Composition for preventing, ameliorating or treating obesity and diabetes comprising zinc gluconate and cyclo-hispro as active ingredients

Abstract

To provide a composition having remarkably improved anti-obesity and anti-diabetic effects, and a method of preventing, ameliorating or treating obesity and diabetes using the composition.SOLUTION: The present invention provides a composition for preventing, ameliorating or treating obesity and diabetes, comprising zinc gluconate and cyclo-hispro (CHP) as active ingredients. The zinc cation or zinc element component of the zinc gluconate and the cyclo-hispro or a pharmaceutically acceptable salt thereof are included at a weight ratio of 1 to 5:1 to 4, wherein the zinc cation or zinc element component is contained at a higher weight ratio than the cyclo-hispro or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for preventing, improving or treating obesity and diabetes, comprising zinc gluconate salt and cyclo-hispro (CHP) as active ingredients. More specifically, the type of zinc salt and / or the content ratio of zinc component are optimized, and the composition with significantly improved anti-obesity and anti-diabetic effects, and a method for preventing, improving or treating obesity and diabetes using the same are provided.

Background Art

[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 therapy, and weight control can be helpful in managing the disease. However, as the disease progresses, oral drug treatment or insulin therapy is often required. Currently available oral drugs such as hypoglycemic agents or insulin sensitizers including sulfonylurea (SU), biguanide, thiazolidinedione (TZD), dipeptidyl peptidase-4 (DPP-4) inhibitor, and sodium-glucose cotransporter 2 (SGLT2) inhibitor have been used to regulate blood glucose levels for a long time. However, some drugs have limited efficacy and can induce various side effects. For example, patients taking SU have an increased risk of weight gain and hypoglycemia, and patients taking biguanide are exposed to the potential risk of lactic acidosis. TZD is not recommended for patients with existing edema, heart failure, or acute liver disease. The most frequent adverse reaction of DPP-4 inhibitor is upper respiratory tract infection, and SGLT2 inhibitor is associated with urinary and genital infections. Therefore, the development of safer and more effective drugs with various mechanisms of action is necessary.

[0003] Lysine acetylation plays an important role in maintaining energy homeostasis in various metabolic pathways. In particular, various enzymes involved in glucose and lipid metabolism are regulated by acetylation of lysine residues. The sirtuin family of enzymes consists of increased levels of β-nicotinamide adenine dinucleotide (NAD + )-dependent deacetylases that regulate the activity of many other enzymes. 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 protein kinase (AMPK) kinase involved in lipid synthesis and fatty acid oxidation. Sirtuins are also considered as new targets for the treatment of chronic metabolic diseases, and enhancement of Sirt1 activity has been reported to reverse the pathological effects of T2DM.

[0004] In Patent Document 1, a composition containing zinc ions and cyclo-hispro (CHP) is disclosed as a composition useful for weakening diabetic symptoms in mammals, but the optimized form of zinc salt for improving the therapeutic effects on obesity and diabetes has not been clarified.

[0005] Under such a background, the inventors of the present invention confirmed that the combination of zinc gluconate salt and CHP exhibits significantly superior anti-obesity and anti-diabetic effects compared to the combination of other zinc salts and CHP, and completed the present invention.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Accordingly, 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 in need thereof.

Means for Solving the Problems

[0009] 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-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 in need thereof.

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

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

[0014] In the present invention, the zinc gluconate salt and cyclo-hispro or a pharmaceutically or food-scientifically acceptable salt thereof may be contained or administered at 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 an increase in 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 of zinc salt and / or the content ratio of the zinc component are 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]

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Mode for Carrying Out the Invention

[0019] Hereinafter, the present invention will be described in more detail. All technical terms used in the present invention are used in the same meaning as commonly understood by those of ordinary skill in the relevant field of the present invention, unless otherwise defined. Also, although preferred methods and samples are described in this specification, those that are the same or equivalent are also included in the scope of the present invention.

[0020] As described above, an optimized form of zinc salt for improving the therapeutic effects on obesity and diabetes in the combination of zinc salt and CHP is not known. Therefore, the present inventors searched for a solution to the above-described problem by experimentally verifying that the combination of zinc gluconate salt and CHP exhibits excellent obesity-improving and diabetes-treatment effects as compared with the combination of other zinc salts and CHP.

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

[0022] As used herein, the term "cyclo-hisPro (CHP)" refers to a naturally occurring cyclic dipeptide composed of histidine-proline, which is a metabolite of thyrotropin-releasing hormone (TRH), or a bioactive dipeptide that may also be synthesized de novo in the body during the TRH metabolic process, and is a substance widely distributed throughout the brain, spinal cord, gastrointestinal tract, etc.

[0023] In the composition of the present invention, the cyclo-hisPro can be synthesized and used, or a commercially available product can be used. Also, it can be purified and used from substances containing cyclo-hisPro, such as prostate extracts and soy hydrolysates.

