Composition for the prevention, improvement, or treatment of obesity and diabetes, containing zinc gluconate and cyclo-hyspro as active ingredients.

Abstract

The present invention provides compositions for the prevention, improvement, or treatment of obesity and diabetes. [Solution] A therapeutic composition is provided that contains zinc gluconate and cyclo-hispro (CHP) as active ingredients.

Description

[Technical Field]

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of obesity and diabetes, comprising zinc gluconate and cyclo-hispro (CHP) as active ingredients. More specifically, the invention provides a composition in which the type of zinc salt and / or the content ratio of zinc components are optimized, resulting in significantly improved anti-obesity and anti-diabetic effects, and a method for preventing, improving, or treating obesity and diabetes using the same. [Background technology]

[0002] Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia and insulin resistance in various organs. A healthy lifestyle, including exercise, appropriate diet, and weight control, can help manage the disease. However, as the disease progresses, oral medication and insulin therapy are often necessary. Currently available oral medications such as hypoglycemic agents or insulin sensitizers, including sulfonylureas (SUs), biguanides, thiazolidinediones (TZDs), dipeptidyl peptidase-4 (DPP-4) inhibitors, and sodium-glucose cotransporter 2 (SGLT2) inhibitors, have been used to regulate blood glucose levels over the long term. However, some medications have limited efficacy and can induce a variety of side effects. For example, patients taking SUs have an increased risk of weight gain and hypoglycemia, and patients taking biguanides are exposed to a potential risk of lactic acidosis. TZDs are not recommended for patients with pre-existing edema, heart failure, or acute liver disease. The most frequent adverse reaction to DPP-4 inhibitors is upper respiratory tract infection, while SGLT2 inhibitors are associated with urinary tract and genital infections. Therefore, there is a need to develop safer and more effective drugs with diverse mechanisms of action.

[0003] Lysine acetylation plays a crucial role in maintaining energy homeostasis across diverse metabolic pathways. In particular, various enzymes involved in glucose and lipid metabolism are regulated by acetylation of lysine residues. Sirtuin family enzymes regulate the activity of many other enzymes, including β-nicotinamide adenine dinucleotide (NAD). + It consists of increased levels of )-dependent deacetylase. PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), a positive regulator of mitochondrial biogenesis, is regulated by sirtuin 1 (Sirt1). Sirt1 also regulates the acetylation of liver kinase B1 (LKB1), a major AMP-activated kinase (AMPK) kinase involved in lipid synthesis and fatty acid oxidation. Sirtuins are also considered a novel target for the treatment of chronic metabolic diseases, and enhancement of Sirt1 activity has been reported to reverse the pathological effects of T2DM.

[0004] While Patent Document 1 discloses a composition containing zinc ions and cyclo-hispro (CHP) as a useful composition for mitigating diabetic symptoms in mammals, it does not specify an optimized form of zinc salt for improving the therapeutic effect of obesity and diabetes.

[0005] Against this backdrop, the inventors confirmed that the combination of zinc gluconate and CHP exhibits significantly superior anti-obesity and anti-diabetic effects compared to other zinc salt and CHP combinations, and thus completed the present invention. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Korean Published Patent Publication No. 10-2001-0022786 [Overview of the project]

Problems to be Solved by the Invention

[0007] Therefore, an object of the present invention is to provide a composition for preventing, improving or treating obesity and diabetes, which contains zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof as active ingredients.

[0008] Another object of the present invention is to provide a method for preventing, improving or treating obesity and diabetes, which includes administering zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual who needs it.

Means for Solving the Problems

[0009] In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating obesity and diabetes, which contains zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof as active ingredients.

[0010] The present invention also provides a health functional food composition for preventing or improving obesity and diabetes, which contains zinc gluconate and cyclo-hispro or a food pharmaceutically acceptable salt thereof as active ingredients.

[0011] In addition, the present invention provides a method for preventing, improving or treating obesity and diabetes, which includes administering zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual who needs it.

[0012] In the present invention, the zinc cation or zinc elemental component of the zinc gluconate salt and cyclo-hispro or its pharmaceutically or food-scientifically acceptable salt may be contained or administered in a weight ratio of 1 to 5:1 to 4, where the zinc cation or zinc elemental component may be contained or administered in a higher weight ratio than cyclo-hispro or its pharmaceutically or food-scientifically acceptable salt.

[0013] In the present invention, the zinc gluconate salt and cyclo-hispro or its pharmaceutically or food-scientifically acceptable salt may be contained or administered in a dosage of 15 to 250 mg.

[0014] In the present invention, the zinc gluconate salt and cyclo-hispro or its pharmaceutically or food-scientifically acceptable salt may be contained or administered in a weight ratio of 7 to 35:1 to 4.

[0015] In the present invention, the diabetes may be type 2 diabetes accompanied by obesity.

[0016] In the present invention, the composition can exhibit anti-obesity and anti-diabetic effects through increasing the synthesis of NAD that regulates Sirt1 deacetylase activity in the liver and visceral adipose tissue. +

Effects of the Invention

[0017] When the composition according to the present invention is used in combination with CHP, the type and / or content ratio of the zinc salt is optimized so that the anti-obesity and anti-diabetic effects are maximized, and it can be utilized as a therapeutic agent and a health functional food for the prevention, improvement or treatment of obesity, diabetes and diabetes accompanied by obesity.

