Peptides and peptide complexes with antidiabetic activity and their uses

Abstract

This invention relates to peptide complexes with anti-diabetic activity and their uses. The peptide complexes of this invention promote cellular glucose uptake, inhibit insulin resistance signaling, promote insulin sensitivity signaling, and inhibit apoptosis of pancreatic β-cells, which are insulin-producing cells, thereby exhibiting a blood glucose-lowering effect. Furthermore, this invention also relates to peptides with anti-diabetic and anti-obesity activities and their uses. The peptides of this invention have activities that inhibit insulin resistance signaling, promote insulin sensitivity signaling, and promote lipolysis in adipocytes.

Description

Technical Field

[0001] This invention relates to peptide complexes with anti-diabetic activity and their uses. Furthermore, this invention relates to peptides with anti-obesity and anti-diabetic activity and their uses. Background Technology

[0002] Diabetes is a metabolic disease characterized by impaired or absent insulin secretion and function, marked by hyperglycemia (high blood sugar), which causes various symptoms and signs and results in glucose being excreted in the urine. In recent years, the incidence of diabetes has exploded due to rising obesity rates, particularly abdominal obesity. Diabetes is mainly divided into type 1 and type 2 diabetes. Type 1 diabetes is insulin-dependent, while type 2 diabetes is non-insulin-dependent. Type 2 diabetes is characterized by hyperglycemia, insulin resistance, and relatively impaired insulin secretion.

[0003] When food is ingested, glucose from the food is absorbed through the digestive tract and stimulates the beta cells of the pancreas to secrete insulin. This insulin promotes glucose uptake by muscles. Insulin also partially participates in glucose uptake by the liver, but primarily inhibits glucose production in the liver. Insulin lowers blood glucose levels by inhibiting glucose production in the liver and promoting glucose uptake in peripheral tissues, including muscles. Insulin resistance is a condition where a given insulin concentration produces a lower than normal blood glucose response. Insulin regulates blood glucose by promoting glucose uptake in muscles or inhibiting glucose production in the liver. Insulin resistance refers to a reduced effect of insulin even in the absence of insulin deficiency. Insulin receptors on cell membranes are involved in the uptake of glucose by cells in peripheral tissues, and a decrease in the number of insulin receptors or intracellular defects after receptor binding leads to insulin resistance. Although insulin receptor defects have been found in type 2 diabetes, it is known that post-receptor intracellular defects, specifically defects in insulin-regulated phosphorylation / dephosphorylation, play a greater role. Among these mechanisms, it is known that a deficiency in phosphatidylinositol-3-kinase (PI3K) signaling reduces the translocation of glucose transporter GLUT-4 (glucose transporter type 4) to the cell membrane.

[0004] Today, blood sugar levels can be controlled through lifestyle modifications (dietary therapy, exercise therapy) and medication. However, dietary therapy or exercise therapy is difficult to strictly manage and practice, and its effectiveness is limited. Therefore, most people with diabetes rely on medication (such as insulin, insulin secretagogues, insulin sensitizers, and hypoglycemic agents) and lifestyle modifications to control their blood sugar.

[0005] Obesity refers to a condition where excess energy is accumulated as body fat due to an imbalance between food intake and 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, with at least three million clinically obese, and the obesity epidemic has increased significantly 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. Furthermore, overweight and obesity are factors that increase the incidence of various adult diseases, such as arteriosclerosis, hypertension, hyperlipidemia, and heart disease, even in children and adolescents as well as adults.

[0006] Currently, representative anti-obesity drugs approved and widely prescribed by the US FDA include drugs that act on the central nervous system to suppress appetite, and orlistat (Xenical), an inhibitor of the pancreatic digestive enzyme lipase. Regarding drugs acting on the central nervous system, many, such as sibutramine, have been withdrawn due to cardiovascular and psychological side effects. Orlistat has limitations due to its various side effects and the fact that its effectiveness varies depending on the amount of fat intake. Meanwhile, liraglutide, an endocrine peptide-targeting drug, has been approved for use on the glucagon-like peptide-1 (GLP-1) receptor promoter, but it carries an increased risk of thyroid cancer.

[0007] [Existing Technical Documents]

[0008] [Patent Literature]

[0009] (Patent Document 1) WO2016-175362

[0010] (Patent Document 2) WO2018-074682 Summary of the Invention

[0011] Technical issues

[0012] The inventors conducted research and efforts to find active substances with improved efficacy and ensured safety, which can treat diabetes by inhibiting insulin resistance and increasing insulin sensitivity to promote glucose uptake and protect pancreatic beta cells from the effects of free fatty acids. Therefore, the inventors experimentally discovered that a complex of two types of peptides and a peptide with a novel amino acid sequence meet the above requirements, thus completing this invention.

[0013] Therefore, one object of the present invention is to provide a peptide complex having anti-diabetic activity.

[0014] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes, the composition comprising a peptide complex having the above-described activity as an active ingredient.

[0015] Another object of the present invention is to provide a composition for controlling blood glucose levels, the composition comprising a peptide complex having the above-described activity as an active ingredient.

[0016] Another object of the present invention is to provide novel peptides with anti-diabetic and anti-obesity activities.

[0017] Another object of the present invention is to provide pharmaceutical compositions and compositions for the prevention, treatment or relief of diabetes, the compositions comprising novel peptides having the above-described activities as active ingredients.

[0018] Another object of the present invention is to provide pharmaceutical compositions and compositions for the prevention, treatment or relief of obesity, the compositions comprising novel peptides having the above-described activities as active ingredients.

[0019] Technical solutions

[0020] According to one aspect of the invention, a peptide complex is provided 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.

[0021] Furthermore, according to another aspect of the invention, a pharmaceutical composition for the prevention or treatment of diabetes is provided, the composition comprising a peptide complex as an active ingredient.

[0022] Furthermore, according to another aspect of the invention, a composition for preventing or alleviating diabetes is provided, the composition comprising a peptide complex having the above-described activity as an active ingredient.

[0023] Furthermore, according to another aspect of the invention, a peptide comprising the amino acid sequence of SEQ ID NO: 2 is provided.

[0024] Furthermore, according to another aspect of the invention, a pharmaceutical composition for the prevention or treatment of diabetes is provided, the composition comprising a peptide as an active ingredient.

[0025] Furthermore, according to another aspect of the invention, a pharmaceutical composition for the prevention or treatment of obesity is provided, the composition comprising peptides as active ingredients.

[0026] Furthermore, according to another aspect of the invention, a composition for controlling blood glucose levels is provided, the composition comprising peptides as active ingredients.

[0027] Furthermore, according to another aspect of the invention, a composition for preventing or alleviating obesity is provided, the composition comprising peptides as active ingredients.

[0028] The present invention will now be described in detail.