[0024] The use of the term "purified" is intended to mean that cyclo-hisPro is in a concentrated form compared to the form that can be obtained from natural sources such as prostate extracts. The purified component can be concentrated from these natural sources or obtained through chemical synthesis methods.

[0025] As used herein, the zinc salt and cyclo-hisPro may also be referred to as "CycloZ", and when the form of the zinc salt is zinc gluconate, it can be referred to as "gluconic acid-CycloZ". The zinc gluconate and cyclo-hisPro can be included in the composition of the present invention in the form of a single complex or as individual components. Therefore, the zinc gluconate and cyclo-hisPro may be administered in the form of a single complex, or as individual components simultaneously, separately, or sequentially.

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

[0027] In the present invention, zinc gluconate and cyclo-his-pro (gluconic acid-Cyclo-Z) can be included in a dosage of 15 to 250 mg in the form of a single complex when applied clinically. When zinc gluconate and cyclo-his-pro are included as individual components, they may be included in dosages calculated according to the weight ratio of zinc gluconate:cyclo-his-pro defined in the present invention within the above dosage range. At this time, if the total dosage of zinc gluconate and cyclo-his-pro (gluconic acid-Cyclo-Z) is less than 15 mg, the dosage may be too low and the therapeutic effects on obesity and diabetes may not appear. If it exceeds 250 mg, there may be problems with toxicity.

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

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

[0030] The composition of the present invention shows beneficial effects on both obesity and diabetes from the perspectives of prevention and therapeutics. Young KK-Ay mice are used as a mild hyperglycemia model for the preventive treatment of type 2 diabetes accompanied by obesity. The hyperglycemia and hyperinsulinemia of KK-Ay mice worsen with age. Therefore, in the present invention, young KK-Ay mice and old KK-Ay mice with progressive hyperglycemia were used in separate studies to verify the therapeutic effect of gluconic acid-Cyclo-Z in a mild hyperglycemia model and a severe diabetes model.

[0031] According to a specific embodiment of the present invention, gluconic acid-CycloZ exhibits anti-obesity and anti-diabetic effects through the regulation of protein acetylation in the liver and VAT. The increase in VAT mass is one of the main risk factors for various metabolic diseases, and the administration of gluconic acid-CycloZ significantly decreased the liver and VAT masses in KK-Ay mice. In addition, the deacetylation of PGC-1α induced by the administration of gluconic acid-CycloZ induced the 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 biogenesis and glucose / fatty acid metabolism. Specifically, the decreased activity of PGC-1α is associated with altered lipid oxidation, and the expression of PGC-1α in adipose tissue is downregulated in type 2 diabetes patients.

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

[0033] In yet another specific embodiment of the present invention, it was confirmed that gluconic acid-CycloZ regulates the expression of enzymes involved in NAD + synthesis. The mRNA expression of Nampt increased in both the liver and VAT of gluconic acid-CycloZ-treated mice. Since NAMPT is a rate-limiting enzyme, the treatment with gluconic acid-CycloZ increased the NAD + level and then increased the Sirt1 activity. The NAD + / NADH ratio and the amount of NAD + increased only in the liver and VAT, but not in the muscle. This indicates that gluconic acid-CycloZ can regulate NAD + synthesis in a tissue-specific manner.

[0034] In yet another specific embodiment 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 which is essential for Sirt1 activity + levels and NAD + / NADH ratio are increased, so it can be inferred that Sirt1 activity can be increased.

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

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

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

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

[0039] As used herein, the term "treatment" 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 eliminating or reversing the symptoms of obesity and diabetes.

[0040] As used herein, the term "pharmaceutically acceptable salt" means any organic or inorganic addition salt of cyclo-hispro that is relatively non-toxic to patients, has a concentration with a harmless and effective effect, and the side effects caused by this salt do not reduce the beneficial efficacy of cyclo-hispro. As these salts, as the free acid, inorganic acids and organic acids can be used. As inorganic acids, hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc. can be used. 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. can be used. Further, these salts include alkali metal salts (such as sodium salts and potassium salts) and alkaline earth metal salts (such as calcium salts and magnesium salts), etc. For example, as acid addition salts, acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, maleate, maleate, malonate, mesylate, methylsulfate, 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. may be included.

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

[0042] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of suppositories, witepsol, macrogol, tween 61, cocoa butter, laurin fat, glycerol, gelatin, etc. can be used.

[0043] The pharmaceutical composition of the present invention can be administered via any common route capable of reaching the target tissue or cells in an individual or sample. The administration may include systemic or local administration, and may include, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration, etc. The dosage of the pharmaceutical composition can vary depending on the age, weight, gender, administration form, health status and disease degree of the patient.

[0044] The term "patient" means any individual unit that requires treatment, including humans, cows, dogs, guinea pigs, rabbits, chickens, insects, etc. Also included are any subjects who participated in a clinical research trial showing no clinical findings of any disease, or subjects who participated in a mechanical study, or subjects used in a control group.