Brief Description of the Drawings

[0018] [Figure 1] Figure 1 is a diagram showing the weight loss effect by administration of CycloZ containing various forms of zinc salts in KKAy mice, which are diabetes and obesity models. [Figure 2] ​Figure 2 shows the effect of administering CycloZ, which contains various zinc salt forms, on reducing glycated hemoglobin (HbA1c) in KKAy mice, a model of diabetes and obesity. [Figure 3a] Figures 3a and 3b show the changes in fasting blood glucose (a) and the blood glucose concentration 2 hours after oral glucose tolerance test (b) in KKAy mice, which are diabetic and obese models, after administration of CycloZ containing various zinc salt forms. [Figure 3b] Figures 3a and 3b show the changes in fasting blood glucose (a) and the blood glucose concentration 2 hours after oral glucose tolerance test (b) in KKAy mice, which are diabetic and obese models, after administration of CycloZ containing various zinc salt forms. [Figure 4a] Figures 4a and 4b show the results of examining changes in fasting blood glucose (a) and blood glycated hemoglobin (HbA1c) (b) in KKAy mice, a model of diabetes and obesity, after individual or combined administration of zinc gluconate and CHP. [Figure 4b] Figures 4a and 4b show the results of examining changes in fasting blood glucose (a) and blood glycated hemoglobin (HbA1c) (b) in KKAy mice, a model of diabetes and obesity, after individual or combined administration of zinc gluconate and CHP. [Figure 4c] Figures 4c to 4f show the results of examining the changes in calorie intake (c), changes in blood low-density lipoprotein cholesterol levels (d), changes in blood total cholesterol levels (e), and the size frequency of adipocytes in white adipose tissue (f) in KKAy mice, which are diabetic and obese models, respectively, after administration of a combination of zinc gluconate and CHP (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 4d]Figures 4c to 4f show the results of examining the changes in calorie intake (c), changes in blood low-density lipoprotein cholesterol levels (d), changes in blood total cholesterol levels (e), and the size frequency of adipocytes in white adipose tissue (f) in KKAy mice, which are diabetic and obese models, respectively, after administration of a combination of zinc gluconate and CHP (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 4e] Figures 4c to 4f show the results of examining the changes in calorie intake (c), changes in blood low-density lipoprotein cholesterol levels (d), changes in blood total cholesterol levels (e), and the size frequency of adipocytes in white adipose tissue (f) in KKAy mice, which are diabetic and obese models, respectively, after administration of a combination of zinc gluconate and CHP (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 4f] Figures 4c to 4f show the results of examining the changes in calorie intake (c), changes in blood low-density lipoprotein cholesterol levels (d), changes in blood total cholesterol levels (e), and the size frequency of adipocytes in white adipose tissue (f) in KKAy mice, which are diabetic and obese models, respectively, after administration of a combination of zinc gluconate and CHP (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 5a] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5b] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5c] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5d]Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5e] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5f]Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5g] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5h]Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5i] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5j]Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5k] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5l]Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 5m] Figures 5a to 5m show, in order, the changes in body weight (a), changes in the weight of each organ (b), oral glucose tolerance test results (c), changes in fasting blood glucose (d), changes in plasma glycated hemoglobin (HbA1c) (e), changes in plasma insulin concentration (f), changes in plasma free fatty acid concentration (g), changes in plasma triglyceride concentration (h), changes in plasma high-density lipoprotein concentration (i), changes in plasma adiponectin levels (j), changes in mRNA expression levels of genes related to fatty acid and cholesterol synthesis in the liver (k), hematoxylin and eosin staining results of the liver (l), and hematoxylin and eosin staining results of epididymal adipose tissue (EAT) (m) in KKAy mice, which are diabetic and obese models (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 6a]Figures 6a to 6e show, in order, changes in mRNA expression levels of genes associated with inflammatory cytokines and infiltrated mononuclear cells in the liver and mesenteric adipose tissue following combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), changes in TNFα and MCP-1 protein levels in the liver (b), changes in TNFα and MCP-1 protein levels in EAT (c), changes in TNFα, MCP-1, F4 / 80, and CD11b expression in the liver as measured by immunohistochemistry (d), and changes in TNFα, MCP-1, F4 / 80, and CD11b expression in EAT as measured by immunohistochemistry (e) (aP≦0.05, bP≦0.01, cP≦0.0001). [Figure 6b] Figures 6a to 6e show, in order, changes in mRNA expression levels of genes associated with inflammatory cytokines and infiltrated mononuclear cells in the liver and mesenteric adipose tissue following combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), changes in TNFα and MCP-1 protein levels in the liver (b), changes in TNFα and MCP-1 protein levels in EAT (c), changes in TNFα, MCP-1, F4 / 80, and CD11b expression in the liver as measured by immunohistochemistry (d), and changes in TNFα, MCP-1, F4 / 80, and CD11b expression in EAT as measured by immunohistochemistry (e) (aP≦0.05, bP≦0.01, cP≦0.0001). [Figure 6c]Figures 6a to 6e show, in order, changes in mRNA expression levels of genes associated with inflammatory cytokines and infiltrated mononuclear cells in the liver and mesenteric adipose tissue following combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), changes in TNFα and MCP-1 protein levels in the liver (b), changes in TNFα and MCP-1 protein levels in EAT (c), changes in TNFα, MCP-1, F4 / 80, and CD11b expression in the liver as measured by immunohistochemistry (d), and changes in TNFα, MCP-1, F4 / 80, and CD11b expression in EAT as measured by immunohistochemistry (e) (aP≦0.05, bP≦0.01, cP≦0.0001). [Figure 6d] Figures 6a to 6e show, in order, changes in mRNA expression levels of genes associated with inflammatory cytokines and infiltrated mononuclear cells in the liver and mesenteric adipose tissue following combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), changes in TNFα and MCP-1 protein levels in the liver (b), changes in TNFα and MCP-1 protein levels in EAT (c), changes in TNFα, MCP-1, F4 / 80, and CD11b expression in the liver as measured by immunohistochemistry (d), and changes in TNFα, MCP-1, F4 / 80, and CD11b expression in EAT as measured by immunohistochemistry (e) (aP≦0.05, bP≦0.01, cP≦0.0001). [Figure 6e]Figures 6a to 6e show, in order, changes in mRNA expression levels of genes associated with inflammatory cytokines and infiltrated mononuclear cells in the liver and mesenteric adipose tissue following combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), changes in TNFα and MCP-1 protein levels in the liver (b), changes in TNFα and MCP-1 protein levels in EAT (c), changes in TNFα, MCP-1, F4 / 80, and CD11b expression in the liver as measured by immunohistochemistry (d), and changes in TNFα, MCP-1, F4 / 80, and CD11b expression in EAT as measured by immunohistochemistry (e) (aP≦0.05, bP≦0.01, cP≦0.0001). [Figure 7a] Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 7b]Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 7c] Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 7d]Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 7e] Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 7f]Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 7g] Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 7h]Figures 7a to 7h show, in order, changes in acetylated lysine levels of p65 in the liver and white adipose tissue (WAT) after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (e) (f) (g [Figure 8a] Figures 8a to 8e show, in order, the total acetyl-lysine in liver tissue after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), mitochondrial DNA (mtDNA) in the liver as measured by mitochondrial D-loop PCR (b), increased NAD+ / NADH ratio in skeletal muscle (c), Sirt1 protein expression in the liver and EAT (d), and changes in Sirt1 mRNA expression levels in the liver and EAT (aP ≤ 0.05, bP ≤ 0.01). [Figure 8b] Figures 8a to 8e show, in order, the total acetyl-lysine in liver tissue after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), mitochondrial DNA (mtDNA) in the liver as measured by mitochondrial D-loop PCR (b), increased NAD+ / NADH ratio in skeletal muscle (c), Sirt1 protein expression in the liver and EAT (d), and changes in Sirt1 mRNA expression levels in the liver and EAT (aP ≤ 0.05, bP ≤ 0.01). [Figure 8c]Figures 8a to 8e show, in order, the total acetyl-lysine in liver tissue after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), mitochondrial DNA (mtDNA) in the liver as measured by mitochondrial D-loop PCR (b), increased NAD+ / NADH ratio in skeletal muscle (c), Sirt1 protein expression in the liver and EAT (d), and changes in Sirt1 mRNA expression levels in the liver and EAT (aP ≤ 0.05, bP ≤ 0.01). [Figure 8d] Figures 8a to 8e show, in order, the total acetyl-lysine in liver tissue after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), mitochondrial DNA (mtDNA) in the liver as measured by mitochondrial D-loop PCR (b), increased NAD+ / NADH ratio in skeletal muscle (c), Sirt1 protein expression in the liver and EAT (d), and changes in Sirt1 mRNA expression levels in the liver and EAT (aP ≤ 0.05, bP ≤ 0.01). [Figure 8e] Figures 8a to 8e show, in order, the total acetyl-lysine in liver tissue after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a), mitochondrial DNA (mtDNA) in the liver as measured by mitochondrial D-loop PCR (b), increased NAD+ / NADH ratio in skeletal muscle (c), Sirt1 protein expression in the liver and EAT (d), and changes in Sirt1 mRNA expression levels in the liver and EAT (aP ≤ 0.05, bP ≤ 0.01). [Figure 9a]Figures 9a to 9d show, in order, the NAD+ / NADH ratio in the liver and EAT after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 9b] Figures 9a to 9d show, in order, the NAD+ / NADH ratio in the liver and EAT after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 9c] Figures 9a to 9d show, in order, the NAD+ / NADH ratio in the liver and EAT after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 9d] Figures 9a to 9d show, in order, the NAD+ / NADH ratio in the liver and EAT after combined administration of zinc gluconate and CHP in KKAy mice, a diabetic and obese model (a) (b) (c) (d) (aP≦0.05, bP≦0.01, cP≦0.001). [Figure 10a]Figures 10a to 10g show, in order, changes in glycated hemoglobin (HbA1c) in KKAy mice, a diabetic and obese model (a), changes in body weight after administration of zinc gluconate and CHP in KKAy mice (b), cumulative calorie intake (c), weight of each organ (d), changes in mRNA expression levels of genes related to mitochondrial biodevelopment in the liver (e), changes in mRNA expression levels of genes related to lipid oxidation in the liver (f), and total acetyl-lysine in the liver (g) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 10b] Figures 10a to 10g show, in order, changes in glycated hemoglobin (HbA1c) in KKAy mice, a diabetic and obese model (a), changes in body weight after administration of zinc gluconate and CHP in KKAy mice (b), cumulative calorie intake (c), weight of each organ (d), changes in mRNA expression levels of genes related to mitochondrial biodevelopment in the liver (e), changes in mRNA expression levels of genes related to lipid oxidation in the liver (f), and total acetyl-lysine in the liver (g) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 10c] Figures 10a to 10g show, in order, changes in glycated hemoglobin (HbA1c) in KKAy mice, a diabetic and obese model (a), changes in body weight after administration of zinc gluconate and CHP in KKAy mice (b), cumulative calorie intake (c), weight of each organ (d), changes in mRNA expression levels of genes related to mitochondrial biodevelopment in the liver (e), changes in mRNA expression levels of genes related to lipid oxidation in the liver (f), and total acetyl-lysine in the liver (g) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 10d]Figures 10a to 10g show, in order, changes in glycated hemoglobin (HbA1c) in KKAy mice, a diabetic and obese model (a), changes in body weight after administration of zinc gluconate and CHP in KKAy mice (b), cumulative calorie intake (c), weight of each organ (d), changes in mRNA expression levels of genes related to mitochondrial biodevelopment in the liver (e), changes in mRNA expression levels of genes related to lipid oxidation in the liver (f), and total acetyl-lysine in the liver (g) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 10e] Figures 10a to 10g show, in order, changes in glycated hemoglobin (HbA1c) in KKAy mice, a diabetic and obese model (a), changes in body weight after administration of zinc gluconate and CHP in KKAy mice (b), cumulative calorie intake (c), weight of each organ (d), changes in mRNA expression levels of genes related to mitochondrial biodevelopment in the liver (e), changes in mRNA expression levels of genes related to lipid oxidation in the liver (f), and total acetyl-lysine in the liver (g) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 10f] Figures 10a to 10g show, in order, changes in glycated hemoglobin (HbA1c) in KKAy mice, a diabetic and obese model (a), changes in body weight after administration of zinc gluconate and CHP in KKAy mice (b), cumulative calorie intake (c), weight of each organ (d), changes in mRNA expression levels of genes related to mitochondrial biodevelopment in the liver (e), changes in mRNA expression levels of genes related to lipid oxidation in the liver (f), and total acetyl-lysine in the liver (g) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 10g]Figures 10a to 10g show, in order, changes in glycated hemoglobin (HbA1c) in KKAy mice, a diabetic and obese model (a), changes in body weight after administration of zinc gluconate and CHP in KKAy mice (b), cumulative calorie intake (c), weight of each organ (d), changes in mRNA expression levels of genes related to mitochondrial biodevelopment in the liver (e), changes in mRNA expression levels of genes related to lipid oxidation in the liver (f), and total acetyl-lysine in the liver (g) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 11a] Figures 11a to 11f show, in order, the results of oral glucose tolerance tests (a), changes in glycated hemoglobin (HbA1c) (b), acetylated lysine levels for PGC-1α and LKB1 in the liver (c), phosphorylated AMPK (T172) in the liver (d), increased NAD+ / NADH ratio and NAD+ quantification results in the liver (e), and changes in mRNA expression levels of genes related to NAD+ synthesis in the liver (f) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 11b] Figures 11a to 11f show, in order, the results of oral glucose tolerance tests (a), changes in glycated hemoglobin (HbA1c) (b), acetylated lysine levels for PGC-1α and LKB1 in the liver (c), phosphorylated AMPK (T172) in the liver (d), increased NAD+ / NADH ratio and NAD+ quantification results in the liver (e), and changes in mRNA expression levels of genes related to NAD+ synthesis in the liver (f) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 11c]Figures 11a to 11f show, in order, the results of oral glucose tolerance tests (a), changes in glycated hemoglobin (HbA1c) (b), acetylated lysine levels for PGC-1α and LKB1 in the liver (c), phosphorylated AMPK (T172) in the liver (d), increased NAD+ / NADH ratio and NAD+ quantification results in the liver (e), and changes in mRNA expression levels of genes related to NAD+ synthesis in the liver (f) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 11d] Figures 11a to 11f show, in order, the results of oral glucose tolerance tests (a), changes in glycated hemoglobin (HbA1c) (b), acetylated lysine levels for PGC-1α and LKB1 in the liver (c), phosphorylated AMPK (T172) in the liver (d), increased NAD+ / NADH ratio and NAD+ quantification results in the liver (e), and changes in mRNA expression levels of genes related to NAD+ synthesis in the liver (f) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 11e] Figures 11a to 11f show, in order, the results of oral glucose tolerance tests (a), changes in glycated hemoglobin (HbA1c) (b), acetylated lysine levels for PGC-1α and LKB1 in the liver (c), phosphorylated AMPK (T172) in the liver (d), increased NAD+ / NADH ratio and NAD+ quantification results in the liver (e), and changes in mRNA expression levels of genes related to NAD+ synthesis in the liver (f) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Figure 11f]Figures 11a to 11f show, in order, the results of oral glucose tolerance tests (a), changes in glycated hemoglobin (HbA1c) (b), acetylated lysine levels for PGC-1α and LKB1 in the liver (c), phosphorylated AMPK (T172) in the liver (d), increased NAD+ / NADH ratio and NAD+ quantification results in the liver (e), and changes in mRNA expression levels of genes related to NAD+ synthesis in the liver (f) (aP≦0.05, bP≦0.01, cP≦0.001, dP≦0.0001). [Modes for carrying out the invention]