[0029] 1. Peptides, peptide complexes, and their activities

[0030] According to one aspect of the invention, a peptide complex is provided comprising: (i) a peptide comprising the amino acid sequence shown in SEQ ID NO: 1; and (ii) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2.

[0031] According to another aspect of the invention, a peptide comprising the amino acid sequence shown in SEQ ID NO: 2 is provided.

[0032] As used in this article, the term "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds.

[0033] The peptides of the present invention containing the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 can be used without modification, but can also be amino acid variants or fragments with different sequences by means of deletion, insertion and substitution of amino acid residues or combinations thereof, without affecting the original activity of the original peptide (such as antidiabetic activity).

[0034] Without altering the peptide activity, the peptides of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc.

[0035] The peptides of the present invention comprise peptides containing amino acid sequences substantially identical to those of the peptide containing the amino acid sequence of SEQ ID NO: 1 or the peptide containing the amino acid sequence of SEQ ID NO: 2, and variants thereof or their active fragments. A substantially identical amino acid sequence means an amino acid sequence having 75% or higher, for example, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 97% or higher sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. Furthermore, the peptide may further comprise a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

[0036] To select specific regions of the amino acid sequence and enhance their activity, the peptides of the present invention may be modified at the N-terminus or C-terminus. Such N-terminal and / or C-terminal modifications can significantly improve the stability of the peptides of the present invention. For example, the half-life can be increased when the peptide is administered in vivo. The term "stability" is intended to include both storage stability (e.g., stability during storage at room temperature) and in vivo stability that protects the peptides of the present invention from attack by protein-lysing enzymes in vivo.

[0037] N-terminal modification can involve attaching a protecting group selected from the group consisting of: acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearoyl, and polyethylene glycol (PEG). C-terminal modification can involve attaching a hydroxyl (-OH), amino (-NH2), hydrazide (-NHNH2), etc., to the C-terminus of the peptide, but is not limited to these.

[0038] The peptides of the present invention can be prepared using various methods well known in the art to which this invention pertains. For example, the peptides of the present invention can be prepared using chemical synthesis methods known in the art, especially solid-phase synthesis (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 (US Patent No. 5,516,891).

[0039] The peptide complex of the present invention comprises: 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.

[0040] The peptide complex of the present invention may refer to a mixture, wherein the peptide comprising the amino acid sequence of SEQ ID NO: 1 is mixed with a peptide comprising the amino acid sequence of SEQ ID NO: 2.

[0041] In the peptide complex of the present invention, the ratio of the peptide containing the amino acid sequence of SEQ ID NO: 1 to the peptide containing the amino acid sequence of SEQ ID NO: 2 is not limited to a specific range. For example, a suitable range can be selected from the weight ratio range of 1:0.1 to 100 regarding the above ratio.

[0042] The peptide complex of the present invention has anti-diabetic activity.

[0043] The peptide complex of this invention has the activity of promoting glucose uptake by cells. The cells can be adipocytes, muscle cells, or hepatocytes.

[0044] The peptide complex of the present invention has the activity of promoting the expression of one or more groups selected from the group consisting of: leptin, adiponectin, insulin receptor substrate 1 (IRS-1), glucose transporter type 4 (GLUT4), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), acyl-CoA oxidase 1 (ACOX-1), peroxisome proliferator-activated receptor-α (PPAR-α), and carnitine palmitoyltransferase 1α (CPT-1α).

[0045] The peptide complex of the present invention has the activity of inhibiting insulin resistance signals.

[0046] The peptide complex of the present invention has the activity of inhibiting phosphorylation of the insulin receptor substrate (IRS) Ser302 or phosphorylation of c-Jun N-terminal kinase (JNK).

[0047] The peptide complex of the present invention has the activity of inhibiting the expression of the TNF-α gene, the mammalian target of rapamycin (mTOR) gene, or the p70S6K gene in an environment that induces insulin resistance.

[0048] The peptide complex of the present invention has the activity of promoting insulin-sensitive signaling.

[0049] The peptide complex of the present invention has the activities of enhancing the phosphorylation of the insulin receptor substrate (IRS) Tyr632, promoting the activation of phosphatidylinositol 3-kinase (PI3K), promoting the activation of AKT, or promoting the activation of AMP-activated protein kinase (AMPK).

[0050] The peptide complex of the present invention has the activity of inhibiting 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.

[0051] The peptide complex of the present invention has the activity of inhibiting apoptosis of pancreatic β cells induced by free fatty acids.

[0052] Because the peptide complex of the present invention has the above-mentioned activity, it can exhibit excellent effects in the treatment of diabetes.

[0053] The peptide of the present invention containing the amino acid sequence SEQ ID NO: 2 has antidiabetic activity.

[0054] The peptide of the present invention containing the amino acid sequence SEQ ID NO: 2 has the activity of inhibiting insulin resistance signals or promoting insulin sensitivity signals.

[0055] Specifically, the peptide comprising the amino acid sequence SEQ ID NO: 2 of the present invention can promote insulin-sensitive signaling by promoting the phosphorylation of Tyr632 of the insulin receptor substrate (IRS), promoting the activation of phospho-AKT, or promoting the phosphorylation of AMPK. The peptide comprising the amino acid sequence SEQ ID NO: 2 of the present invention can inhibit insulin resistance signaling by inhibiting the phosphorylation of Ser302 of the IRS in an environment that induces insulin resistance.

[0056] The peptide of the present invention containing the amino acid sequence SEQ ID NO: 2 has anti-obesity activity.

[0057] The peptide of the present invention, comprising the amino acid sequence SEQ ID NO: 2, has lipolysis-promoting activity in adipocytes.

[0058] Specifically, the peptide of the present invention containing the amino acid sequence of SEQ ID NO: 2 can increase the expression of lipid triglyceride lipase (ATGL), phosphorylated hormone-sensitive lipase (pHSL), or perilipin (PLIN, lipid droplet-associated protein) as lipase proteins in adipocytes.

[0059] Because the peptide of the present invention containing the amino acid sequence SEQ ID NO: 2 has the above-mentioned activity, it can exhibit excellent effects in treating, preventing or alleviating diabetes and obesity.

[0060] 2. A composition for the prevention, treatment or relief of diabetes.

[0061] Pharmaceutical Composition

[0062] According to another aspect of the invention, a pharmaceutical composition for the prevention or treatment of diabetes is provided, the composition comprising a peptide complex as an active ingredient, the 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.

[0063] As described above, the peptide complex of the present invention has activities that promote glucose uptake, inhibit insulin resistance, promote insulin sensitivity, and protect pancreatic β cells, thereby having excellent effects in the treatment or prevention of diabetes.

[0064] In this invention, diabetes can be type 1 diabetes or type 2 diabetes, especially type 2 diabetes.

[0065] In pharmaceutical compositions used to prevent or treat diabetes, peptide complexes can promote glucose uptake.

