Composition for preventing, ameliorating or treating diseases caused by protein nitrification, containing peptide having tyrosine at terminal as active ingredient

Abstract

The peptide with tyrosine at the terminal has an excellent effect of inhibiting protein nitration; the composition has an excellent effect of preventing, improving or treating disease symptoms in a chronic body-bound stress depression / cognitive disorder inducing model, an Alzheimer disease dementia model, an epileptic seizure model, a cerebral apoplexy model, a type 2 diabetes mellitus model, an acute renal failure model or a hyperammonemia model.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of Chinese invention patent application number 202280016506.4, filed on February 23, 2022, entitled "Compositions comprising a peptide having a terminal tyrosine as an active ingredient for preventing, ameliorating, or treating diseases caused by protein nitration." This application claims priority to KR10-2021-0024072 (February 23, 2021). Technical Field

[0003] The present invention relates to a composition for preventing, improving or treating diseases caused by protein nitration, comprising a peptide having tyrosine at the end as an effective ingredient. Background Art

[0004] Oxidative stress, a condition in which oxygen generated during metabolism acts as free radicals, can damage cells and lead to various diseases. In particular, when reactive oxygen species (ROS) or reactive nitrogen species (RNS) in cells generate nitrogen peroxide (peroxynitrite, ONOO-) at tyrosine residues in proteins, proteins become nitrated. This protein nitration is known to be a side effect of the oxidative process. When certain proteins are nitrated, their structural changes lead to decreased activity or inability to function properly, leading to various diseases. Protein nitration is reported to be closely linked to intracellular signaling, inflammatory diseases, neurodegenerative diseases, and aging. Recently, it has also been reported to affect asthma, diabetes, cancer, chronic stress-induced depression, type 2 diabetes, and acute kidney disease. Therefore, research on protein nitration has important biological and clinical implications, and the development of substances that inhibit protein nitration is warranted.

[0005] On the other hand, Korean Patent No. 1897400 discloses a “composition for inhibiting tyrosine decarboxylase activity and a method for preparing a fermented food using the same,” and Korean Patent Publication No. 2018-0021746 discloses a “method for purifying nitrated aromatic compounds from a nitration process,” but there is no description of the present invention’s “composition for preventing, ameliorating, or treating diseases caused by protein nitration, comprising a peptide having a terminal tyrosine as an active ingredient.” Summary of the Invention

[0006] Technical issues

[0007] The present invention has been made in response to the above-mentioned requirements. The inventors provide a composition for preventing, ameliorating, or treating diseases caused by protein nitration, comprising a peptide having a terminal tyrosine as an active ingredient. The inventors have confirmed that the peptide having a terminal tyrosine as an active ingredient of the present invention can inhibit protein nitration and can prevent, ameliorate, or treat disease symptoms in a chronic physical restraint stress depression / cognitive impairment-induced model, an Alzheimer's dementia model, an epileptic seizure model, a stroke model, a type 2 diabetes model, an acute renal failure model, or a hyperammonemia model, thereby completing the present invention.

[0008] Solutions to the Problem

[0009] In order to solve the above technical problems, the present invention provides a health functional food composition for preventing or improving diseases caused by protein nitration, wherein the composition comprises a peptide having a terminal tyrosine or a food-acceptable salt thereof as an active ingredient.

[0010] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by protein nitration, wherein the composition comprises a peptide having a terminal tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] Furthermore, the present invention provides a composition for inhibiting nitration of tyrosine in a protein, the composition comprising a peptide having a terminal tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Furthermore, the present invention provides a method for removing a nitro group from nitrated tyrosine by treating a protein containing nitrated tyrosine with a peptide having a tyrosine residue at the terminal end.

[0013] Furthermore, the present invention provides a health functional food composition for preventing or ameliorating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, wherein the composition comprises a peptide having a terminal tyrosine or a food-acceptable salt thereof as an active ingredient.

[0014] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, the composition comprising a peptide having a terminal tyrosine or a food-acceptable salt thereof as an active ingredient.

[0015] Effects of the Invention

[0016] The peptide having a tyrosine residue at the end of the present invention has an excellent effect of inhibiting protein nitration, reducing the tyrosine nitration level in glutamine synthetase increased by stress, and increasing the activity of glutamine synthetase decreased by stress. It has an excellent effect of preventing, improving or treating disease symptoms in a chronic physical restraint stress depression / cognitive impairment induced model, an Alzheimer's dementia model, an epileptic seizure model, a stroke model, a type 2 diabetes model, an acute renal failure model or a hyperammonemia model. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure shows the process of performing an object recognition test (ORT) and an object location recognition test (OLT) in order to analyze the long-term memory ability of animals fed with the peptide diet according to the present invention.

