Example 8: Inhibition of ROS generation induced by palmitate Whether the peptide conjugate prepared in Preparation Example 1 inhibits the generation of reactive oxygen species (ROS) induced by palmitate, a type of free fatty acid, was confirmed by intracellular ROS detection assay (FACS).

[0133] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were cultured in a 6-well plate. 5 Cells were seeded at 1 cell / well and used in the experiment. Two days after reaching confluence, the medium was changed to differentiation induction medium to induce differentiation into adipocytes, and two days after induction, the cells were cultured in medium containing 10% FBS and 10ug / mL insulin for two days, and then changed to medium containing 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the medium was changed to serum-free medium and cultured for 4 hours to induce starvation. The control group was treated with rhTNF-α (2nM), insulin (1ug / ml), and peptide complex (2ug / ml) and cultured for 24 hours, and DCF-DH was treated for 30 minutes and oxidative activity was measured by the degree of fluorescence using FACS (Becton Dickinson & Company (BD), USA). As a result, as shown in the graphs of FIG. 8A and FIG. 8B, it was confirmed that reactive oxygen species (ROS) induced by palmitic acid was reduced by the peptide complex.

[0134] Experimental Example 9: Inhibition of TNF-α gene expression induced by palmitic acid Whether the peptide complex prepared in Preparation Example 1 inhibits inflammation induced by palmitate, a type of free fatty acid, was confirmed through gene expression analysis (RT-PCR).

[0135] INS-1 cells were starved in serum-free media RPMI 1640 (Gibco, New York, USA) and pretreated with 25 uM palmitic acid for 2 hours, then treated with peptide complexes (0.2, 2, 20 ug / ml). After 24 hours, RNA was extracted using easy-BLUETM 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 TNF-α and GAPDH genes, PCR was performed using a PCR machine (Eppendorf, Germany). The mRNA expression pattern was then determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiment are shown in Table 4 below.

[0136] [Table 4]

[0137] As a result of the experiment, as shown in FIG. 9A and FIG. 9B, it was confirmed that gene expression of TNF-α, an inflammatory protein induced by palmitic acid, was significantly decreased by treatment with the peptide complex.

[0138] Experimental Example 10: Inhibition of inflammatory cytokine expression induced by palmitic acid Western blot analysis was performed to confirm whether the peptide complex prepared in Preparation Example 1 inhibits the expression of inflammatory cytokines induced by palmitic acid, a type of free fatty acid.

[0139] INS-1 cells (rat pancreatic beta cells) were pretreated with 25uM palmitic acid for 2 hours after starvation in serum-free media RPMI 1640 (Gibco, New York, USA) and treated with peptide complexes (0.2, 2, 20ug / ml). After 24 hours, the cells were washed once with PBS, and then dissolved on ice for 30 minutes in lysis buffer (Millipore, Darmstadt, Germany) containing 10mM Tris (pH7.5), 100mM NaCl, 1% NP-40, and protease inhibitor. Proteins were quantified using a BCA kit (Thermo Fisher Scientific, Waltham, USA) after centrifugation at 13,000 rpm for 10 minutes at 4℃. Equal amounts of proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred to a polyvinylidene difluoride membrane. To block nonspecific binding of antibodies, the membrane with the attached proteins was treated with 5% skim milk (BD, New Jersey, USA) for 1 hour and then reacted with the primary antibody overnight at 4℃. After washing with PBS-T, the membrane was reacted with the secondary antibody for 1-2 hours at room temperature, washed again with PBS-T, and the proteins were visualized on X-ray film using enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Waltham, USA). The antibodies used in the experiments 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).As a result of the experiment, as shown in Figures 10A and 10B, it was confirmed that the expression levels of TNF-α and IL-1β proteins, which were increased by palmitic acid treatment in INS-1 (Rat pancreatic beta cell) cells, were significantly decreased again by peptide complex treatment.

[0140] Experimental Example 11: Inhibition of pancreatic β-cell death induced by palmitic acid A cell cytotoxicity assay was performed to confirm whether the peptide conjugate prepared in Preparation Example 1 inhibits 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 pretreated with 25uM palmitic acid for 2 hours after starvation was induced in serum-free media RPMI 1640 (Gibco, New York, USA) and treated with peptide complexes (0.2, 2, 20ug / ml). After 24 hours, CCK-8 solution (Dojindo, Kumamoto, Japan) was added to 1 / 10 of the culture volume, and the absorbance was measured at intervals of 30 minutes or 1 hour. The experiment was terminated when the average absorbance value of the control group reached 1.0. The absorbance of the reaction product was measured at 450nm wavelength using a microplate reader. As a result, as shown in Figure 11, it was confirmed that the increase in INS-1 cell death induced by palmitic acid treatment was reduced again by treatment with peptide complexes, and the cell viability was increased.