[0066] In pharmaceutical compositions used to prevent or treat diabetes, peptide complexes can inhibit insulin resistance signals or promote insulin sensitivity signals.

[0067] In pharmaceutical compositions used for the prevention or treatment of diabetes, the peptide complex inhibits phosphorylation of Ser302 of the insulin receptor substrate (IRS) or phosphorylation of c-Jun N-terminal kinase (JNK).

[0068] In pharmaceutical compositions used for the prevention or treatment of diabetes, peptide complexes can inhibit the expression of the TNF-α gene, the mammalian target of rapamycin (mTOR) gene, or the p70S6K gene in an environment that induces insulin resistance.

[0069] In pharmaceutical compositions used for the prevention or treatment of diabetes, the peptide complex can enhance phosphorylation of Tyr632 of the insulin receptor substrate (IRS), promote activation of phosphatidylinositol 3-kinase (PI3K), activation of AKT, or activation of AMP-activated protein kinase (AMPK).

[0070] In pharmaceutical compositions used for the prevention or treatment of diabetes, the peptide complex can promote the expression of one or more groups 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.

[0071] In pharmaceutical compositions used for the prevention or treatment of diabetes, peptide complexes can inhibit the production 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 can inhibit apoptosis of pancreatic β cells induced by free fatty acids.

[0072] According to another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of diabetes is provided, the composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

[0073] As described above, since the peptide of the present invention containing the amino acid sequence of SEQ ID NO: 2 has the activity of inhibiting insulin resistance signaling and promoting insulin sensitivity, it has excellent effects in the treatment or prevention of diabetes.

[0074] In this invention, diabetes can be type 1 diabetes or type 2 diabetes, especially type 2 diabetes.

[0075] In pharmaceutical compositions used to prevent or treat diabetes, peptides can inhibit insulin resistance signals or promote insulin sensitivity signals.

[0076] In pharmaceutical compositions used for the prevention or treatment of diabetes, peptides can promote phosphorylation of the insulin receptor substrate (IRS) Tyr632, promote activation of phospho-AKT, or promote phosphorylation of AMPK.

[0077] In pharmaceutical compositions used for the prevention or treatment of diabetes, peptides can inhibit the phosphorylation of Ser302 in the IRS in an environment that induces insulin resistance.

[0078] According to another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of obesity is provided, the composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

[0079] In pharmaceutical compositions used to prevent or treat obesity, peptides can promote lipolysis in adipocytes.

[0080] In pharmaceutical compositions used to prevent or treat obesity, peptides can increase the expression of lipid triglyceride lipase (ATGL), phosphorylated hormone-sensitive lipase (pHSL), or perilipin (PLIN, lipid droplet-associated protein) proteins that act as lipases in adipocytes.

[0081] The pharmaceutical compositions of the present invention may comprise a therapeutically effective amount of a peptide complex or peptide, and a pharmaceutically acceptable carrier.

[0082] As used herein, the term "therapeutic effective amount" means an amount sufficient to achieve the activity or efficacy of the active ingredient of the pharmaceutical composition of the present invention, such as an amount sufficient to achieve the efficacy of treating or preventing diabetes or obesity.

[0083] Pharmaceutically acceptable carriers are those commonly used in formulations, including but not limited to lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0084] In addition to the above-mentioned components, the pharmaceutical compositions of the present invention may further include lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. However, the present invention is not limited thereto.

[0085] Suitable pharmaceutically acceptable carriers and reagents are described in detail in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0086] The pharmaceutical compositions of the present invention can be administered via any suitable route to treat diabetes or obesity, such as by oral administration or parenteral administration. Parenteral administration can include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, local application, and transdermal application.

[0087] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg daily. The pharmaceutical composition may be prescribed in various ways depending on factors such as preparation method, route of administration, patient age, weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and responsiveness.

[0088] According to methods readily practiced by those skilled in the art, the pharmaceutical compositions of the present invention can be prepared into unit dosage forms or incorporated into multi-dosage form containers using pharmaceutically acceptable carriers and / or excipients. In this document, formulations may be in the form of solutions, suspensions, or emulsions in oily or aqueous media, or may be in the form of extracts, powders, granules, tablets, or capsules, and may further contain dispersants or stabilizers.

[0089] Composition

[0090] According to another aspect of the invention, a composition for controlling blood glucose levels is provided, the composition comprising a peptide complex as an active ingredient, the 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.

[0091] In the compositions of the present invention, controlling blood glucose levels can be achieved by controlling blood glucose levels in diabetic patients or patients at high risk of prediabetes.

[0092] In the compositions of the present invention, diabetes can be type 1 diabetes or type 2 diabetes, especially type 2 diabetes.

[0093] In the composition of the present invention, controlling blood glucose levels can be achieved by lowering blood glucose levels.

[0094] In the compositions of the present invention, an appropriate amount of peptide complex may be included in the range of 0.0001 wt% to 10 wt%, based on the total weight of the composition.

[0095] According to another aspect of the invention, a composition for controlling blood glucose levels is provided, the composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

[0096] In the composition of the present invention, controlling blood glucose levels can be done by controlling the blood glucose levels of diabetic patients or patients at high risk of prediabetes.

[0097] In the compositions of the present invention, diabetes can be type 1 diabetes or type 2 diabetes, especially type 2 diabetes.

[0098] In the composition of the present invention, controlling blood glucose levels can be achieved by lowering blood glucose levels.

[0099] In compositions used to control blood glucose levels, peptides can inhibit insulin resistance signals or promote insulin sensitivity signals.

[0100] In compositions used to control blood glucose levels, peptides can promote phosphorylation of the insulin receptor substrate (IRS) Tyr632, promote activation of phospho-AKT, or promote phosphorylation of AMPK.

[0101] In compositions used to control blood glucose levels, peptides can inhibit the phosphorylation of Ser302 in the IRS in an environment that induces insulin resistance.

[0102] According to another aspect of the invention, a composition for preventing or alleviating obesity is provided, the composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

[0103] In compositions used to prevent or alleviate obesity, peptides can promote lipolysis in adipocytes.

[0104] In compositions used to prevent or alleviate obesity, peptides can increase the expression of lipase proteins, triglyceride lipase (ATGL), phosphorylated hormone-sensitive lipase (pHSL), or perilipoplasmin (PLIN, lipid droplet-associated protein) in adipocytes.

[0105] In the compositions of the present invention, an appropriate amount of peptide may be included, selected from 0.0001 wt% to 10 wt%, based on the total weight of the composition.

[0106] In one embodiment, the composition of the present invention may comprise a bromatologically effective amount of a peptide complex and an acceptable carrier.