[0018] Figure 2 Graphs confirming the protein nitration inhibitory effect of the peptide of the present invention. (A) shows the results of Western blotting of nitrotyrosine proteins in PFC (prefrontal cortex) tissue, and (B) shows the results of Western blotting of nitrotyrosine proteins in liver tissue.

[0019] Figure 3 Shown are the results of Western blotting of insulin receptor β and phosphorylated insulin receptor β proteins in liver tissue for confirming the protein expression level regulating effect of the peptide of the present invention.

[0020] Figure 4 Results show the inhibitory effects of peptides containing terminal tyrosine on the nitration of Cu / ZnSOD (Part (A)) and MnSOD (Part (B)). PN is peroxynitrite, which induces protein nitration. * and *** indicate statistically significant increases in Cu / ZnSOD or MnSOD activity in the peptide-treated group compared to the PN-only group; * indicates p < 0.05, and *** indicates p < 0.001.

[0021] Figure 5 Results show the inhibitory effect of a peptide containing a terminal tyrosine on nitration of glutamine synthetase. PN is peroxynitrite, which induces protein nitration. ** indicates a statistically significant increase in glutamine synthetase activity in the peptide-treated group compared to the PN-only-treated group (p < 0.01).

[0022] Figure 6 The results of Western blotting confirming the inhibitory effect of peptides having terminal tyrosine on catalase nitration are shown.

[0023] Figure 7 The results show the results of confirming the inhibitory effect of nitration on heat shock protein 60 (HSP60) by measuring the refolding activity of peptides with terminal tyrosine. RLU represents the difference in absorbance (relative light unit) measured at time 0 (Time 0) and time 60 (Time 60).

[0024] Figure 8 The results are shown for confirming GS activity (Part (A)), GS expression (Part (B)), and tyrosine nitration levels in GS (Parts (C) and (D)) in the chronic physical restraint stress-induced group (STR) in response to the tyrosine-glutamine peptide diet (PD).

[0025] Figure 9 The results are shown for confirming GS activity (Part (A)), GS expression (Part (B)), and tyrosine nitration levels in GS (Parts (C) and (D)) in the chronic physical restraint stress-induced group (STR) in response to the glutamine-tyrosine peptide diet (PD).

[0026] Figure 10 Shown are the results of confirming the plasma corticosterone concentration (Part (A)), sucrose preference (Part (B)), plasma ROS / RNS concentration (Part (C)), and PFC tissue ROS / RNS concentration (Part (D)) according to the tyrosine-glutamine peptide diet (PD) in the chronic physical restraint stress-induced group (STR).

[0027] Figure 11 Shown are the results of confirming the plasma corticosterone concentration (Part (A)), sucrose preference (Part (B)), plasma ROS / RNS concentration (Part (C)), and PFC tissue ROS / RNS concentration (Part (D)) in the chronic physical restraint stress-induced group (STR) according to the glutamine-tyrosine peptide diet (PD).

[0028] Figure 12Figure 2 shows the discrimination index of memory performance in an animal model of chronic physical restraint stress, following either a tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptide diet. (A) shows the object recognition test (ORT) results for the 1xYQ peptide diet, (B) shows the object recognition test (ORT) results for the 2xYQ peptide diet, and (C) shows the object location recognition test (OLT) results for the 1xYQ or 1xYW peptide diet. * and ** indicate statistically significant decreases in the object recognition index or object location recognition index in the stress-treated group (STR) compared to the control group (CTL). * indicates p < 0.05, ** indicates p < 0.01. # indicates statistically significant increases in the object recognition index in the peptide diet group (1xYQ or 2xYQ) compared to the normal diet group (ND).

[0029] Figure 13 The results of the object recognition test (ORT) in an animal model of Alzheimer's disease, following a tyrosine-glutamine (YQ) peptide diet, are shown. (A) Results are from 2-month-old animals, (B) Results are from 8-month-old animals, (C) Results are from 2- and 8-month-old animals, and (D) Results show cognitive decline over a 6-month period. * and ** indicate statistically significant decreases in the object recognition index in the dementia model (3xTG) compared to the normal control group (WT). * indicates p < 0.05, ** indicates p < 0.01. # indicates statistically significant decreases in the object recognition index at 8 months of age compared to 2 months of age.