[0142] Experimental Example 12: Promotion of expression of glucose absorption-related genes Whether the peptide complex prepared in Preparation Example 1 promotes the expression of glucose absorption-related genes, 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) genes was confirmed by gene expression analysis (RT-PCR).

[0143] When HepG2 hepatoma cells reached 80% confluence, 2x10 5 Cells were seeded at 1000 cells / well and used for the experiment. The next day, the medium was replaced with serum-free media and starvation was induced for 4 hours. After 4 hours, the control group and the peptide complexes (0.2, 2, 20ug / ml) were treated, and after 24 hours, RNA was extracted and RT-PCR was performed. RNA was extracted using easy-BLUETM 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 PGC-1α, ACOX-1, PPAR-α, or CPT-1α genes, PCR was performed using a PCR machine (Eppendorf, Germany). The mRNA expression pattern was then determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are shown in Table 5 below.

[0144] [Table 5]

[0145] As a result of the experiment, as shown in FIG. 12A and FIG. 12B, it was confirmed that the expression of FFA beta oxidation-related genes, PGC-1α, ACOX-1, PPAR-α, and CPT-1α genes, in HepG2 cells was promoted by the peptide complex.

[0146] Experimental Example 13: Promotion of lipolytic enzyme protein expression in adipocytes Western blot analysis was performed to determine whether the peptide having the amino acid sequence of SEQ ID NO: 2 prepared in Preparation Example 1 promotes the expression of lipolytic enzyme protein in adipocytes.

[0147] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 5Cells were seeded at 1 cell / well and used in the experiment. Two days after reaching confluence, the medium was changed to a differentiation induction medium [DMEM medium containing 10% FBS, 1ug / ml insulin, 0.5mM isobutylmethylxanthine (IBMX) (Sigma, USA) and 1uM dexamethasone (Sigma, USA)] to induce differentiation into adipocytes, and two days after the induction, the medium was cultured for two days in a medium containing 10% FBS and 10ug / mL insulin, and then changed to a medium containing 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the medium was changed to serum-free medium and cultured for 4 hours to induce starvation. The cells were divided into a group without any treatment (negative control group), a group treated with the peptide of Preparation Example 1 (2, 20ug / ml), and a group treated with TNFα (20nM) (positive control group), and the cells were treated with the peptide at a specified concentration and cultured for 1 hour, after which the cells of each treatment group were dissolved by adding a lysis buffer. After dissolution, the cells were centrifuged at 12,000 rpm at 4℃ for 30 minutes, and the proteins obtained were quantified using a BCA kit. The proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred to a membrane. The membrane with the attached proteins was blocked by treating it with 5% skim milk, and then reacted with the primary antibody overnight at 4℃. After washing with PBS-T, the secondary antibody was incubated at room temperature for 1 hour, and then washed again with PBS-T. The sections were visualized with a 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 a result of the experiment, as shown in Figures 13A and 13b, it was confirmed that when the peptide of the present invention was treated, the expression of lipolytic enzyme proteins ATGL (Adipose triglyceride lipase), pHSL (phosphorylated hormone-sensitive lipase), and PLIN (Perilipin, lipid droplet-associated protein) was increased in adipocytes.

[0148] Experimental Example 14: Promotion of lipolysis in fat cells To determine whether the peptide having the amino acid sequence of SEQ ID NO: 2 prepared in Preparation Example 1 promotes lipolysis in adipocytes, the amount of glycerol released, which is a product of lipolysis, was analyzed.

[0149] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were cultured in a 6-well plate. 5Cells were seeded at 1 cell / well and used in the experiment. Two days after reaching confluence, the medium was changed to a differentiation induction medium [DMEM medium containing 10% FBS, 1ug / ml insulin, 0.5mM isobutylmethylxanthine (IBMX) (Sigma, USA) and 1uM dexamethasone (Sigma, USA)] to induce differentiation into adipocytes, and two days after the induction, the medium was cultured for two days in a medium containing 10% FBS and 10ug / mL insulin, and then changed to a medium containing 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the medium was changed to serum-free medium and cultured for 4 hours to induce starvation. The cells were divided into a group without any treatment (negative control group), a group treated with the peptide of Preparation Example 1 (2, 20ug / ml), and a group treated with TNF-α (20nM) (positive control group), and the cells were treated with the above substances at a specified concentration and cultured for 48 hours to obtain a supernatant. The amount of glycerol released was compared by performing a glycerol release assay on the obtained supernatant using a "Glycerol Colormetric assay kit" (Cayman Chemical, USA). As a result of the glycerol release assay, as shown in Figure 14, it was confirmed that when the peptide of the present invention was treated, the amount of glycerol released from adipocytes, which is a product of lipolysis, increased depending on the concentration of the treated peptide.