[0019] The present invention will be described in more detail below. All technical terms used in this invention, unless otherwise defined, are used in the same sense as those generally understood by those skilled in the art in the relevant field. Furthermore, preferred methods and samples are described herein, and similar or equivalent methods and samples are also included within the scope of this invention.

[0020] As mentioned above, no optimized zinc salt form is known for improving the therapeutic effects of zinc salts and CHP in combinations of zinc salts and CHP. Therefore, the inventors sought a solution to the above problem by experimentally verifying that the combination of zinc gluconate and CHP shows superior effects in improving obesity and treating diabetes compared to other zinc salt and CHP combinations.

[0021] Accordingly, the present invention relates in a first aspect to a pharmaceutical composition for the prevention or treatment of obesity and diabetes, comprising zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof as active ingredients.

[0022] In this specification, the term "cyclo-hisPro (CHP)" refers to a naturally occurring cyclic dipeptide composed of histidine-proline, a metabolite of thyrotropin-releasing hormone (TRH), or a physiologically active dipeptide that may be synthesized in the body de novo through the TRH metabolic process, and is a substance widely distributed throughout the brain, spinal cord, and digestive tract.

[0023] In the composition of the present invention, cyclo-hyspro can be synthesized or commercially available. Alternatively, cyclo-hyspro can be purified from substances containing it, such as prostate extract and soy hydrolysate.

[0024] The use of the term "purified" is intended to imply that cyclo-hyspro is in a more concentrated form compared to forms that can be obtained from natural sources, such as prostate extract. Purified components can be obtained by concentrating these natural sources or through chemical synthesis methods.

[0025] In this specification, the zinc salt and cyclo-hyspro may also be referred to as "CycloZ," and if the zinc salt is zinc gluconate, it may be referred to as "gluconate-CycloZ." Zinc gluconate and cyclo-hyspro may be included in the composition of the present invention in the form of a single compound or as individual components. Therefore, zinc gluconate and cyclo-hyspro may be administered in the form of a single compound, or as individual components simultaneously, separately, or sequentially.