[0045] The second aspect of the present invention relates to a health - functional food composition for preventing or improving obesity and diabetes, containing zinc gluconate salt and cyclo - hispro or a pharmaceutically acceptable salt thereof as active ingredients.

[0046] In the health - functional food composition of the present invention, the description of the composition and effects of the zinc gluconate salt and cyclo - hispro contained as active ingredients is the same as that described above, so the description thereof is omitted.

[0047] In the present invention, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable organic acids, inorganic acids or bases.

[0048] In the present invention, the term "health - functional food" fully includes the meanings of "functional food" and "health food".

[0049] In the present invention, the term "functional food" is the same term as "food for special health use (FoSHU)", and means a food with high medical and therapeutic effects that is processed so that its biological regulatory function can be efficiently manifested in addition to nutrient supply.

[0050] In the present invention, the term "health food" means a food having a positive health maintenance or promotion effect compared to general food, and "Health supplement food" means a food for health supplement purposes. In some cases, the terms functional food, health food, and health supplement food are used interchangeably. The food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. in order to obtain useful effects for the prevention or improvement of obesity and diabetes.

[0051] As a specific example of such a functional food, using the zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof of the present invention, while taking advantage of the characteristics of agricultural products, livestock products or fishery products and modifying them, a processed food with improved storability can be manufactured.

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

[0053] The food composition of the above type can be manufactured in various forms by ordinary methods known in the art. General foods include, but are not limited to, 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., hams, 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, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., miso, soy sauce, sauce, etc.), etc. The zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof of the present invention can be added to manufacture them.

[0054] In addition, as nutritional supplements, although not limited thereto, zinc gluconate salt and cyclo-hispro or a food-acceptable salt thereof of the present invention can be added to capsules, tablets, pills, etc. for production.

[0055] In addition, as functional foods, although not limited thereto, for example, zinc gluconate salt and cyclo-hispro or a food-acceptable salt thereof of the present invention can be produced in the form of tea, juice, and drinks, and can be ingested in a liquefied, granulated, encapsulated, or powdered form for drinking (health drinks). In addition, for using zinc gluconate salt and cyclo-hispro or a food-acceptable salt thereof of the present invention in the form of food additives, they can be produced and used in the form of powders or concentrated solutions. In addition, zinc gluconate salt and cyclo-hispro or a food-acceptable salt thereof of the present invention can be mixed with known active ingredients known to be effective in preventing or improving obesity and diabetes and produced in the form of a composition.

[0056] When the food composition of the present invention is used as a health drink composition, the health drink composition can contain various flavoring agents or natural carbohydrates, etc. as additional components like ordinary drinks. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract; synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

[0057] The zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof of the present invention may be contained as an active ingredient in a health functional food composition for preventing or improving obesity and diabetes, and the amount thereof is an amount effective for obtaining the preventive or improving effect, for example, preferably 0.01 to 100% by weight based on 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 it with a known active ingredient known to be effective in preventing or improving obesity and diabetes together with the zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof.

[0058] In addition to the above, the health functional food of the present invention can contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol or carbonating agents, etc. In addition, the health food of the present invention can contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. Such components can be used independently or in combination. The ratio of such additives is not of great importance, but is generally selected in the range of 0.01 to 0.1 part 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, which comprises administering a zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0060] In the method 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, cow, cat, guinea pig or rodent). Such terms do not indicate a specific age or sex. Thus, it is intended to include not only female / female, male / male, adult / adult and neonatal subjects, but also fetuses. A patient refers to a subject having a disease or disorder. The term patient includes human and veterinary subjects.

[0061] In the method of the present invention, the description regarding the composition including the effects, administration routes, number of administrations, dosage, etc. of zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof administered to the individual is the same as that described above, and thus the description thereof is omitted.

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

[0063] When administered sequentially, each active ingredient may be administered at intervals as an individual formulation, and the administration order may be determined by a physician or a person having ordinary knowledge in the art.

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

[0065] The fourth aspect of the present invention relates to the use of a composition comprising a zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof for the manufacture of a medicament 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 comprising the zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof, and the constitution including the administration route, the number of administrations, the dosage, etc. is the same as that described above, and thus the description thereof is omitted.

[0067] The fifth aspect of the present invention relates to a method for increasing β-nicotinamide adenine dinucleotide (NAD + ), which comprises administering a zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need thereof.

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

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

[0070] The description of the effects of the composition comprising the zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof, and the constitution including the administration route, the number of administrations, the dosage, etc. is the same as that described above, and thus the description thereof is omitted.

[0071] The sixth aspect of the present invention relates to a method for increasing Sirt1 deacetylase activity, which comprises administering a zinc gluconate salt and cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need thereof. In connection with the fifth aspect, 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 an activator of Sirt1 deacetylase activity.