[0107] The compositions of the present invention may contain not only peptide complexes or peptides as active ingredients, but also commonly added ingredients such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of carbohydrates include: monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; oligosaccharides; common sugars such as polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of flavorings may include natural flavorings, sematrandrine, stevia extracts (e.g., stevia diglycoside A, glycyrrhizin, etc.), and synthetic flavorings (saccharin, aspartame, etc.). The proportion of carbohydrates may be, but is not limited to, about 1 g to 20 g per 100 g of the composition of the present invention, preferably about 5 g to 12 g.

[0108] In addition to the above-mentioned components, the compositions of the present invention may also contain various nutrients, vitamins, minerals (electrolytes), flavorings (such as synthetic and natural flavorings), colorants and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc.

[0109] For example, the compositions of the present invention, in addition to the peptide complex as an active ingredient, may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, licorice extract, etc.

[0110] According to another aspect of the present invention, the present invention provides a method for treating diabetes, the method comprising: administering to a diabetic patient a therapeutically effective amount of the above-mentioned peptide complex, the 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.

[0111] According to another aspect of the present invention, the present invention provides a method for controlling blood glucose levels, the method comprising: administering a therapeutically effective amount of the above-described peptide complex to a subject in need, the 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.

[0112] According to another aspect of the present invention, a method for treating, preventing or alleviating diabetes is provided, the method comprising: administering to a diabetic patient a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 2 described above.

[0113] According to another aspect of the invention, a method for controlling blood glucose is provided, comprising: administering a therapeutically effective amount of a peptide to a subject in need, the peptide comprising the amino acid sequence of SEQ ID NO: 2 described above.

[0114] According to another aspect of the present invention, a method for treating, alleviating or preventing obesity is provided, the method comprising: administering a therapeutically effective amount of a peptide to a subject in need of treating obesity, the peptide comprising the amino acid sequence of SEQ ID NO: 2 described above.

[0115] Beneficial effects

[0116] The peptide complexes or peptides of the present invention promote cellular glucose uptake, inhibit insulin resistance signals, promote insulin sensitivity signals, and inhibit apoptosis of pancreatic β cells (insulin-producing cells), thereby exhibiting a blood glucose-lowering effect. The peptide complexes or peptides of the present invention can be used to treat, prevent, and alleviate diabetes, and can be effectively used to lower blood glucose levels in diabetic patients or those at high risk of prediabetes.

[0117] However, the effects of the present invention are not limited to those described above, and those skilled in the art will clearly understand other effects not mentioned through the following description. Attached Figure Description

[0118] Figure 1a and Figure 1b The experimental results show that the peptide complex of the present invention promotes glucose uptake by adipocytes.

[0119] Figure 2a and Figure 2bThe experimental results show that the peptide complex of the present invention promotes glucose uptake by myoblasts.

[0120] Figure 3a and Figure 3b The experimental results show that the peptide complex of the present invention has the effect of promoting the activity of phospho-AMPK and phospho-ACC, wherein phospho-AMPK and phospho-ACC are insulin-sensitizing factors.

[0121] Figure 4a and Figure 4b The experimental results show that the peptide complex of the present invention has the effect of inhibiting the phosphorylation of IRS Serine302 and JNK, wherein IRS Serine302 and JNK are insulin resistance signaling factors.

[0122] Figure 5a and Figure 5b The experimental results show that the peptide complex of the present invention reduces the increase in gene expression of the insulin resistance-inducing cytokine TNF-α and the insulin resistance-promoting signaling factors mTOR and p70S6K induced by rhTNF-α treatment.

[0123] Figure 6a and Figure 6b The experimental results show that when adipocytes that had previously been treated with rhTNF-α to induce insulin resistance were treated with the peptide complex of the present invention, phosphorylation of IRS Tyrosine632 (an insulin sensitivity promoting factor) was enhanced, and activation of PI3K, AKT and AMPK was promoted.

[0124] Figure 7a and Figure 7b The experimental results show that treatment with the peptide complex of the present invention increases the expression of glucose uptake-related genes leptin, adiponectin, IRS-1 and GLUT4 in adipocytes, which were previously reduced by rhTNF-α treatment.

[0125] Figure 8a and Figure 8b The experimental results show that the peptide complex of the present invention reduces palmitic acid-induced reactive oxygen species (ROS).

[0126] Figure 9a and Figure 9b The experimental results show that treatment with the peptide complex of the present invention significantly reduces the gene expression of TNF-α (an inflammatory protein induced by palmitic acid).

[0127] Figure 10a and Figure 10bThe experimental results show that treatment with the peptide complex of the present invention significantly reduces the gene expression levels of TNF-α and IL-1β proteins in INS-1 cells (rat pancreatic β cells) that were originally increased by palmitic acid treatment.

[0128] Figure 11 The experimental results show that treatment with the peptide complex of the present invention reduced palmitic acid-induced increased INS-1 cell death again, thereby improving cell survival.

[0129] Figure 12a and Figure 12b The experimental results show that the peptide complex of the present invention promotes the expression of PGC-1α, ACOX-1, PPAR-α or CPT-1α genes in HepG2 cells, wherein PGC-1α, ACOX-1, PPAR-α or CPT-1α are genes related to FFA β-oxidation.

[0130] Figure 13a and Figure 13b The peptides of the present invention demonstrate that they increase the activity of lipase proteins, including triglyceride lipase (ATGL), phosphorylated hormone-sensitive lipase (pHSL), and perilipid-associated protein (PLIN) in adipocytes.

[0131] Figure 14 This demonstrates a trend in which the release of glycerol (lipolysis product) increases in a concentration-dependent manner when adipocytes are treated with the peptides of the present invention.

[0132] Figure 15a and Figure 15b It has been shown that treatment of adipocytes with the peptides of the present invention promotes phosphorylation of Tyr632 (a factor that promotes insulin sensitivity) of the insulin receptor substrate (IRS) and activation of phosphorylated AKT, and increases phosphorylation of the signaling protein AMPK. Meanwhile, Figure 15a and 15b The study showed reduced phosphorylation of Ser302, an insulin receptor substrate (IRS), in an environment that induces insulin resistance. Detailed Implementation

[0133] The present invention will now be described in detail through several embodiments. However, the following embodiments specifically describe the present invention, and the content of the present invention is not limited to the following embodiments.

[0134] Example

[0135] Preparation Example 1: Preparation of peptides and peptide complexes

[0136] The peptides with the amino acid sequence of SEQ ID NO: 1 and the peptides with the amino acid sequence of SEQ ID NO: 2, as described in Table 1 below, were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA). The synthesized peptides were completely separated by C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The chromatographic column used was an ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA).

[0137] [Table 1]

[0138]

[0139] The peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were mixed in equal amounts to prepare a peptide complex, and its efficacy was evaluated. In addition, the effect of the prepared peptide of SEQ ID NO: 2 was also evaluated.