[0026] In the present invention, the ratio of the zinc cation or zinc element component of the zinc gluconate salt to cyclo-hyspro or a pharmaceutically acceptable salt thereof may be 1 to 10:1 to 5 by weight, preferably 1 to 5:1 to 4, 1 to 5:1 to 3, or 1 to 5:1 to 2 by weight.

[0027] In the present invention, zinc gluconate and cyclo-hyspro (gluconate-Cyclo-Z) may be included in a single compound form at a dose of 15 to 250 mg when applied clinically. When zinc gluconate and cyclo-hyspro are included as individual components, they may be included in a dose calculated by the weight ratio of zinc gluconate:cyclo-hyspro as defined in the present invention, within the aforementioned dose range. In this case, if the total dose of zinc gluconate and cyclo-hyspro (gluconate-Cyclo-Z) is less than 15 mg, the dose may be too low to produce an effect in treating obesity and diabetes, and if it exceeds 250 mg, toxicity issues may arise.

[0028] In the present invention, the zinc gluconate salt and cyclo-hyspro or a pharmaceutically acceptable salt thereof may be included in a weight ratio of 7-35:1-4, preferably 7-35:1-3 or 1-5:1-2. If the zinc gluconate salt and cyclo-hyspro are included in amounts deviating from the above weight ratios, the therapeutic effects on obesity and diabetes may be reduced.

[0029] In the present invention, the diabetes may be type 2 diabetes or type 2 diabetes accompanied by obesity.

[0030] The composition of the present invention exhibits beneficial effects on both obesity and diabetes from both preventive and therapeutic viewpoints. Young KK-Ay mice are used as a mild hyperglycemia model for the preventive treatment of type 2 diabetes with obesity. Hyperglycemia and hyperinsulinemia in KK-Ay mice worsen with age. Therefore, in this invention, young KK-Ay mice and aged KK-Ay mice with progressive hyperglycemia were used in separate studies to verify the therapeutic effects of gluconate-Cyclo-Z in mild hyperglycemia models and severe diabetes models.

[0031] According to a specific embodiment of the present invention, gluconate-CycloZ exhibits anti-obesity and anti-diabetic effects through the regulation of protein acetylation in the liver and VAT. Increased VAT mass is one of the main risk factors for various metabolic diseases, and administration of gluconate-CycloZ significantly reduced liver and VAT mass in KK-Ay mice. Furthermore, PGC-1α deacetylation by gluconate-CycloZ administration induced transcriptional regulation of genes related to mitochondrial function in the liver and VAT. A close relationship is known to exist between PGC-1α activity and the development of type 2 diabetes, which is related to mitochondrial biosynthesis and glucose / fatty acid metabolism. Specifically, reduced PGC-1α activity is associated with altered lipid oxidation, and PGC-1α expression in adipose tissue is downwardly regulated in patients with type 2 diabetes.

[0032] Chronic inflammation accompanied by abnormally elevated cytokine levels and immune cell infiltration is observed in many metabolic disorders and contributes to disease progression. Therefore, in a specific embodiment of the present invention, we observed reduced inflammation in the liver and VAT of KK-Ay mice treated with gluconate-CycloZ. We confirmed that acetylation of p65, a core NF-κB subunit, was reduced upon administration of gluconate-CycloZ.

[0033] In yet another specific embodiment of the present invention, gluconic acid-CycloZ is NAD + We confirmed that it modulates the expression of enzymes involved in synthesis. NAMPT mRNA expression was increased in both the liver and VAT of gluconate-CycloZ treated mice. Since NAMPT is a rate-limiting enzyme, gluconate-CycloZ treatment modifies NAD + The level was increased, and then Sirt1 activity was increased. NAD + / NADH ratio and NAD + The amount increased only in the liver and VAT, but not in the muscles. This is because gluconate-CycloZ increases NAD in a tissue-specific manner. + This demonstrates that synthesis can be controlled.

[0034] In yet another specific example of the present invention, it was confirmed that gluconic acid-CycloZ increases Sirt1 mRNA and protein expression in the liver and EAT of KK-Ay mice. In the present invention, NAD that is essential for Sirt1 activity + levels and NAD + / NADH ratio increase, so it can be inferred that Sirt1 activity can increase.

[0035] Overall, gluconic acid-CycloZ increases NAD + levels and regulates the activity of NAD-dependent deacetylase such as sirtuin, thereby reducing protein acetylation. +

[0036] Consequently, gluconic acid-CycloZ activated the Sirt1 / PGC-1α / LKB1 / AMPK signaling axis and showed excellent anti-obesity and anti-diabetic properties and safety profile. Based on such data, gluconic acid-CycloZ is a novel NAD + booster and Sirt1 deacetylase activator with a mechanism of action different from existing type 2 diabetes drugs.

[0037] The term "prevention" as used in the present invention means all acts that suppress or delay the onset of a disease or medical condition. In the present invention, it means delaying the onset time or suppressing the onset of obesity and diabetes.

[0038] The term "amelioration" as used in the present invention means all acts that improve or beneficially change a disease or medical condition, and in the present invention, it means improving the symptoms of obesity and diabetes.

[0039] The term "treatment" as used in the present invention means all acts that delay, interrupt or reverse the progression of a disease or medical condition, and in the present invention, it means reducing, alleviating or removing or reversing the symptoms of obesity and diabetes.

[0040] As used in this invention, the term "pharmaceutically acceptable salt" means any organic or inorganic addition salt of cyclohyspro at a concentration that is relatively non-toxic to the patient, has no harmful active effect, and the side effects caused by this salt do not diminish the beneficial efficacy of cyclohyspro. These salts can be made using inorganic or organic acids as free acids. As inorganic acids, hydrochloric acid, bromate, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc., and as organic acids, citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, gluconic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid, etc. Furthermore, these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, acid addition salts include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hypenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, maleate, maleate, malonate, mesylate, It may also contain methyl sulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, etc.

[0041] The pharmaceutical compositions of the present invention may further contain a pharmaceutically acceptable carrier. The pharmaceutical compositions containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, they can be prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, or other diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, lozenges, etc. Such solid formulations can be prepared by mixing one or more compounds of the present invention with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium, stearate, and talc can also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, or syrups, and may contain various excipients in addition to water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweeteners, fragrances, and preservatives.

[0042] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, glycerol, and gelatin.

[0043] The pharmaceutical composition of the present invention can be administered via any common route that can reach the target tissue or cells in an individual or sample. Such administration may include, but is not limited to, systemic or local administration, and may include, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, intranasal, intrapulmonary, or rectal administration. The dosage of the pharmaceutical composition may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity.

[0044] The term "patient" refers to any single individual requiring treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, etc. It also includes any subject participating in a clinical research trial or a biomechanical study that does not exhibit any disease-related clinical findings, or any subject used as a control group.

[0045] A second aspect of the present invention relates to a functional food composition for the prevention or improvement of obesity and diabetes, comprising zinc gluconate and cyclo-hispro or a food-grade salt thereof as active ingredients.

[0046] The description of the composition and effects of zinc gluconate and cyclo-hyspro, which are included as active ingredients in the health functional food composition of the present invention, is the same as described above, so that description is omitted.

[0047] In this invention, the term "food-grade salt" includes salts derived from food-grade organic acids, inorganic acids, or bases.

[0048] In this invention, the term "health functional food" encompasses the meanings of both "functional food" and "health food."

[0049] In this invention, the term "functional food" is the same as "food for special health use (FoSHU)," and refers to a food that has high medical and therapeutic effects, processed to efficiently exhibit biological regulatory functions in addition to providing nutrients.