[0030] Figure 14 Figure 2 shows ROS / RNS concentrations in an animal model of Alzheimer's disease based on a tyrosine-glutamine (YQ) peptide diet. * indicates a statistically significant increase in ROS / RNS concentrations in the dementia animal model (3xTG) compared to the normal control group (WT), with p < 0.05. # indicates a statistically significant decrease in ROS / RNS concentrations in the peptide diet group (YQ) compared to the normal diet group (ND) in the dementia animal model, with p < 0.05. DCF stands for 2',7'-dichlorofluorescin.

[0031] Figure 15Figure 1 shows the results of an animal model for Alzheimer's disease dementia with fluorescently labeled glutamatergic neurons. (A) shows the results of confirming that glutamatergic neurons can be labeled with red. (B) shows the activity of glutamatergic neurons in response to a tyrosine-glutamine (YQ) peptide diet. * indicates a statistically significant decrease in glutamatergic neuron activity in the glutamatergic neuron fluorescently labeled Alzheimer's disease dementia animal model (3xTG-vGluT2-Cre::tdTomato) compared to the normal diet group (WT), with p < 0.05. # indicates a statistically significant increase in glutamatergic neuron activity in the peptide diet group (YQ) compared to the normal diet group (ND) in this animal model for Alzheimer's disease dementia with fluorescently labeled glutamatergic neurons, with p < 0.05.

[0032] Figure 16 Shown are seizure activity levels in an animal model of epilepsy following treatment with tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptides (Parts (A) and (B)), as well as the areas under the curves (Parts (C) and (D)) for Parts (A) and (B). *, **, and *** indicate statistically significant reductions in seizure activity in the peptide-treated groups (5xL-Tyr, 3xYQ, YQ ip, 1xYW, or 3xYQ) compared to the diet group (ND); * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0033] Figure 17 Figure 2 shows ROS / RNS concentrations in an animal model of epileptic seizures following treatment with tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptides. * indicates a statistically significant increase in ROS / RNS concentrations in the kainic acid-treated group compared to the control group (CTL), with p < 0.05. # indicates a statistically significant decrease in ROS / RNS concentrations in the peptide diet group (3xYW or 3xYQ) compared to the normal diet group (ND), with p < 0.05.

[0034] Figure 18 Figure 2 shows glutamine synthetase activity in an animal model of epileptic seizures following treatment with tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptides. *** indicates a statistically significant decrease in glutamine synthetase activity in the kainic acid-treated group compared to the control group (CTL), with p < 0.001. # and ## indicate statistically significant increases in glutamine synthetase activity in the kainic acid-treated group (3xYQ, YQ ip, 1xYW, 3xYW, or 3xYQ) compared to the normal diet group (ND). # indicates p < 0.05, and ## indicates p < 0.01.

[0035] Figure 19Shown are the neuronal cell number (Part (A)), SOD activity (Part (B)), and catalase activity (Part (C)) in an ischemic stroke animal model following treatment with the tyrosine-glutamine (YQ) peptide. Sham represents the sham-operated group, and vehicle represents the 0.9% saline-administered group. * and *** indicate statistically significant decreases in neuronal cell number, SOD activity, or catalase activity in the ischemic stroke animal model compared to the sham-operated group (* p < 0.05, *** p < 0.001). # indicates statistically significant increases in neuronal cell number, SOD activity, or catalase activity in the peptide-treated group (YQ) compared to the 0.9% saline-administered group (vehicle) in the ischemic stroke animal model (p < 0.05).

[0036] Figure 20 Shown are the results of immunohistochemical staining used to confirm neuronal cell death in an ischemic stroke animal model following treatment with tyrosine-glutamine (YQ) peptide. "Sham" indicates a sham-operated group; "Vehicle" indicates a 0.9% saline-administered group; and "Ischemia" indicates an ischemic stroke animal model. NeuN stains for pyramidal neurons, Iba-1 stains for astrocytes, and GFAP stains for microglia.

[0037] Figure 21 To examine the changes in body weight (Part (A)) and food intake (Part (B)) in a high-fat diet-fed type 2 diabetes animal model treated with tyrosine-glutamine (YQ) peptide at different time periods. ND represents the normal diet group, and HFD represents the high-fat diet group.