[0140] Experiment Example 1: Promoting glucose uptake by adipocytes

[0141] The peptide complex prepared in Example 1 was tested using adipocytes to determine whether it promoted cellular glucose uptake.

[0142] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶ cells. 3 Cells were seeded in 96-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium, and differentiation into adipocytes was induced. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / ml insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with glucose-free medium, and the cells were cultured for 16 hours, and then cultured in 100 μL of KRPH buffer containing 2% BSA for 40 minutes to induce starvation. Cells were treated with rhTNF-α (2 nM), insulin (10 μg / ml), and peptide complex (2 μg / ml and 20 μg / ml) for 1 hour, and then treated with 80 μM 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 cells was imaged using a fluorescence microscope (ECLIPSE 80i, Nikon, Japan).

[0143] like Figure 1a and Figure 1b As shown, this experiment demonstrates that treatment with the peptide complex increases glucose uptake by cells inhibited by TNF-α. Furthermore, Experiment 3, described below, confirms that this effect occurs through the AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) signaling pathways. These experimental results indicate that the peptide complex again significantly increases glucose uptake by cells reduced due to inflammation.

[0144] Experiment Example 2: Promoting Glucose Uptake by Muscle Cells

[0145] The myoblast assay was used to determine whether the peptide complex prepared in Example 1 promoted cellular glucose uptake.

[0146] C2C12 myoblasts were charged at 5 × 10⁻⁶ 3 Cells were seeded in 96-well plates and cultured for two days in DMEM medium containing 10% FBS. The medium was then replaced with DMEM medium containing 2% horse serum, and C2C12 myoblasts were cultured for 6 days to induce differentiation into myotubes. Cultured myotubes were treated with rhTNF-α (2 nM), insulin (10 μg / ml), and peptide complexes (2 μg / ml and 20 μg / ml) for 1 hour, followed by treatment with 80 μM 2-NBDG and culturing for another 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 imaged using a fluorescence microscope (ECLIPSE80i, Nikon, Japan). Figure 2a and Figure 2b As shown, experiments have demonstrated that the peptide complex of the present invention increases glucose uptake by myoblasts inhibited by rhTNF-α treatment.

[0147] Experimental Example 3: Signaling Pathways of Peptide Complexes in Promoting Glucose Uptake

[0148] Protein expression analysis (Western blot analysis) was used to confirm whether the peptide complex prepared in Example 1 had the effect of promoting the activity of insulin sensitivity promoting factor.

[0149] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶. 3Cells were seeded in 96-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium, and cells were induced to differentiate into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / ml insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and the cells were cultured for 4 hours to induce starvation. The peptide complexes of Preparation Example 1 (0.2 μg / ml, 2 μg / ml, and 20 μg / ml) were added to the cells, and the cells were cultured for 30 minutes. Lysis buffer was added to lyse the cells. The lysate was then centrifuged at 4°C and 12,000 rpm for 30 minutes. The proteins thus obtained were quantified using a BCA kit. The proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a membrane. The membrane to which the proteins were attached was blocked with 5% skim milk, and then the primary antibody was reacted overnight at 4°C. The membrane was washed with PBS-T, and the secondary antibody was allowed to react at room temperature for 1 hour. The membrane was then washed again with PBS-T, and visualization was performed using Western spectroscopy assays (Elpis Biotech, Daejeon, Korea) via a Gel Doc system (Bio-Rad, Hercules, CA, USA). The antibodies used in the experiment were: anti-phospho-AMPK antibody (Cellsignaling technology (CST), USA), anti-phospho-ACC antibody (Cell signaling technology (CST), USA), and anti-α-tubulin antibody (Santa Cruz Biotechnology). Figure 3a and Figure 3b As shown, experiments have demonstrated that the peptide complex of the present invention has the effect of promoting the activity of phospho-AMPK and phospho-ACC, wherein phospho-AMPK and phospho-ACC are insulin sensitivity promoting factors.

[0150] Experimental Example 4: Inhibition of Insulin Resistance Signals by Peptide Complexes

[0151] Protein expression analysis (Western blot analysis) was used to confirm whether the peptide complex prepared in Preparation Example 1 had the effect of inhibiting insulin resistance signals.

[0152] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶. 5Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium, and cells were induced to differentiate into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / ml insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and the cells were cultured for 4 hours to induce starvation. Cells were treated with rhTNF-α (2 nM), insulin (10 μg / ml), and peptide complexes (0.2 μg / ml, 2 μg / ml, and 20 μg / ml) and cultured for 30 minutes. Lysis buffer was added to the cells of each treatment group to lyse the cells. The lysate was then centrifuged at 4°C and 12,000 rpm for 30 minutes. The proteins thus obtained were quantified using a BCA kit. The proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a membrane. The membrane containing the protein was blocked by treatment with 5% skim milk, followed by incubation of the primary antibody at 4°C overnight. The membrane was washed with PBS-T, and the secondary antibody was incubated at room temperature for 1 hour. The membrane was then washed again with PBS-T and visualized using a Western spectroscopy assay (Elpis Biotech, Daejeon, Korea) via a Gel Doc system (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). Figure 4a and Figure 4b As shown, experiments demonstrate that the peptide complex of the present invention inhibits the phosphorylation of Serine302 and c-Jun N-terminal kinase (JNK) of the insulin receptor substrate (IRS), wherein Serine302 and c-Jun N-terminal kinase (JNK) of the IRS are insulin resistance signaling factors, and their phosphorylation is induced by TNF-α treatment.

[0153] Experimental Example 5: Inhibition of Insulin Resistance-Induced Gene Expression by Peptide Complexes

[0154] The study tested whether the peptide complex prepared in Example 1 inhibited the expression of insulin resistance-inducing genes. Adipocytes were treated with rhTNF-α to induce insulin resistance, then treated with the peptide complex, and the expression levels of mTOR and p70S6K genes were measured.

[0155] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium, and cells were induced to differentiate into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / ml insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and the cells were cultured for 4 hours to induce starvation. Cells were treated with rhTNF-α (2 nM), insulin (1 μg / ml), and peptide complexes (0.2 μg / ml, 2 μg / ml, and 20 μg / ml). 24 hours later, RNA was extracted from the untreated control and treated groups and RT-PCR was performed. Easy-BLUE was used. TM RNA was extracted from 3T3-L1 cells using a 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, mammalian target of rapamycin (mTOR), p70S6K, or GAPDH genes, and PCR was performed using a PCR instrument (Eppendorf, Germany). The mRNA expression pattern was subsequently determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiment are shown in Table 2 below.

[0156] [Table 2]

[0157]

[0158] like Figure 5a and Figure 5b As shown, the experiment demonstrated that rhTNF-α treatment increased the expression of the TNF-α gene, as well as the mTOR and p70S6K genes. However, after treatment with the peptide complex, the expression of these genes decreased again. The TNF-α gene is an inflammatory cytokine that induces insulin resistance, while the mTOR and p70S6K genes are signaling factors that promote insulin resistance.