[0072] Explanation regarding the effects of the composition comprising the zinc gluconate and cyclo-hispro or a pharmaceutically acceptable salt thereof, and its configuration including the administration route, the number of administrations, the dosage, etc. is the same as that described above, and thus the description thereof is omitted.

Example

[0073] Hereinafter, the present invention will be described in more detail based on examples. It will be apparent to those having ordinary knowledge in the art that these examples are merely for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

[0074] [Example 1] Confirmation of the effect of improving obesity depending on the type of zinc salt 1-1. Experimental Animals and Administered Drugs Five-week-old male Kkay mice from CLEA Japan Inc. were purchased from Seron Bio Co., Ltd. Breeding was carried out under certain conditions (temperature: 22 ± 2°C, relative humidity: 55 ± 10%, one cycle: 12 hours), and Purina feed was used for feed and distilled water was used for drinking water. The mice were used in the experiment after a one-week adaptation period following purchase. CHP was purchased from Angene, zinc gluconate was purchased from Captek Softgel International, and zinc acetate, zinc chloride, and zinc sulfate were purchased from Sigma-Aldrich.

[0075] After 5-week-old Kkay mice were pre-fed for one week, they were randomly divided into groups with the same average weight. The administration conditions for each group are as shown in Table 1. The control group was orally administered distilled water daily. In experimental group 1, Glu-CycloZ, which was a mixture of zinc gluconate and CHP at a weight ratio of 2:1 (based on zinc element), was administered. In experimental group 2, Ac-CycloZ, which was a mixture of zinc acetate salt and CHP at a weight ratio of 2:1 (based on zinc element), was administered. In experimental group 3, Cl-CycloZ, which was a mixture of zinc chloride salt and CHP at a weight ratio of 2:1 (based on zinc element), was administered. In experimental group 4, Sulf-CycloZ, which was a mixture of zinc sulfate salt and CHP at a weight ratio of 2:1 (based on zinc element), was orally administered once a day, and the body weight was measured weekly.

[0076]

Table 1

[0077] 1-2. Confirmation of Weight Loss Effect The body weight was measured weekly during the 15 weeks when drug administration was in progress, and the body weights were compared at the 15th week. As confirmed from Figure 1, the average body weight, which was about 26.6 g at the start of the experiment, increased to 46.6 g in the control group, while the body weights decreased to 42.9 g in experimental group 1, 44 g in experimental group 2, 44.7 g in experimental group 3, and 45.9 g in experimental group 4. The body weight reduction effects compared to the control group appeared as a 7.9%, 5.6%, 4.1%, and 1.5% decrease in the order of the experimental groups depending on the type of zinc salt, and it was confirmed that the reduction effect and significance were most significant in experimental group 1. Thus, it was confirmed that Glu-CycloZ in the form containing zinc gluconate salt was the most effective in improving obesity compared to CycloZ in the form containing other zinc salts.

[0078] [Example 2] Confirmation of the diabetes improvement effect depending on the type of zinc salt 2-1. Oral Glucose Tolerance Test and Measurement of Glycated Hemoglobin To confirm the diabetes-improving effect according to the type of zinc salt, an oral glucose tolerance test and a glycated hemoglobin measurement were performed. First, for the oral glucose tolerance test (OGTT), mice were fasted for 16 hours, and 2 g / kg of glucose was orally administered through a gastric tube. After glucose administration, blood was collected from the tail vein at 15, 30, 60, 90, and 120 minutes. The blood glucose level was immediately measured using a blood glucose meter (AGM-4000, Allmedicus, Anyang, Korea). To measure glycated hemoglobin (HbA1c), blood was collected from the tail vein. HbA1c was measured using a DCA vantage® analyzer (Siemens, Munich, Germany).

[0079] As shown in Figure 2, the glycated hemoglobin value in the blood, which is a standard index for diabetes, showed a decrease in glycated hemoglobin of approximately 13.5% in experimental group 1 only compared to the control group, and significance was confirmed. Also, as shown in Figures 3a and 3b, a decrease in fasting blood glucose was confirmed in experimental group 1, and it was confirmed that blood glucose decreased most rapidly 2 hours after glucose administration. The oral glucose tolerance test values 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 compared to the control group, and in experimental group 4, no decrease effect was observed compared to the control group.

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

[0081] Statistical Analysis The statistical significance for the experimental results of Examples 1 and 2 was analyzed by the t-test statistical method against the control group for each experimental group. *p < 0.05, **p < 0.01.