[0050] In this invention, the term "health food" refers to food that has a more active effect on maintaining or promoting health compared to general foods, and "health supplement food" refers to food intended for health supplementation. The terms functional food, health food, and health supplement food may be used interchangeably depending on the context. The aforementioned foods can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills to obtain effects useful in preventing or improving obesity and diabetes.

[0051] As a specific example of such functional foods, the zinc gluconate salt and cyclo-hyspro or a food-grade salt of the present invention can be used to produce processed foods that modify agricultural, livestock, or marine products while preserving their characteristics and improving their storability.

[0052] The health functional food composition of the present invention can also be manufactured in the form of nutritional supplements, food additives, and animal feed, and is intended for consumption by humans or animals, including livestock.

[0053] The aforementioned types of food compositions can be manufactured in various forms by conventional methods known to the industry. General foods, though not limited to these, include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and their processed foods (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candies, dairy products (e.g., butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., miso, soy sauce, sauces, etc.), which can be manufactured by adding the zinc gluconate salt and cyclo-hyspro or a food-grade salt of the present invention.

[0054] Furthermore, as a nutritional supplement, it can be manufactured by adding the zinc gluconate salt and cyclo-hyspro or a food-safe salt thereof of the present invention to capsules, tablets, pills, etc., although this is not limited to the present invention.

[0055] Furthermore, as a health functional food, although not limited to these, for example, the zinc gluconate salt and cyclo-hyspro or a food-grade salt of the present invention can be liquefied, granulated, encapsulated, and powdered for consumption in the form of tea, juice, and drinks (health beverages). In addition, the zinc gluconate salt and cyclo-hyspro or a food-grade salt of the present invention can be used as a food additive by being manufactured and used in the form of a powder or concentrated liquid. Furthermore, the zinc gluconate salt and cyclo-hyspro or a food-grade salt of the present invention can be manufactured in the form of a composition by mixing them with known active ingredients known to be effective in preventing or improving obesity and diabetes.

[0056] When the food composition of the present invention is used as a health beverage composition, the health beverage composition may contain various flavorings or natural carbohydrates as further ingredients, as with ordinary beverages. The natural carbohydrates mentioned above may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates is generally about 0.01 to 0.04 g per 100 mL of the composition of the present invention, preferably about 0.02 to 0.03 g.

[0057] The zinc gluconate salt and cyclo-hyspro or a food-grade acceptable salt of the present invention may be included as active ingredients in a health functional food composition for the prevention or improvement of obesity and diabetes, and the amount thereof is an amount effective in obtaining the aforementioned preventive or ameliorative effect, preferably, for example, 0.01 to 100% by weight of the total weight of the whole composition, but is not particularly limited thereto. The health functional food composition of the present invention can be produced in the form of a composition by mixing the zinc gluconate salt and cyclo-hyspro or a food-grade acceptable salt together with a known active ingredient known to be effective in preventing or improving obesity and diabetes.

[0058] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavorings, colorings, pectin, salts of pectin, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, the health food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. Such components can be used individually or in combination. The proportion of such additives is not of great importance, but it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0059] A third aspect of the present invention relates to a method for preventing, improving or treating obesity and diabetes, comprising administering zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need.

[0060] In the methods of the present invention, the term “individual” includes, but is not limited to, any animal (e.g., human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cattle, cattle, guinea pig, or rodent). Such terms do not indicate a specific age or sex. Thus, it is intended to include fetuses as well as female / female, male / male, adult / mature and neonatal subjects. “Patient” refers to a subject with a disease or disability. The term patient includes human and veterinary subjects.

[0061] In the method of the present invention, the description of the configuration, including the effects, route of administration, number of administrations, and dosage of zinc gluconate salt and cyclo-hyspro or pharmaceutically acceptable salts thereof administered to the individual, is the same as described above, and therefore will be omitted.

[0062] In the method of the present invention, zinc gluconate and cyclo-hyspro, when administered in an effective amount, can provide desirable preventive, ameliorative, or therapeutic effects on obesity and diabetes. For desirable effects, the combination of zinc gluconate and cyclo-hyspro or a pharmaceutically acceptable salt of the present invention can be administered once or several times at regular time intervals. In this case, the zinc gluconate and cyclo-hyspro may be administered simultaneously, separately, or sequentially. For example, zinc gluconate and cyclo-hyspro or a pharmaceutically acceptable salt of the present invention may be co-formulated and administered simultaneously in a combined unit dose formulation, or administered simultaneously or sequentially as separate formulations.

[0063] When administered sequentially, each active ingredient may be administered as an individual formulation with time intervals between doses, and the order of administration may be determined by a physician or a person with ordinary knowledge in the field.

[0064] Furthermore, it can be used in combination with other methods for the prevention, improvement, or treatment of obesity and diabetes.

[0065] A fourth aspect of the present invention relates to the use of compositions comprising zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof for the manufacture of agents for the prevention, improvement or treatment of obesity and diabetes.

[0066] In the use of the present invention, the description of the effects of the composition containing the zinc gluconate salt and cyclo-hyspro or a pharmaceutically acceptable salt thereof, as well as its configuration including the route of administration, number of administrations, and dosage, is the same as described above, and therefore will be omitted.

[0067] A fifth aspect of the present invention involves administering zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need, wherein β-nicotinamide adenine dinucleotide (NAD + Regarding methods for increasing it.

[0068] In relation to the fifth aspect described above, the present invention relates to β-nicotinamide adenine dinucleotide (NAD + The present invention provides the use of a composition comprising zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof for the manufacture of a booster.

[0069] In the present invention, the NAD + The increase may be in the liver and / or visceral adipose tissue.

[0070] The description of the effects of the composition containing the aforementioned zinc gluconate salt and cyclo-hyspro or a pharmaceutically acceptable salt thereof, as well as its composition including the route of administration, number of administrations, and dosage, is the same as described above and is therefore omitted.

[0071] A sixth aspect of the present invention relates to a method for increasing Sirt1 deacetylase activity, comprising administering zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need. In connection with the fifth aspect described above, the present invention provides the use of a composition comprising zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof for producing a Sirt1 deacetylase activator.

[0072] The description of the effects of the composition containing the aforementioned zinc gluconate salt and cyclo-hyspro or a pharmaceutically acceptable salt thereof, as well as its composition including the route of administration, number of administrations, and dosage, is the same as described above and is therefore omitted. [Examples]

[0073] The present invention will be described in more detail below based on examples. It will be obvious to those ordinary in the art that these examples are for illustrative purposes only and that the scope of the present invention is not limited by these examples.

[0074] [Example 1] Confirmation of the effects of different types of zinc salts on improving obesity. 1-1. Experimental animals and administered drugs CLEA Japan Inc. purchased 5-week-old male Kkay mice from Celon Bio Co., Ltd. The mice were reared under controlled conditions (temperature: 22±2℃, relative humidity: 55±10%, cycle: 12 hours), fed with Purina feed and drinking water (distilled water). The mice were used in experiments after a one-week adaptation period following purchase. CHP was purchased from Angene, zinc gluconate from Captek Softgel International, and zinc acetate, zinc chloride, and zinc sulfate from Sigma-Aldrich.

[0075] Five-week-old Kkay mice were pre-fed for one week, then randomly divided into groups based on similar average body weight. The administration conditions for each group are shown in Table 1. The control group received distilled water orally daily. Experimental group 1 received gluconate-CycloZ, a mixture of zinc gluconate and CHP in a 2:1 weight ratio (based on zinc element). Experimental group 2 received acetate-CycloZ, a mixture of zinc acetate and CHP in a 2:1 weight ratio (based on zinc element). Experimental group 3 received chloride-CycloZ, a mixture of zinc chloride and CHP in a 2:1 weight ratio (based on zinc element). Experimental group 4 received sulfate-CycloZ, a mixture of zinc sulfate and CHP in a 2:1 weight ratio (based on zinc element). Body weight was measured weekly.