[0159] Experimental Example 6: Promotion of Insulin Sensitivity Signals by Peptide Complexes

[0160] The test was conducted to determine whether the peptide complex prepared in Example 1 promoted insulin-sensitive signals.

[0161] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium, and cells were induced to differentiate into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / mL insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and the cells were cultured for 4 hours to induce starvation. Cells were treated with rhTNF-α (2 nM), insulin (1 μg / mL), and peptide complexes (0.2 μg / mL, 2 μg / mL, and 20 μg / mL) and cultured for 30 minutes. Lysis buffer was added to the cells of each treatment group to lyse the cells. The lysate was then centrifuged at 4 °C and 12,000 rpm for 30 minutes. The proteins thus obtained were quantified using a BCA kit. The proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a membrane. The membrane containing protein attachment was blocked by treatment with 5% skim milk, followed by incubation of the primary antibody at 4°C overnight. The membrane was washed with PBS-T, and the secondary antibody was incubated at room temperature for 1 hour. The membrane was then washed again with PBS-T and visualized using a Western spectroscopy assay (Elpis Biotech, Daejeon, Korea) via Gel Doc (Bio-Rad, Hercules, CA, USA). The antibodies used in the experiment were: anti-phospho-IRS (Tyr632) antibody (Cellsignaling technology (CST), USA), anti-phospho-PI3K antibody (Cellsignaling technology (CST), USA), anti-phospho-AKT antibody (Cellsignaling technology (CST), USA), anti-phospho-AMPK antibody (Cellsignaling technology (CST), USA), and anti-α-tubulin antibody (Santa Cruz Biotechnology, USA). Figure 6a and Figure 6b As shown, experiments indicate that in adipocytes with insulin resistance induced by rhTNF-α treatment, peptide complex treatment increases phosphorylation of insulin receptor substrate (IRS) Tyrosine632 (a factor that promotes insulin sensitivity), and thus promotes the activation of phosphatidylinositol 3-kinase (PI3K), AKT, and AMP-activated protein kinase (AMPK).

[0162] Experiment Example 7: Expression of genes related to promoting glucose uptake

[0163] The expression analysis of leptin, adiponectin, insulin receptor substrate 1 (IRS-1), and glucose transporter type 4 (GLUT4) genes was used to confirm whether the peptide complex prepared in Preparation Example 1 promoted the expression of glucose uptake-related genes.

[0164] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium, and cells were induced to differentiate into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / mL insulin, then the medium was replaced every two days with medium containing 10% FBS until preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and cells were cultured for 4 hours to induce starvation. Cells were treated with rhTNF-α (2 nM), insulin (1 μg / ml), and peptide complexes (0.2 μg / ml, 2 μg / ml, and 20 μg / ml). 24 hours later, RNA was extracted from the untreated control and treated groups and RT-PCR was performed. Easy-BLUE was used. TM RNA was extracted from 3T3-L1 cells using a 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 each gene, and PCR was performed using a PCR instrument (Eppendorf, Germany). Subsequently, mRNA expression patterns were determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiments are shown in Table 3 below.

[0165] [Table 3]

[0166]

[0167] like Figure 7a and Figure 7b As shown, the experiment demonstrated that in adipocytes, peptide complex treatment increased the expression of glucose uptake-related genes leptin, adiponectin, IRS-1, and GLUT4, which were reduced by rhTNF-α treatment.

[0168] Experimental Example 8: Inhibition of Palmitic Acid-Induced ROS Production

[0169] The intracellular ROS detection analysis (FACS) was used to confirm whether the peptide complex prepared in Preparation Example 1 inhibited the production of reactive oxygen species (ROS) induced by palmitic acid (a free fatty acid).

[0170] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium, and differentiation into adipocytes was induced. Two days after induction, preadipocytes were cultured in medium containing 10% FBS and 10 μg / ml insulin for two days, then the medium was replaced every two days with medium containing 10% FBS until preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and cells were cultured for 4 hours to induce starvation. Cells were treated with rhTNF-α (2 nM), insulin (1 μg / ml), and a peptide complex (2 μg / ml). The untreated control and treated groups were cultured for 24 hours and then treated with DCF-DH for 30 minutes. Oxidative activity was then measured under fluorescence using FACS (Becton Dickinson & Company (BD), USA). Figure 8a and Figure 8b As shown, the measurement results indicate that the peptide complex reduced the amount of reactive oxygen species (ROS) induced by palmitic acid.

[0171] Experimental Example 9: Inhibition of Palmitic Acid-Induced TNF-α Gene Expression

[0172] Gene expression analysis (RT-PCR) was used to confirm whether the peptide complex prepared in Preparation Example 1 inhibited inflammation induced by palmitic acid (a free fatty acid).

[0173] INS-1 cells induced to starve on serum-free RPMI 1640 (Gibco, New York, USA) medium with 25 μM palmitic acid were pretreated for 2 h and then treated with peptide complexes (0.2 μg / ml, 2 μg / ml, and 20 μg / ml). After 24 hours, they were analyzed using easy-BLUE. TMRNA was extracted using a 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, and PCR was performed using a PCR instrument (Eppendorf, Germany). The mRNA expression pattern was subsequently determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiment are shown in Table 4 below.

[0174] [Table 4]

[0175]

[0176] like Figure 9a and Figure 9b As shown, the experiment demonstrated that peptide complex treatment significantly reduced the gene expression of TNF-α (palmitic acid-induced inflammatory protein).

[0177] Experimental Example 10: Inhibition of Palmitic Acid-Induced Inflammatory Cytokine Expression

[0178] Protein performance analysis (Western proteolysis) was used to confirm whether the peptide complex prepared in Example 1 inhibited the expression of palmitic acid (a free fatty acid)-induced inflammatory cytokines.

[0179] INS-1 cells (rat pancreatic β cells) induced with serum-free RPMI 1640 medium (Gibco, New York, USA) and starved for 2 h were pretreated with 25 μM palmitic acid and treated with peptide complexes (0.2 μg / ml, 2 μg / ml, and 20 μg / ml). After 24 hours, the cells were washed once with PBS and lysed on ice for 30 min with lysis buffer (Millipore, Darmstadt, Germany) containing 10 mM Tris (pH 7.5), 100 mM NaCl, 1% NP-40, and a protease inhibitor. The lysate was then centrifuged at 4 °C and 13,000 rpm for 10 min. The proteins thus obtained were quantified using a BCA kit (Thermo Fisher Scientific, Waltham, USA). Equal volumes of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to a polyvinylidene fluoride membrane. To block non-specific antibody binding, the protein-linked membrane was treated with 5% skim milk (BD, New Jersey, USA) and blocked for 1 hour, followed by incubation of the primary antibody at 4°C overnight. The membrane was washed with PBS-T, and the secondary antibody was incubated at room temperature for 1 to 2 hours. The membrane was then washed again with PBS-T, and enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Waltham, USA) was used to visualize the protein on X-ray film. The antibodies used in the experiment were: 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). Figure 10a and Figure 10b As shown, the experiment demonstrated that the increased expression levels of TNF-α and IL-1β proteins in palmitic acid-treated INS-1 cells (rat pancreatic β cells) were significantly reduced again by peptide complex treatment.