[0082] [Example 3] Confirmation of the effect of Glu-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. (Nishishinbashi, Japan) were individually caged and bred in a room with an air conditioner set at a temperature of 23 ± 3°C under a 12-hour light-dark cycle. They were allowed free access to distilled water and laboratory chow. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Pohang Center for Advanced Biomaterials and Convergence (ABCC201712). All animals were used in the experiments after a 1-week acclimation period. For the preventive study, the animals were divided into two groups. The control group was administered water as a vehicle. KK-Ay mice were orally gavaged with CycloZ gluconate (CHP 5 mg / kg and zinc gluconate 70 mg / kg) daily for 20 weeks. For the treatment study, the animals were divided into a control group and an experimental group at 12 weeks of age. KK-Ay mice were orally gavaged with water or CycloZ gluconate daily for 8 weeks. At the end of each experiment, all mice were anesthetized with isoflurane using an 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 test and glycated hemoglobin measurement were performed in the same manner as in Example 2-1. As confirmed from FIGS. 4a and 4b, the administration of CycloZ gluconate had a higher effect on improving glucose tolerance than the treatment with individual compounds. Also, as confirmed through FIG. 5c, CycloZ gluconate improved the glucose tolerance of KK-Ay mice as measured by the results of the OGTT. As shown in FIGS. 5d, 5e, and 5f, it was confirmed that the fasting blood glucose, HbA1c level, and plasma insulin concentration were significantly decreased by the administration of CycloZ gluconate. These results demonstrate that CycloZ gluconate improved glucose metabolism and insulin sensitivity.

[0084] 3-3. Measurement of Body Weight, Food Intake and Weights of Organs As confirmed from FIGS. 5a and 4c, the weight gain of the mice in the gluconic acid-CycloZ treatment group gradually decreased at the end of the experiment compared to the control group, and there was no noticeable difference in food intake. Also, as confirmed from FIG. 5b, gluconic acid-CycloZ administration significantly suppressed the mass increase of the liver and visceral adipose tissue (VAT) such as epididymal adipose tissue (EAT) and mesenteric adipose tissue (MAT), but not subcutaneous adipose tissue. Gluconic acid-CycloZ treatment also decreased the liver and VAT weights of the mice.

[0085] 3-4. Analysis of Blood Biochemical Parameters Whole blood was collected via cardiac puncture and centrifuged at 2,000 xg for 10 minutes to separate plasma. Next, it was stored at -80 °C until plasma 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 an Enzy-Chrom Free Fatty Acid Assay Kit (EFFA-100, BioAssay System, Hayward, CA, USA).

[0086] As shown in Table 2, it was confirmed that gluconic acid-CycloZ administration is safe and has excellent drug resistance as it does not induce changes in AST, BUN, and creatinine.

[0087]

Table 2

[0088] The blood lipid profiles of mice were also investigated using a biochemical analyzer. As confirmed from Figures 5g, 5h, and 5i, in mice treated with gluconic acid-CycloZ, the free fatty acid and triglyceride levels decreased, and the HDL-C level increased. However, as shown in Figures 4d and 4e, it was observed that the total cholesterol level remained unchanged.

[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 in Table 3 and IQ SYBR Green Supermix (BR1708882, Bio-Rad). RT-qPCR was carried out with the following amplification reaction cycle: 10 seconds at 95 °C, 10 seconds at 60 °C, and 30 seconds at 72 °C. The 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 Example 3-4 is explained as a decrease in the 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 The liver and adipose tissue were fixed with 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 then 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). The stained sections were examined with an optical microscope (Olympus BX53 upright microscope, Olympus, Tokyo, Japan).

[0093] As confirmed from Figure 5l, gluconic acid-CycloZ administration also improved hepatic lipid deposition, whereas the control group showed the pathology of fatty liver such as steatosis. Also, as confirmed from Figure 5m and Figure 4f, the adipocyte area of EAT was significantly decreased in the gluconic acid-CycloZ-treated group compared with the control group.

[0094] 3-7. Western Blot Tissues and cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (89901, Thermo Scientific, Waltham, MA, USA) containing Halt protease and phosphatase inhibitor cocktail (78440, Thermo Scientific) for protein experiments. For SDS-PAGE, Bolt 4%–12% Bis-Tris Plus Gel (Thermo Scientific) was used, and transfer was performed using the 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 confirmed from Fig. 5j, adiponectin levels were significantly increased in the blood of gluconic acid-CycloZ-treated mice.

[0096] Through the above results, it was confirmed that the weight loss caused by gluconic acid-CycloZ administration was due to fat loss through the regulation of lipid and cholesterol metabolism in the liver and VAT.

[0097] [Example 4] Confirmation of the effect of gluconic acid-CycloZ administration on improving inflammation and reducing immune cell infiltration in diabetic and obese animal models 4-1. mRNA Expression Analysis Since hepatic and VAT obesity-induced insulin resistance is closely associated with chronic inflammation in many tissues, it was investigated whether gluconic acid-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] As confirmed from Figure 6a, the expression levels of inflammatory cytokine genes and F4 / 80 and MCP-1 in the liver and MAT were significantly decreased by gluconic acid-CycloZ administration. Consistently, it can be confirmed through Figures 6b and 6c that gluconic acid-CycloZ treatment also caused a significant decrease in TNFα and MCP-1 protein levels in the liver and EAT.