[0076] [Table 1]

[0077] 1-2. Confirmation of weight loss effect Weight was measured weekly for 15 weeks during drug administration, and compared at the 15th week. As can be seen in Figure 1, the average body weight, which was approximately 26.6g at the start of the experiment, increased to 46.6g in the control group, while experimental group 1 decreased to 42.9g, experimental group 2 to 44g, experimental group 3 to 44.7g, and experimental group 4 to 45.9g. The weight reduction effect compared to the control group was 7.9%, 5.6%, 4.1%, and 1.5% in the experimental groups, depending on the type of zinc salt, with experimental group 1 showing the greatest reduction effect and statistical significance. This confirms that gluconate-CycloZ containing zinc gluconate is the most effective in improving obesity compared to other forms of CycloZ containing zinc salts.

[0078] [Example 2] Confirmation of the effect of different types of zinc salts on improving diabetes. 2-1. Oral glucose tolerance test and measurement of glycated hemoglobin To confirm the effect of different types of zinc salts on improving diabetes, oral glucose tolerance tests and glycated hemoglobin measurements were performed. First, mice were fasted for 16 hours for the oral glucose tolerance test (OGTT), and 2 g / kg of glucose was administered orally via a gastric tube. Blood was collected from the tail vein at 15, 30, 60, 90, and 120 minutes after glucose administration. Blood glucose levels were measured immediately using a blood glucose meter (AGM-4000, Allmedicus, Anyang, South Korea). Blood was also collected from the tail vein to measure glycated hemoglobin (HbA1c). HbA1c was measured using a DCA Vantage® analyzer (Siemens, Munich, Germany).

[0079] As shown in Figure 2, the blood glycated hemoglobin level, a standard indicator of diabetes, showed a statistically significant decrease of approximately 13.5% in experimental group 1 compared to the control group. Furthermore, as shown in Figures 3a and 3b, a decrease in fasting blood glucose was observed in experimental group 1, and it was confirmed that blood glucose decreased most rapidly 2 hours after glucose administration. Compared to the control group, glucose tolerance test results showed decreases of 18.8% (experimental group 1), 2.5% (experimental group 2), and 8.4% (experimental group 3), depending on the type of zinc salt used, while experimental group 4 showed no reduction compared to the control group.

[0080] Through this study, it was confirmed that gluconate-CycloZ, in the form containing zinc gluconate, is the most effective for improving blood glucose regulation and diabetes compared to other zinc salt-containing forms of CycloZ.

[0081] statistical analysis The statistical significance of the experimental results in Examples 1 and 2 was analyzed for each experimental group using t-test statistics against the control group. *p<0.05, **p<0.01.

[0082] [Example 3] Confirmation of the effects of gluconate-CycloZ administration on improving T2DM and obesity in diabetic and obese animal models. 3-1. Experimental animals and administered drugs Five-week-old male KK-Ay mice purchased from CLEA Japan Inc. (Nishi-Shinbashi, Japan) were raised in individual cages in an air-conditioned room with a 12-hour light-dark cycle and a temperature of 23±3°C. Distilled water and laboratory food were provided free access. All animal experiments were approved by the Ethics Review Committee (ABCC201712) of the Pohang Advanced Bio-Fusion Center, South Korea. All animals were used in experiments one week after adaptation. For preventive studies, the animals were divided into two groups. The control group was administered water as an excipient. KK-Ay mice were given gluconate-CycloZ (CHP 5 mg / kg and zinc gluconate 70 mg / kg) daily via gastric tube for 20 weeks. For therapeutic studies, the animals were divided into a control group and an experimental group at 12 weeks of age. KK-Ay mice were given water or gluconate-CycloZ daily via gastric tube for 8 weeks in each group. After each experiment, all mice were anesthetized with isopullulan using the RC2 rodent circuit controller anesthesia system (Vetequip, Pleasanton, CA, USA). Blood was collected via cardiac puncture, and plasma was separated. The separated adipose tissue, liver, and plasma were stored at -80°C until analysis.

[0083] 3-2. Oral glucose tolerance test and measurement of glycated hemoglobin Oral glucose tolerance tests and glycated hemoglobin measurements were performed using the same method as in Example 2-1. As confirmed in Figures 4a and 4b, gluconate-CycloZ administration showed a greater improvement in glucose tolerance compared to individual compound treatments. Furthermore, as measured by the OGTT results, gluconate-CycloZ improved glucose tolerance in KK-Ay mice, as confirmed in Figure 5c. As shown in Figures 5d, 5e, and 5f, gluconate-CycloZ administration significantly reduced fasting blood glucose, HbA1c levels, and plasma insulin concentration. These results demonstrate that gluconate-CycloZ improved glucose metabolism and insulin sensitivity.

[0084] 3-3. Measurement of body weight, food intake, and weight of each organ As can be seen in Figures 5a and 4c, the weight gain in the gluconate-CycloZ-treated mice gradually decreased compared to the control group at the end of the experiment, and there was no noticeable difference in food intake. Furthermore, as can be seen in Figure 5b, gluconate-CycloZ administration significantly suppressed the mass increase of liver and visceral adipose tissue (VAT), such as epididymal adipose tissue (EAT) and mesenteric adipose tissue (MAT), but not subcutaneous adipose tissue. Gluconate-CycloZ treatment also reduced the liver and VAT weight in mice.

[0085] 3-4. Analysis of blood biochemical parameters Whole blood was collected via cardiac puncture, and plasma was separated by centrifugation at 2,000xg for 10 minutes. The plasma was then stored at -80°C until analysis. Aspartate aminotransferase (AST), alanine aminotransferase, alkaline phosphatase, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, creatinine, and blood urea nitrogen (BUN) were measured using a biochemical analyzer (BS-390, Mindray Bio-medical Electronics Co. Ltd., Shenzhen, China). Free fatty acids were quantified using the Enzy-Chromium Free Fatty Acid Assay Kit (EFFA-100, BioAssay System, Hayward, CA, USA).

[0086] As shown in Table 2, gluconate-CycloZ administration did not induce changes in AST, BUN, and creatinine, confirming its safety and excellent drug tolerance.

[0087] [Table 2]

[0088] We also investigated the blood lipid profiles of mice using a biochemical analyzer. As can be seen in Figures 5g, 5h, and 5i, mice treated with gluconic acid-CycloZ showed a decrease in free fatty acid and triglyceride levels and an increase in HDL-C levels, but total cholesterol levels remained unchanged, as shown in Figures 4d and 4e.

[0089] 3-5. RNA extraction, cDNA synthesis, and mRNA expression analysis Total RNA was extracted from tissues and cells using NucleoZOL reagent (740404.200, Macherey-Nagel, Allentown, PA, USA). 1 μg of total RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (1708891, Bio-Rad, Hercules, CA, USA). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using the gene-specific primers shown in Table 3 and IQ SYBR Green Supermix (BR1708882, Bio-Rad). RT-qPCR was performed using the following amplification cycles: 10 seconds at 95°C, 10 seconds at 60°C, and 30 seconds at 72°C. Expression levels were normalized to the expression levels of β-actin or GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

[0090] [Table 3]

[0091] The decrease in lipid levels in Examples 3-4 is explained as a decrease in mRNA expression of genes related to fatty acid and cholesterol synthesis, including Srebf1 (sterol regulatory-element binding transcription factor 1), Fasn (fatty acid synthase), Srebf2, and Hmgcr (3-hydroxy-3-methylglutaryl-CoA reductase), as shown in Figure 5k.