[0180] Experimental Example 11: Inhibition of Palmitic Acid-Induced Apoptosis in Pancreatic β-Cells

[0181] The cell cytotoxicity assay was used to confirm whether the peptide complex prepared in Preparation Example 1 inhibited palmitic acid (a free fatty acid)-induced apoptosis of pancreatic β cells.

[0182] INS-1 cells (rat pancreatic β cells) induced with serum-free RPMI 1640 (Gibco, New York, USA) and starved for 2 hours were pretreated with 25 μM palmitic acid and then treated with peptide complexes (0.2 μg / ml, 2 μg / ml, and 20 μg / ml). After 24 hours, CCK-8 solution (Dojindo, Kumamoto, Japan) was added until the medium volume was 1 / 10, and absorbance was measured every 30 minutes or 1 hour. The experiment was terminated when the average absorbance value of the control group was 1.0. The absorbance of the reaction products was measured at 450 nm using a microplate reader. Figure 11 As shown in the figure, the experiment demonstrated that treatment with the peptide complex reduced the palmitic acid-induced increase in INS-1 cell death, thereby improving cell survival.

[0183] Experimental Example 12: Expression of genes related to promoting glucose uptake

[0184] Gene expression analysis (RT-PCR) was used to confirm whether the peptide complex prepared in Preparation Example 1 promoted the expression of genes related to glucose uptake, including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), acyl-CoA oxidase 1 (ACOX-1), peroxisome proliferator-activated receptor-α (PPAR-α), and carnitine palmitoyltransferase 1α (CPT-1α).

[0185] When HepG2 liver cancer cells reached 80% confluence, they were divided into groups of 2 × 10⁻⁶ cells. 5 Cells were seeded in 12-well plates and used for experiments. The next day, the medium was replaced with serum-free medium to induce starvation for 4 hours. After 4 hours, cells were treated with a peptide complex (0.2 μg / ml, 2 μg / ml, and 20 μg / ml). After 24 hours, RNA was extracted from both the untreated control and treated groups and used for RT-PCR. Easy-BLUE was used. TM RNA was extracted using a 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 PGC-1α, ACOX-1, PPAR-α, or CPT-1α genes, and PCR was performed using a PCR instrument (Eppendorf, Germany). The mRNA expression pattern was subsequently determined by agarose gel electrophoresis. The nucleotide sequences of the primers used in the experiment are shown in Table 5 below.

[0186] [Table 5]

[0187]

[0188] like Figure 12a and Figure 12b As shown, the experiment demonstrated that the peptide complex promoted the expression of PGC-1α, ACOX-1, PPAR-α, or CPT-1α genes in HepG2 cells, among which PGC-1α, ACOX-1, PPAR-α, and CPT-1α genes are genes associated with FFA β-oxidation.

[0189] Experimental Example 13: Promoting the expression of lipase proteins in adipocytes

[0190] The peptide prepared in Example 1, SEQ ID NO: 2, was tested using protein expression analysis (Western blot analysis) to determine whether it promotes the expression of lipase protein in adipocytes.

[0191] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶ cells. 5Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium [DMEM medium containing 10% FBS, 1 μg / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA)] to induce differentiation into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / ml insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and the cells were cultured for 4 hours to induce starvation. Cells were divided into an untreated group (negative control), a peptide (2 μg / ml, 20 μg / ml)-treated group (prepared in Example 1), and a TNFα (20 nM)-treated group (positive control), and then treated with the predetermined concentrations of the substance and cultured for 1 hour. Cells in each treatment group were then lysed by adding lysis buffer. After lysis, the proteins obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes were quantified using a BCA kit. The proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a membrane. The membrane with protein attachment was blocked by treatment with 5% skim milk, and then the primary antibody was allowed to react overnight at 4°C. The membrane was washed with PBS-T, and the secondary antibody was allowed to react at room temperature for 1 hour. The membrane was washed again with PBS-T, and then visualized using a Gel Doc (Bio-Rad, Hercules, CA, USA) with a Western blot assay kit (Elpis Biotech, Daejeon, Korea). The antibodies used in the experiment were: 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). Figure 13a and Figure 13b As shown, experiments have demonstrated that treatment with the peptides of this invention increases the expression of lipase protein, triglyceride lipase (ATGL), phosphorylated hormone-sensitive lipase (pHSL), and perilipid-associated protein (PLIN) in adipocytes.

[0192] Experimental Example 14: Promoting Lipolysis in Adipocytes

[0193] The amount of glycerol released from the lipolysis product was analyzed to confirm whether the peptide prepared in Example 1 (SEQ ID NO: 2) promotes lipolysis in adipocytes.

[0194] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶ cells. 5 Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium [DMEM medium containing 10% FBS, 1 μg / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA)] to induce differentiation into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / mL insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and the cells were cultured for 4 hours to induce starvation. Cells were divided into an untreated group (negative control), a peptide (2 μg / ml, 20 μg / ml) treatment group (prepared in Example 1), and a TNF-α (20 nM) treatment group (positive control), and then treated with the predetermined concentration of the substance and cultured for 48 hours. The supernatant was then obtained. The obtained supernatant was analyzed for glycerol release using a "Glycerol Colorimetric Assay Kit" (Cayman Chemical, USA), and the amount of glycerol released was compared. Figure 14 As shown, when adipocytes are treated with the peptides of the present invention, glycerol release assays indicate that the release of glycerol (lipolysis product) from adipocytes tends to increase in a concentration-dependent manner relative to the treated peptide.

[0195] Experiment Example 15: Inhibiting Insulin Resistance Signals and Promoting Insulin Sensitivity Signals

[0196] Protein expression was analyzed to confirm whether the peptide of SEQ ID NO: 2 prepared in Preparation Example 1 inhibits insulin resistance in adipocytes and promotes insulin-sensitive signaling.