[0100] 4-2. Histochemistry The expression of TNFα, MCP-1, F4 / 80 and CD11b in the liver and EAT was investigated through immunohistochemistry. Histochemistry was performed in the same manner as in Example 3-6. As confirmed from Figures 6d and 6e, inflammatory cytokine production and mononuclear cell infiltration completely disappeared with gluconic acid-CycloZ administration. Overall, such results indicate that after gluconic acid-CycloZ administration, the improvement of tissue insulin resistance was accompanied by a reduction in inflammation.

[0101] [Example 5] Confirmation of the effect of gluconic acid-CycloZ administration on mitochondrial biogenesis and inflammation improvement in diabetic and obese animal models 5-1. Western Blot Infiltrating macrophages play an important role in developing insulin resistance in metabolic organs. These activities are partially regulated by the deacetylation of transcription factors such as the p65 subunit of NF-κB. Also, lysine acetylation regulates the activities of many metabolic enzymes and transcription factors. According to existing reports, it has been revealed that the overall lysine acetylation profile increases 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 metabolism 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 overall acetyl-lysine level in the liver of mice in the gluconic acid-CycloZ treatment group.

[0102] As shown in FIG. 7a, it can be seen that the acetylation of p65 was strongly decreased in the liver and EAT of gluconic acid-CycloZ-treated mice. Also, as shown in FIG. 8a, it was confirmed that the overall acetyl-lysine level in the liver of mice in the gluconic acid-CycloZ treatment group was significantly decreased compared to the level of the control group. Furthermore, as confirmed from FIGS. 7b and 7c, the acetylation of PGC-1α and LKB1 was significantly decreased in the liver and EAT of gluconic acid-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 similarly performed as in Examples 3-7. As a result, as confirmed from Figure 7d, it was shown that AMPK phosphorylation increased in the liver and EAT of gluconic acid-CycloZ-treated mice.

[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 confirmed from Fig. 7e, the expressions 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) required for mitochondrial biogenesis increased in the liver and MAT of mice in the gluconic acid-CycloZ treatment group compared to the control group. Similarly, as shown in Fig. 7f, the expressions of Ppara (peroxisome proliferator-activated receptor alpha), Cpt1a (carnitine palmitoyltransferase 1A), and Ppargc1a (carnitine palmitoyltransferase 1A) related to hepatic lipid oxidation also increased in the gluconic acid-CycloZ treatment group, and the Acox1 (acyl-CoA oxidase 1), Mcad (medium-chain acyl-CoA dehydrogenase), Pparα, Cpt1a, and Ppargc1a levels increased in the MAT of gluconic acid-CycloZ-treated mice.

[0107] The increased mitochondrial biogenesis upon gluconic acid-CycloZ administration was evidenced by the increased mitochondrial DNA (mtDNA) content in the liver of gluconic acid-CycloZ-treated mice as shown in Fig. 8b. This means that gluconic acid-CycloZ improves mitochondrial biogenesis and lipid oxidation activation that increase mitochondrial function.

[0108] 5-3. Measurement of Oxygen Consumption Rate Mitochondrial respiration and OCR were confirmed in AML12 mouse hepatocytes through the following method. The oxygen consumption rate (OCR) was measured using an XF96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA) based on the manufacturer's protocol. AML12 cells were seeded in an XF-96 tissue culture plate at a density of 1×10 4 cells / well. The next day, after the medium was replaced with XF base 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 GlutaMAXTM (35050, Gibco, Waltham, MA, USA), the appropriate drugs were treated. To evaluate the 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), rotenone (1 μM, R8875, Sigma-Aldrich). To normalize to the number of cells, 4′,6-diamino-2-phenylindole (DAPI)-stained cells were automatically counted using ImageXpress Micro Confocus Microscopy (Molecular Devices, San Jose, CA, USA).

[0109] As confirmed from Figure 7g, compared with the palmitate treatment group, the gluconate-CycloZ treatment group had a significant improvement in OCR, ATP-linked respiration, and maximal respiratory capacity.

[0110] 5-4. MitoTracker Staining 0.5x10 5Cells were plated on glass coverslips in 12-well plates and cultured for 24 hours. The cells were treated with 200 μM palmitate in serum-free medium in the presence or absence of gluconic acid-CycloZ for 24 hours. The cells were treated with 500 nM MitoTracker Deep Red FM (M22426, Invitrogen, Carlsbad, CA, USA) diluted in serum-free medium and cultured for 30 minutes. The cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 40 minutes. After Hoechst staining, the coverslips were mounted on glass slides. Images (7 - 10) per group were taken using a Leica confocal laser scanning microscope. Fluorescence was measured in 5 randomly selected regions from each image using the Leica LAS AF program (Leica, Wetzlar, Germany).

[0111] The MitoTracker Deep Red FM staining results in Fig. 7h show an increase in mitochondrial mass in gluconic acid-CycloZ-treated AML12 cells. Such results indicate that gluconic acid-CycloZ regulates the acetylation state and improves mitochondrial biogenesis and function.