[0150] Experimental Example 15: Inhibition of insulin resistance signaling and promotion of insulin sensitivity signaling An experiment was carried out by protein expression analysis to determine whether the peptide having the amino acid sequence of SEQ ID NO: 2 prepared in Preparation Example 1 suppresses insulin resistance in adipocytes and promotes insulin sensitivity signal transduction.

[0151] When 3T3-L1 preadipocytes reached 80% confluence, 5x10 cells were cultured in a 6-well plate. 5The cells were seeded at 1 cell / well and used in the experiment. Two days after reaching confluence, the medium was changed to a differentiation induction medium [DMEM medium containing 10% FBS, 1ug / ml insulin, 0.5mM isobutylmethylxanthine (IBMX) (Sigma, USA) and 1uM dexamethasone (Sigma, USA)] to induce differentiation into adipocytes, and two days after the induction, the medium was cultured for two days in a medium containing 10% FBS and 10ug / mL insulin, and then changed to a medium containing 10% FBS every two days until differentiation into adipocytes. When differentiation was completed, the medium was changed to a serum-free medium and cultured for 4 hours to induce starvation. Here, rhTNF-α (2nM), insulin (1ug / ml) and the peptide of Preparation 1 (2, 20ug / ml) were treated, and cultured for 1 hour. After 30 minutes of incubation, the cells of each treatment group were dissolved in a lysis buffer and centrifuged at 12,000 rpm at 4℃ for 30 minutes. Proteins were quantified using a BCA kit. Proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membrane with the attached proteins was treated with 5% skim milk for blocking and reacted with a primary antibody overnight at 4℃. After washing with PBS-T, the membrane was reacted with a secondary antibody at room temperature for 1 hour, washed again with PBS-T, and visualized with a 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-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). As a result, as shown in FIG. 15A and FIG. 15B, it was confirmed that the phosphorylation of IRS (Insulin Receptor Substrate) Tyr632, which is an insulin sensitivity promoting factor, and the activation of phosphorylation-AKT were promoted when the peptide of the present invention was treated. In addition, an increase in phosphorylation of AMPK, a signal transduction protein that increases insulin sensitivity, was confirmed. On the other hand, it was confirmed that it reduced phosphorylation of IRS (Insulin Receptor Substrate) Ser302 under an insulin resistance-inducing environment, thereby suppressing insulin resistance.

[0152] Preparation Example 2: Preparation of pharmaceutical composition 2-1. Manufacturing of powders 2g of the peptide complex of the present invention or the peptide of the present invention 1g lactose The ingredients were mixed and filled into a sealed bag to prepare a powder.

[0153] 2-2. Tablet manufacturing 100 mg of the peptide complex of the present invention or the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg The above ingredients were mixed and then compressed into tablets by a conventional tablet manufacturing method.

[0154] 2-3. Capsule manufacturing 100 mg of the peptide complex of the present invention or the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg The above ingredients were mixed and then filled into a gelatin capsule according to a conventional capsule manufacturing method to prepare a capsule.

[0155] 2-4. Ring manufacturing 1 g of the peptide complex of the present invention or the peptide of the present invention Lactose 1.5g 1g glycerin Xylitol 0.5g The above ingredients were mixed and then prepared in a conventional manner so that each ring weighed 4 g.

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

[0157] Production Example 3: Production of functional food composition 3-1. Health food manufacturing 500ug of the peptide complex of the present invention or the peptide of the present invention Vitamin mixture (as needed) Vitamin A Acetate 70mg Vitamin E 1.0mg Vitamin D 0.13 mg Vitamin B2 0.15mg Vitamin B6 0.5mg Vitamin B12 0.2 mg Vitamin C 10mg Biotin 10mg Nicotinamide 1.7mg Folic acid 50mg Calcium pantothenate 0.5mg Appropriate amount of inorganic mixture Ferrous sulfate 1.75mg Zinc oxide 0.82mg Magnesium carbonate 25.3mg Dipotassium phosphate 15mg Dicalcium phosphate 55mg Potassium citrate 90mg Calcium carbonate 100mg Magnesium chloride 24.8mg The composition ratio of the vitamin and mineral mixture is a mixture of ingredients that are relatively suitable for health foods in the preferred embodiment, but the mixing ratio may be modified as desired. After mixing the ingredients using normal health food manufacturing methods, granules can be produced and used to manufacture a health food composition using normal methods.

[0158] 3-2. Manufacturing of health drinks 500ug of the peptide complex of the present invention or the peptide of the present invention Citric acid 1000mg Oligosaccharides 100g Plum concentrate 2g Taurine 1g Add purified water to make a total of 900ml The ingredients were mixed according to a typical method for producing health drinks, then heated and stirred at 85°C for about 1 hour, and the resulting solution was filtered and placed in a sterilized container, sealed and sterilized, then refrigerated and stored, and then used to produce a health drink composition. The above composition ratio is a mixture of ingredients that are relatively suitable for luxury drinks in a preferred embodiment, but the ratio can be freely modified according to regional and ethnic preferences such as demand class, demand country, and use.