[0197] When 3T3-L1 preadipocytes reached 80% confluence, they were divided into groups of 5 × 10⁻⁶ cells. 5Cells were seeded in 6-well plates for experiments. Two days after reaching confluence, the medium was replaced with differentiation-inducing medium [DMEM medium containing 10% FBS, 1 μg / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA)] to induce differentiation into adipocytes. Two days after induction, preadipocytes were cultured for two days in medium containing 10% FBS and 10 μg / mL insulin, and then the medium was replaced every two days with medium containing 10% FBS until the preadipocytes differentiated into adipocytes. When differentiation was complete, the medium was replaced with serum-free medium, and the cells were cultured for 4 hours to induce starvation. rhTNF-α (2 nM), insulin (1 μg / ml), and the peptides of SEQ ID NO: 2 of Preparation Example 1 (2 μg / ml, 20 μg / ml) were added to the cells, and the cells were cultured for 1 hour. Cells were cultured for 30 minutes, and cells in each treatment group were lysed by adding lysis buffer, followed by centrifugation at 4°C and 12,000 rpm for 30 minutes. Proteins were quantified using a BCA kit. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to membranes. The membranes containing protein attachments were blocked with 5% skim milk, and the primary antibody was incubated overnight at 4°C. The membranes were washed with PBS-T, incubated with the secondary antibody at room temperature for 1 hour, washed again with PBS-T, and visualized using a Western spectroscopy assay (Elpis Biotech, Daejeon, Korea) via 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). Figure 15a and Figure 15bAs shown, experiments demonstrate that treatment with the peptides of the present invention promotes phosphorylation of Tyr632, a substrate of the insulin receptor (IRS), and activation of phosphorylated AKT, factors that promote insulin sensitivity. Furthermore, it was confirmed that phosphorylation of AMPK (a signaling protein that increases insulin sensitivity) increased upon treatment with the peptides of the present invention. Simultaneously, it was confirmed that treatment with the peptides of the present invention reduced phosphorylation of Ser302, a substrate of the insulin receptor (IRS), under insulin resistance-inducing conditions, thereby inhibiting insulin resistance.

[0198] Preparation Example 2: Preparation of Pharmaceutical Compositions

[0199] 2-1. Preparation of powder

[0200] The peptide complex or peptide of the present invention, 2g

[0201] Lactose, 1g

[0202] The above ingredients are mixed and filled into a sealed bag to prepare a powder.

[0203] 2-2. Tablet Preparation

[0204] The peptide complex or peptide of the present invention, 100 mg

[0205] Corn starch, 100 mg

[0206] Lactose, 100 mg

[0207] Magnesium stearate, 2 mg

[0208] The above ingredients are mixed and compressed according to conventional tablet preparation methods to provide tablets.

[0209] 2-3. Preparation of capsules

[0210] The peptide complex or peptide of the present invention, 100 mg

[0211] Corn starch, 100 mg

[0212] Lactose, 100 mg

[0213] Magnesium stearate, 2 mg

[0214] The above ingredients are mixed according to conventional capsule preparation methods and filled into gelatin capsules to prepare capsules.

[0215] 2-4. Preparation of pills

[0216] The peptide complex or peptide of the present invention, 1 g;

[0217] Lactose, 1.5 g

[0218] Glycerin, 1g

[0219] Xylitol, 0.5 g

[0220] The above ingredients are mixed and prepared into pills in a manner that yields 4 g of pill weight, according to conventional methods.

[0221] 2-5. Preparation of granules

[0222] The peptide complex or peptide of the present invention, 150 mg

[0223] Soybean extract, 50 mg

[0224] Glucose, 200 mg

[0225] Starch, 600 mg

[0226] Mix the above ingredients, add 100 mg of 30% ethanol, and then dry at 60°C. After forming granules, fill the granules into packaging bags.

[0227] Preparation Example 3: Preparation of Composition

[0228] 3-1. Preparation of the composition

[0229] The peptide complex or peptide of the present invention, 500 μg

[0230] Appropriate amount of vitamin mixture

[0231] Vitamin A acetate, 70 mg

[0232] Vitamin E, 1.0 mg

[0233] Vitamin D, 0.13 mg

[0234] Vitamin B2, 0.15 mg

[0235] Vitamin B6, 0.5 mg

[0236] Vitamin B12, 0.2 mg

[0237] Vitamin C, 10 mg

[0238] Biotin, 10 mg

[0239] Nicotinamide, 1.7 mg

[0240] Folic acid, 50 mg

[0241] Calcium pantothenate, 0.5 mg

[0242] Appropriate amount of mineral mixture

[0243] Ferrous sulfate, 1.75 mg

[0244] Zinc oxide, 0.82 mg

[0245] Magnesium carbonate, 25.3 mg

[0246] Potassium dihydrogen phosphate, 15 mg

[0247] Dipotassium hydrogen phosphate, 55 mg

[0248] Potassium citrate, 90 mg

[0249] Calcium carbonate, 100 mg

[0250] Magnesium chloride, 24.8 mg

[0251] Although, as a preferred embodiment, the components suitable for use in the composition are mixed in a ratio of vitamin mixture to mineral mixture, this mixing ratio can be varied arbitrarily, and the components can be mixed according to general preparation methods, then prepared into granules, which can then be used to prepare the composition according to general methods.

[0252] 3-2. Preparation of the composition

[0253] The peptide complex or peptide of the present invention, 500 μg

[0254] Citric acid, 1000 mg

[0255] Oligosaccharides, 100 g

[0256] Green plum concentrate, 2 g

[0257] Taurine, 1 g

[0258] Add pure water until the total volume reaches 900 ml.

[0259] The above-mentioned components are mixed according to a general preparation method. The mixture is heated at 85°C and stirred for about 1 hour to prepare a solution. The solution is filtered, and the filtrate is collected in a sterile container, then sealed and sterilized, and then refrigerated for storage. It can then be used to prepare the composition. Although, as a preferred embodiment, the above-described proportions are used to mix a variety of components that are relatively suitable for use in a preferred composition, the mixing proportions can be varied according to local and national preferences, such as the level of demand, the country of demand, the purpose of use, etc.

[0260] Although representative embodiments of this application have been described above, the scope of this application is not limited to the specific embodiments described above, and those skilled in the art can modify this application within the scope of the claims.

Claims

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

3. Use of the peptide complex according to claim 1 in the preparation of a medicament for the prevention or treatment of diabetes.

4. A composition for controlling blood glucose levels, comprising the peptide complex according to claim 1 as an active ingredient.

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

2.

6. A pharmaceutical composition for the prevention or treatment of diabetes, comprising the peptide of claim 5 as an active ingredient.

7. Use of the peptide according to claim 5 in the preparation of a medicament for the prevention or treatment of diabetes.

8. A pharmaceutical composition for the prevention or treatment of obesity, comprising the peptide of claim 5 as an active ingredient.

9. Use of the peptide according to claim 5 in the preparation of a medicament for the prevention or treatment of obesity.

10. A composition for controlling blood glucose levels, comprising the peptide of claim 5 as an active ingredient.

11. A composition for preventing or alleviating obesity, comprising the peptide of claim 5 as an active ingredient.

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