[0112] [Example 6] Confirmation of the effect of gluconic acid-CycloZ administration on NAD + synthesis increase in diabetic and obese animal models Deacetylation of non-histone transcription factors PGC-1α, LKB1, and p65 in the liver and VAT was observed. The sirtuin family of deacetylases is known to regulate the acetylation of the said proteins. Sirt1 has the broadest range of substrates and affects various physiological pathways including energy metabolism. Sirt1 uses NAD + as a co-substrate to remove acetyl groups, so the cellular NAD + / NADH ratio reflects Sirt1 enzyme activity. Therefore, gluconic acid-CycloZ affects the NAD + level or the NAD +The hypothesis was put forward that increasing the / NADH ratio would increase the deacetylation activity of Sirt1.

[0113] 6-1. NAD + / β-Nicotinamide Adenine Dinucleotide Quantification NAD + The ratio of NAD and β-nicotinamide adenine dinucleotide (NADH) was measured in tissue lysates based on the manufacturer's protocol using a / NADH quantitative colorimetric kit (K-337-100, Biovision, Milpitas, CA, USA). Briefly, 10 mg of tissue was homogenized in the provided extraction buffer. + To measure the total NAD concentration, 50 μL of the extracted sample was transferred to a 96-well microplate. The remaining extracted sample was heated at 60 °C for 30 minutes to degrade NAD. Consequently, 50 μL of the degraded sample was transferred to a 96-well microplate. After development, the plate was measured at 450 nm. + As confirmed from Figure 9a and Figure 8c, it was found that the / NADH ratio increased by glucuronic acid-CycloZ administration in the liver and EAT, but not in the muscle. The increase in the / NADH ratio in the liver and EAT can be confirmed through Figure 9b to be due to an increase in the total amount of NAD.

[0114] As confirmed from Figure 9a and Figure 8c, it was found that the / NADH ratio increased by glucuronic acid-CycloZ administration in the liver and EAT, but not in the muscle. The increase in the / NADH ratio in the liver and EAT can be confirmed through Figure 9b to be due to an increase in the total amount of NAD. + As confirmed from Figure 9a and Figure 8c, it was found that the / NADH ratio increased by glucuronic acid-CycloZ administration in the liver and EAT, but not in the muscle. The increase in the / NADH ratio in the liver and EAT can be confirmed through Figure 9b to be due to an increase in the total amount of NAD. + / NADH ratio increase + The increase in the / NADH ratio in the liver and EAT can be confirmed through Figure 9b to be due to an increase in the total amount of NAD.

[0115] 6-2. mRNA Expression Analysis NAD + To investigate the reason for the increase in NAD level, the expression of genes involved in NAD 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. + To investigate the reason for the increase in NAD level, the expression of genes involved in NAD 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 of FIGS. 9c and 9d showed that the expression of various genes involved in NAD biosynthesis was significantly upregulated upon gluconic acid-CycloZ administration compared to the control group. Such results indicate that gluconic acid-CycloZ regulated gene expression related to NAD + synthesis and increased NAD + levels.

[0118] [Example 7] Confirmation of the therapeutic effect of gluconic acid-CycloZ administration in diabetic and obese animal models The in vivo data of the above examples showed the preventive effect of gluconic acid-CycloZ in KK-Ay mice administered the drug at an early stage of progression to hyperglycemia. In a clinical setting, T2DM patients start drug treatment only when they are in the pre-diabetes stage or diagnosed with diabetes. Therefore, it is necessary to examine the therapeutic effect of gluconic acid-CycloZ in the late stage of diabetes, which is generally characterized by severe hyperglycemia. According to existing studies, it has been revealed that KK-Ay mice develop increased hyperglycemia and insulin resistance with age (Iwatsuka, Hisashi, Akio Shino, and Ziro Suzuoki. “General survey of diabetic features of yellow KK mice.” Endocrinologia japonica 17.1 (1970): 23-35).

[0119] Indeed, as confirmed from FIG. 10a by measuring the HbA1c level of KK-Ay mice, it was found that hyperglycemia became more severe at 12 weeks of age compared to 8 weeks of age. Therefore, in this example, gluconic acid-CycloZ was administered to 12-week-old mice for 8 weeks to investigate the therapeutic effect.

[0120] 7-1. Oral Glucose Tolerance Test and Measurement of Glycated Hemoglobin As a result of performing an oral glucose tolerance test and measuring glycated hemoglobin in the same manner as in Example 3-2, it was confirmed that glucose tolerance and HbA1c levels were significantly improved by gluconic acid-CycloZ administration, as shown in FIGS. 11a and 11b.

[0121] 7-2. Western Blot Western blotting was performed in the same manner as in Examples 3-7 to examine the acetylation of PGC-1α and LKB1 and the phosphorylation of AMPK. As shown in FIGS. 11c, 11d, and 10g, it was also revealed that the acetylation of PGC-1α and LKB1 decreased and the phosphorylation of AMPK increased, and it was observed that this was completely consistent with the results of the prevention study.