[0159] Although the above describes exemplary embodiments of the present application, the scope of the present application is not limited to the specific embodiments described above, and a person having ordinary knowledge in the field can make appropriate modifications within the scope of the claims of the present application.

Claims

1. 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.

2. The peptide conjugate of claim 1 , wherein the peptide conjugate has antidiabetic activity.

3. A pharmaceutical composition for preventing or treating diabetes, comprising the peptide complex according to claim 1 as an active ingredient.

4. The pharmaceutical composition for preventing or treating diabetes according to claim 3 , wherein the peptide complex promotes glucose absorption.

5. The pharmaceutical composition for preventing or treating diabetes according to claim 3 , wherein the peptide complex suppresses insulin resistance signals or promotes insulin sensitivity signals.

6. 4. The pharmaceutical composition for preventing or treating diabetes according to claim 3, wherein the peptide complex inhibits phosphorylation of Ser302 of IRS (insulin receptor kinase) or phosphorylation of JNK (c-Jun N-terminal kinase).

7. 4. The pharmaceutical composition for preventing or treating diabetes according to claim 3, wherein the peptide complex suppresses expression of TNF-α gene, mTOR (mammalian Target Of Rapamycin) gene, or p70S6K gene under an insulin resistance-inducing environment.

8. The pharmaceutical composition for preventing or treating diabetes according to claim 3, wherein the peptide complex increases phosphorylation of Tyr632 of insulin receptor substrate (IRS), or promotes activation of phosphoinositide 3-kinase (PI3K), activation of ATK, or activation of AMP-activated protein kinase (AMPK).

9. The pharmaceutical composition for preventing or treating diabetes according to claim 3, wherein the peptide complex promotes expression of one or more genes selected from the group consisting of leptin, adiponectin, insulin receptor substrate 1 (IRS-1), glucose transporter type 4 (GLUT4), PGC-1α, ACOX-1, PPAR-α, and CPT-1α.

10. 4. The pharmaceutical composition for preventing or treating diabetes according to claim 3, wherein the peptide complex inhibits free fatty acid-induced production of reactive oxygen species (ROS), TNF-α gene expression, TNF-α protein expression, or IL-1β protein expression, or inhibits free fatty acid-induced death of pancreatic β cells.

11. A functional food composition for regulating blood sugar levels, comprising the peptide complex according to claim 1 as an active ingredient.

12. The functional food composition for regulating blood sugar levels according to claim 11, wherein said peptide complex promotes glucose absorption.

13. The functional food composition for regulating blood sugar levels according to claim 11, wherein said peptide complex inhibits insulin resistance signals or promotes insulin sensitivity signals.

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

2.

15. The peptide of claim 14 , wherein the peptide has antidiabetic or antiobesity activity.

16. A pharmaceutical composition for preventing or treating diabetes, comprising the peptide according to claim 14 as an active ingredient.

17. The pharmaceutical composition for preventing or treating diabetes according to claim 16, wherein the peptide suppresses insulin resistance signals or promotes insulin sensitivity signals.

18. the peptide has an activity of promoting phosphorylation of Tyr632 of an insulin receptor substrate (IRS), promoting activation of phospho-AKT, or promoting phosphorylation of AMPK; Or the pharmaceutical composition for preventing or treating diabetes according to claim 16, which has activity of inhibiting phosphorylation of Sre302 of IRS under an insulin resistance-inducing environment.

19. A pharmaceutical composition for preventing or treating obesity, comprising the peptide according to claim 14 as an active ingredient.

20. The pharmaceutical composition for preventing or treating obesity according to claim 19, wherein the peptide promotes fat breakdown in adipocytes.

21. The pharmaceutical composition for preventing or treating obesity according to claim 19, wherein the peptide increases expression of adipose triglyceride lipase (ATGL), phosphorylated hormone-sensitive lipase (pHSL), or perilipin (PLIN, lipid droplet-associated protein), which is a lipolytic enzyme protein, in adipocytes.

22. A functional food composition for regulating blood sugar levels, comprising the peptide according to claim 14 as an active ingredient.

23. The functional food composition for regulating blood sugar levels according to claim 22, wherein said peptide suppresses insulin resistance signals or promotes insulin sensitivity signals.

24. A functional food composition for preventing or ameliorating obesity, comprising the peptide according to claim 14 as an active ingredient.

25. The functional food composition for preventing or ameliorating obesity according to claim 24, wherein the peptide promotes the breakdown of fat in adipocytes.

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