[0092] 3-6.Histochemistry Liver and adipose tissue were fixed in neutral buffered 10% formalin solution (HT-501128, Sigma, St. Louis, MO, USA) and embedded in paraffin blocks. A series of (4 μm thick) sections were deparaffinized, dehydrated, and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis was performed using antibodies against tumor necrosis factor α (TNFα, Abcam, Cambridge, UK; ab1793), macrophage chemoattractant protein 1 (MCP-1, Abcam, ab25124), F4 / 80 (Abcam, ab111101), and CD11b (Abcam, ab133357). Stained areas were confirmed using a light microscope (Olympus BX53 upright microscope, Olympus, Tokyo, Japan).

[0093] As can be seen in Figure 5l, gluconate-CycloZ administration also improved hepatic lipid deposition, while the control group exhibited fatty liver pathology similar to steatosis. Furthermore, as can be seen in Figures 5m and 4f, the adipocyte area of ​​EAT was significantly reduced in the gluconate-CycloZ treated group compared to the control group.

[0094] 3-7. Western blot For protein experiments, tissues and cells were dissolved in radioactive immunoprecipitation (RIPA) buffer (89901, Thermo Scientific, Waltham, MA, USA) containing a cocktail of Halt protease and phosphatase inhibitors (78440, Thermo Scientific). SDS-PAGE was performed using Bolt 4%~12% Bis-Tris Plus Gel (Thermo Scientific) and a Trans-Blot Turbo system (Bio-Rad). The following antibodies were used to detect the target proteins: PGC-1α (NBP1-04676, Novusbio, Centennial, CO, USA), Ac-Lysine (9814S, Cell Signaling Technology [CST], Danvers, MA, USA), phospho-AMPK (5831S, CST), AMPK (2535S, CST), phospho-Akt (9271S, CST), Akt (9272S, CST), LC3 I / II (4108S, CST), GAPDH (2118S, CST), adiponectin (2789S, CST), and Sirt1 (07-131, EMD Millipore, Burlington, MA, USA).

[0095] As can be seen in Figure 5j, adiponectin levels were significantly increased in the blood of gluconate-CycloZ-treated mice.

[0096] Based on these results, we confirmed that the weight loss induced by gluconate-CycloZ administration was due to fat reduction resulting from the regulation of lipid and cholesterol metabolism in the liver and VAT.

[0097] [Example 4] Confirmation of the effects of gluconate-CycloZ administration on improving inflammation and reducing immune cell infiltration in diabetic and obese animal models. 4-1. mRNA expression analysis Since obesity-induced insulin resistance in the liver and VAT is closely associated with chronic inflammation in many tissues, we investigated whether gluconate-CycloZ reduces the production of pro-inflammatory cytokines (TNFα and MCP-1) and mononuclear cell infiltration (F4 / 80 and CD11b) through mRNA expression analysis. RT-qPCR was performed in the same manner as in Examples 3-5, and the gene-specific primer information used is shown in Table 4.

[0098] [Table 4]

[0099] The expression levels of inflammatory cytokine genes in the liver and MAT, as well as the expression levels of F4 / 80 and MCP-1, were significantly reduced by gluconate-CycloZ administration, as can be seen from Figure 6a. Consistently, gluconate-CycloZ treatment also caused a substantial decrease in TNFα and MCP-1 protein levels in the liver and EAT, as can be seen through Figures 6b and 6c.

[0100] 4-2.Histochemistry The expression of TNFα, MCP-1, F4 / 80, and CD11b in the liver and EAT was investigated by immunohistochemistry. Histochemistry was performed using the same method as in Examples 3-6. As can be seen from Figures 6d and 6e, inflammatory cytokine production and mononuclear cell infiltration completely disappeared with gluconate-CycloZ administration. Taken together, these results indicate that the improvement in tissue insulin resistance after gluconate-CycloZ administration was accompanied by a reduction in inflammation.

[0101] [Example 5] Confirmation of the effects of gluconate-CycloZ administration on mitochondrial biodevelopment and inflammation improvement in diabetic and obese animal models. 5-1. Western blot Permeating macrophages play a crucial role in developing insulin resistance in metabolic organelles. Their activity is partially regulated by the deacetylation of transcription factors such as the p65 subunit of NF-κB. Lysine acetylation also regulates the activity of many metabolic enzymes and transcription factors. Existing reports have shown that the overall lysine acetylation profile is increased in the kidneys and hearts of diabetic patients (Berthiaume, Jessica M., et al. “Methylene blue decreases mitochondrial lysine acetylation in the diabetic heart.” Molecular and Cellular Biochemistry 432(2017):7-24; and Kosanam, Hari, et al. “Diabetes induces lysine acetylation of intermediary metabolic enzymes in the kidney.” Diabetes 63.7(2014):2432-2439). Therefore, in this example, Western blotting was performed as in Examples 3-7 to investigate the total acetyllysine level in the livers of mice treated with gluconate-CycloZ.

[0102] As shown in Figure 7a, gluconate-CycloZ-treated mice showed a strong reduction in p65 acetylation in the liver and EAT. Furthermore, as shown in Figure 8a, the total acetyllysine level in the liver of gluconate-CycloZ-treated mice was significantly reduced compared to the control group. In addition, as can be seen from Figures 7b and 7c, the acetylation of PGC-1α and LKB1 was significantly reduced in the liver and EAT of gluconate-CycloZ-treated mice.

[0103] In addition to deacetylation, PGC-1α requires AMPK-mediated phosphorylation for activation. Therefore, to investigate whether CycloZ affects the AMPK-PGC-1α pathway, Western blotting was performed similarly to that in Examples 3-7. The results showed increased AMPK phosphorylation in the liver and EAT of gluconate-CycloZ-treated mice, as can be seen in Figure 7d.

[0104] 5-2. mRNA expression analysis Next, to investigate the expression of PGC-1α-related genes, RT-qPCR was performed in the same manner as in Examples 3-5, and the gene-specific primer information used is shown in Table 5.

[0105] [Table 5]

[0106] As can be seen in Figure 7e, the expression of Foxo1 (forkhead box O1), Esrra (estrogen-related receptor alpha), Tfam (transcription factor A, mitochondrial), Nrf1 (nuclear respiratory factor 1), and Ucp1 (uncoupling protein 1), which are necessary for mitochondrial development, was increased in the liver and MAT of mice in the gluconate-CycloZ treated group compared to the control group. Similarly, as shown in Figure 7f, the expression of Ppara (peroxisome proliferator-activated receptor alpha), Cpt1a (carnitine palmitoyltransferase 1A), and Ppargc1a (carnitine palmitoyltransferase 1A), which are associated with intrahepatic lipid oxidation, was also increased in the gluconate-CycloZ treated group, and the levels of Acox1 (acyl-CoA oxidase 1), Mcad (medium-chain acyl-CoA dehydrogenase), Pparα, Cpt1a, and Ppargc1a were increased in MAT of gluconate-CycloZ treated mice.

[0107] The increased mitochondrial biodevelopment upon administration of gluconate-CycloZ was evidenced by the increased mitochondrial DNA (mtDNA) content in the livers of gluconate-CycloZ-treated mice, as shown in Figure 8b. This suggests that gluconate-CycloZ enhances mitochondrial biodevelopment and lipid oxidation activation, thereby increasing mitochondrial function.