[0122] 7-3. mRNA Expression Analysis After the therapeutic administration of CycloZ, to investigate the expression of genes related to mitochondrial biogenesis and function and genes involved in NAD + synthesis in the liver, 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 confirmed from the RT-qPCR results in FIGS. 10e and 10f, the expression of mRNA related to mitochondrial biogenesis and function increased, and as confirmed from the RT-qPCR results in FIG. 11f, it was shown that the expression of genes involved in NAD + synthesis also increased.

[0123] 7-4. NAD + / NADH Quantification When gluconic acid-CycloZ was administered in the same manner as in Example 6-1, the amount of NAD + and the NAD + / NADH ratio were quantified. As confirmed from FIG. 11e, both the amount of NAD + and the NAD + / NADH ratio increased. Such results indicate that the administration of gluconic acid-CycloZ is still effective even in a more severe diabetes model.

[0124] Statistical Analysis Statistical analysis was performed using Prism software (GraphPad Prism 6, GraphPad Software Inc., San Diego, CA, USA). All data from the results of Examples 3 to 7 are presented as mean ± standard error of the mean. The significant difference between two groups was analyzed by Student's t-test (two-sided), and multiple comparisons were measured by 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 exclude outliers.

[0125] As described in detail the specific parts of the present invention content above, it will be apparent to those with ordinary knowledge in the art that such specific technologies are merely preferred embodiments and do not limit the scope of the present invention thereby. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Zinc gluconate; and A pharmaceutical composition for preventing or treating obesity and diabetes, comprising cyclo-hispro or a pharma- ceutically acceptable salt thereof as an active ingredient.

2. 2. The pharmaceutical composition for preventing or treating obesity and diabetes according to claim 1, wherein the zinc cation or zinc element component of the zinc gluconate and cyclo-hispro or a pharma- ceutically acceptable salt thereof are contained in a weight ratio of 1-5:1-4, and wherein the zinc cation or zinc element component is contained in a higher weight ratio than cyclo-hispro or a pharma- ceutically acceptable salt thereof.

3. 2. The pharmaceutical composition for preventing or treating obesity and diabetes according to claim 1, wherein the zinc gluconate and cyclo-hispro or a pharma- ceutically acceptable salt thereof are contained in a dose of 15 to 250 mg.

4. 5. The pharmaceutical composition for preventing or treating obesity and diabetes according to claim 4, wherein the zinc gluconate and cyclo-hispro or a pharma- ceutically acceptable salt thereof are contained in a weight ratio of 7-35:1-4.

5. The pharmaceutical composition for preventing or treating obesity and diabetes according to claim 1 , wherein the diabetes is type 2 diabetes accompanied by obesity.

6. The composition is a NADPH-binding protein that regulates Sirt1 deacetylase activity in the liver and visceral adipose tissue. ＋ The pharmaceutical composition for preventing or treating obesity and diabetes according to claim 1, which exhibits anti-obesity and anti-diabetes effects through increased synthesis.

7. Zinc gluconate; and A health food composition for preventing or improving obesity and diabetes, comprising cyclo-hispro or a food-logically acceptable salt thereof as an active ingredient.

8. The health functional food composition for preventing or improving obesity and diabetes according to claim 8, wherein the zinc cation or zinc elemental component of the zinc gluconate and cyclo-hispro or its edible acceptable salt are contained in a weight ratio of 1-5:1-4, and the zinc cation or zinc elemental component is contained in a higher weight ratio than cyclo-hispro or its edible acceptable salt.

9. The health functional food composition for preventing or improving obesity and diabetes according to claim 8, wherein the zinc gluconate and cyclo-hispro or a food-logically acceptable salt thereof are contained in a dose of 15 to 250 mg.

10. The health functional food composition for preventing or improving obesity and diabetes according to claim 11, wherein the zinc gluconate and cyclo-hispro or a food-logically acceptable salt thereof are contained in a weight ratio of 7-35:1-4.

11. The health functional food composition for preventing or ameliorating obesity and diabetes according to claim 7, wherein the diabetes is type 2 diabetes accompanied by obesity.

12. The composition is a NADPH-binding protein that regulates Sirt1 deacetylase activity in the liver and visceral adipose tissue. ＋ The health functional food composition for preventing or improving obesity and diabetes according to claim 7, which exhibits anti-obesity and anti-diabetes effects through increased synthesis.

13. Use of a composition comprising zinc gluconate and cyclo-hispro or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for preventing, ameliorating or treating obesity and diabetes.

14. Use of a composition comprising zinc gluconate and cyclo-hispro, or a pharma- ceutically acceptable salt thereof, for producing a β-nicotinamide adenine dinucleotide (NAD+) booster.

15. Use of a composition comprising zinc gluconate and cyclo-hispro or a pharma- ceutically acceptable salt thereof for the manufacture of a Sirt1 deacetylase activator.

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