[0108] 5-3. Measurement of Oxygen Consumption Rate Mitochondrial respiration and OCR were confirmed in AML12 mouse hepatocytes through the following method. Oxygen consumption rate (OCR) was measured using an XF96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA) according to the manufacturer's protocol. AML12 cells were placed in an XF-96 tissue culture plate at a rate of 1 × 10⁶. 4 Cells were inoculated at a cell / well density. The following day, the medium was replaced with XF basal medium (pH 7.4, Seahorse Biosciences, North Billerica, MA, USA) supplemented with 25 mM D-glucose (G7528, Sigma-Aldrich), 1 mM sodium pyruvate (S8636, Sigma-Aldrich), and 1X GlutaMAX™ (35050, Gibco, Waltham, MA, USA), after which the appropriate drugs were administered. To evaluate OCR, the compounds and metabolites used in this example were as follows: insulin (100 nM, I5556, Sigma-Aldrich), oligomycin A (1 μM, 75351, Sigma-Aldrich), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (CCCP, 2 μM, C2920, Sigma-Aldrich), and rotenone (1 μM, R8875, Sigma-Aldrich). To normalize to cell number, 4',6-diamino-2-phenylindole (DAPI) stained cells were automatically calculated using ImageXpress Micro Confocus Microscopy (Molecular Devices, San Jose, CA, USA).

[0109] As can be seen in Figure 7g, the gluconate-CycloZ treatment group showed significant improvements in OCR, ATP-coupled respiration, and maximum respiratory capacity compared to the palmitate treatment group.

[0110] 5-4.MitoTracker staining 0.5x10 5Cells were plated onto glass coverslips in 12-well plates and cultured for 24 hours. 200 μM palmitate was treated with serum-free medium, with or without gluconate-CycloZ, for 24 hours. 500 nM MitoTracker Deep Red FM (M22426, Invitrogen, Carlsbad, CA, USA), diluted in serum-free medium, was treated and cultured for 30 minutes. Cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 40 minutes. After Hoechst staining, the coverslips were mounted on glass slides. Images per group (7-10) were acquired using a Leica confocal laser scanning microscope. Fluorescence was measured in five randomly selected regions from each image using the Leica LAS AF program (Leica, Wetzlar, Germany).

[0111] The MitoTracker Deep Red FM staining results in Figure 7h show an increase in mitochondrial mass in gluconate-CycloZ-treated AML12 cells. These results suggest that gluconate-CycloZ regulates the acetylation state and improves mitochondrial biogenesis and function.

[0112] [Example 6] NAD levels improved by gluconate-CycloZ administration in diabetic and obese animal models + Confirmation of synthesis increase effect Deacetylation of the non-histone transcription factors PGC-1α, LKB1, and p65 was observed in liver and VAT. The sirtuin family of deacetylases is known to regulate the acetylation of these proteins. Sirt1 has the broadest substrate range and affects various physiological pathways, including energy metabolism. Sirt1 is involved in NAD + Since acetyl groups are removed using as an auxiliary substrate, the NAD of the cell + The / NADH ratio reflects Sirt1 enzyme activity. Therefore, gluconate-CycloZ is NAD + Level or NAD +We hypothesized that increasing the / NADH ratio would increase the deacetylation activity of Sirt1.

[0113] 6-1.NAD + Quantification of β-nicotinamide adenine dinucleotide NAD + The β-nicotinamide adenine dinucleotide (NADH) ratio is determined in tissue lysates based on the manufacturer's protocol. + Total NAD was measured using a colorimetric NADH quantitative kit (K-337-100, Biovision, Milpitas, CA, USA). In summary, 10 mg of tissue was homogenized with the provided extraction buffer. + To measure the concentration, 50 μL of the extracted sample was transferred to a 96-well microplate. To decompose the NAD, the remaining extracted sample was heated at 60°C for 30 minutes. Consequently, 50 μL of the decomposed sample was transferred to a 96-well microplate. After development, the plate was measured at 450 nm.

[0114] As can be seen from Figures 9a and 8c, gluconate-CycloZ administration induced NAD in the liver and EAT. + We found that the / NADH ratio increased, but not in muscle. NAD in liver and EAT + An increase in the / NADH ratio indicates that NAD + This can be confirmed through Figure 9b as being due to an increase in the total amount.

[0115] 6-2. mRNA expression analysis NAD + To investigate the reason for the increase in level, NAD + The expression of genes involved in synthesis was confirmed by RT-qPCR. The experimental method was the same as in Examples 3-5, and the gene-specific primer information used is shown in Table 6.

[0116] [Table 6]

[0117] The results in Figures 9c and 9d show that the expression of various genes involved in NAD biosynthesis was significantly upregulated upon administration of gluconate-CycloZ compared to the control group. These results suggest that gluconate-CycloZ is effective in promoting NAD synthesis. + Regulating gene expression related to synthesis to increase NAD + This indicates that the level has been increased.

[0118] [Example 7] Confirmation of therapeutic effects of gluconate-CycloZ administration in diabetic and obese animal models. The in vivo data from the above example demonstrate the preventive effect of gluconate-CycloZ in KK-Ay mice administered the drug at an early stage of progression to hyperglycemia. In a clinical setting, T2DM patients only begin drug treatment when diagnosed with prediabetes or diabetes. Therefore, it is necessary to investigate the therapeutic effect of gluconate-CycloZ in the later stages of diabetes, which are generally characterized by severe hyperglycemia. Existing studies have shown that hyperglycemia and insulin resistance increase with age in KK-Ay mice (Iwatsuka, Hisashi, Akio Shino, and Ziro Suzuoki. "General survey of diabetic features of yellow KK mice." Endocrinologia japonica 17.1(1970):23-35).

[0119] In fact, when we measured the HbA1c levels of KK-Ay mice, we found that hyperglycemia became more severe at 12 weeks of age compared to 8 weeks of age, as can be seen in Figure 10a. Therefore, in this example, we administered gluconate-CycloZ to 12-week-old mice for 8 weeks and investigated the therapeutic effect.

[0120] 7-1. Oral glucose tolerance test and measurement of glycated hemoglobin Oral glucose tolerance tests and glycated hemoglobin measurements were performed using the same method as in Example 3-2. As a result, as shown in Figures 11a and 11b, it was confirmed that glucose tolerance and HbA1c levels were significantly improved with gluconate-CycloZ administration.

[0121] 7-2. Western blot Western blotting was performed in the same manner as in Example 3-7, and the acetylation and AMPK phosphorylation of PGC-1α and LKB1 were investigated. As shown in Figures 11c, 11d, and 10g, it was clear that the acetylation of PGC-1α and LKB1 decreased and AMPK phosphorylation increased, which was in complete agreement with the results of the preventive study.

[0122] 7-3. mRNA expression analysis After therapeutic administration of CycloZ, genes and NAD related to mitochondrial biosynthesis and function in the liver + To investigate the expression of genes involved in synthesis, RT-qPCR was performed in the same manner as in Examples 3-5, and the gene-specific primer information used is shown in Tables 5 and 6. As can be seen from the RT-qPCR results in Figures 10e and 10f, the expression of mRNA related to mitochondrial biosynthesis and function increased, and as can be seen from the RT-qPCR results in Figure 11f, NAD + It was also shown that the expression of genes involved in synthesis increased.

[0123] 7-4.NAD + / NADH quantification In the same manner as in Example 6-1, when gluconate-CycloZ was administered, NAD + The amount and NAD + As can be seen in Figure 11e, the quantification of the / NADH ratio shows that NAD + The amount and NAD + Both the / NADH ratio increased. These results indicate that gluconate-CycloZ administration remains effective even in more severe diabetic models.

[0124] statistical analysis Statistical analysis was performed using Prism software (GraphPad Prism 6, GraphPad Software Inc., San Diego, CA, USA). All data from Examples 3-7 are presented as mean ± standard error of the mean. Significant differences between two groups were analyzed using a Student's t-test (two-sided), and multiple comparisons were measured using Tukey's post-hoc test after one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant. Grubb's test was applied to remove outliers.

[0125] Having described in detail certain aspects of the present invention, it will be clear to those with ordinary skill in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

[Claim 1] Zinc gluconate; and A pharmaceutical composition for the prevention or treatment of obesity and diabetes, comprising cyclo-hispro or a pharmaceutically acceptable salt thereof as an active ingredient.

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