[0038] Figure 22 This study examined blood glucose levels in a high-fat diet animal model of type 2 diabetes at different time points after treatment with tyrosine-glutamine (YQ) peptide. ND represents the normal diet group, and HFD represents the high-fat diet group. *** and **** indicate statistically significant increases in blood glucose levels in the high-fat diet group (HFD) compared to the normal diet group (ND); *** indicates p < 0.001, **** indicates p < 0.0001. ## and ### indicate statistically significant decreases in blood glucose levels in the YQ peptide-supplemented high-fat diet group (HFD + 1xYQ or HFD + 3xYQ) compared to the high-fat diet group (HFD); ## indicates p < 0.01, ### indicates p < 0.001.

[0039] Figure 23To confirm the results of a glucose tolerance test (GTT) in an animal model of type 2 diabetes induced by a high-fat diet, treated with a tyrosine-glutamine (YQ) peptide. (A) shows the changes in blood glucose levels over time, and (B) shows the area under the curve (AUC) for (A). *** indicates a statistically significant increase in glucose levels in the high-fat diet (HFD) group compared to the normal diet (ND) group; *** indicates p < 0.001. ## indicates a statistically significant decrease in glucose levels in the YQ peptide-supplemented high-fat diet group (HFD + 3xYQ) compared to the high-fat diet (HFD) group; p < 0.01.

[0040] Figure 24 To confirm insulin sensitivity (insulin tolerance test, ITT) results in a high-fat diet type 2 diabetes animal model treated with tyrosine-glutamine (YQ) peptide. (A) shows the changes in blood glucose levels over time, and (B) shows the area under the curve (AUC) for (A). *** indicates a statistically significant increase in glucose levels in the high-fat diet (HFD) group compared to the normal diet (ND) group (p < 0.001). #### indicates a statistically significant decrease in glucose levels in the YQ peptide-supplemented high-fat diet group (HFD + 3xYQ) compared to the high-fat diet (HFD) group (p < 0.0001).

[0041] Figure 25 This study examined urine output (Part (A)) and fat mass (Part (B)) in an animal model of type 2 diabetes induced by a high-fat diet, using tyrosine-glutamine (YQ) peptide. * indicates a statistically significant increase in urine output or fat mass in the high-fat diet (HFD) group compared to the normal diet (ND) group (p < 0.05). ## and #### indicate a statistically significant decrease in urine output or fat mass in the YQ peptide-supplemented high-fat diet group (HFD + 3xYQ) compared to the high-fat diet (HFD) group (## indicates p < 0.01, and #### indicates p < 0.0001).

[0042] Figure 26This study examined the results of plasma insulin (Part (A)), alanine aminotransferase (ALT) (Part (B)), and ROS / RNS (Part (C)) concentrations in an animal model of type 2 diabetes induced by a high-fat diet, based on treatment with a tyrosine-glutamine (YQ) peptide. **, ***, and **** indicate statistically significant increases in plasma insulin, ALT, or ROS / RNS concentrations in the high-fat diet (HFD) group compared to the normal diet (ND) group (**: p < 0.01, ***: p < 0.001, ****: p < 0.0001). ## indicates statistically significant decreases in plasma insulin, ALT, or ROS / RNS concentrations in the YQ peptide-supplemented high-fat diet group (HFD+3xYQ) compared to the high-fat diet (HFD) group (p < 0.01).

[0043] Figure 27 H&E staining results confirm changes in liver tissue following treatment with tyrosine-glutamine (YQ) peptide in a high-fat diet type 2 diabetes animal model. ND represents the normal diet group, and HFD represents the high-fat diet group.

[0044] Figure 28 This study examined the expression of insulin receptor β (IRβ) in an animal model of type 2 diabetes induced by a high-fat diet, based on treatment with a tyrosine-glutamine (YQ) peptide. * indicates a statistically significant decrease in IRβ expression in the high-fat diet (HFD) group compared to the normal diet (ND) group (p < 0.05). ## indicates a statistically significant increase in IRβ expression in the YQ peptide-supplemented high-fat diet group (HFD + 3xYQ) compared to the high-fat diet (HFD) group (p < 0.01).

[0045] Figure 29 To examine the effects of tyrosine-glutamine (YQ) peptide treatment on the urinary albumin / creatinine ratio (Part (A)), liver tissue neutral fat content (Part (B)), and plasma neutral fat content (Part (C)) in an animal model of type 2 diabetes induced by a high-fat diet. * and ** indicate statistically significant increases in the urinary albumin / creatinine ratio, liver tissue neutral fat content, or plasma neutral fat content in the high-fat diet (HFD) group compared with the normal diet (ND) group; * indicates p < 0.05, ** indicates p < 0.01. # and ## indicate statistically significant decreases in the urinary albumin / creatinine ratio, liver tissue neutral fat content, or plasma neutral fat content in the YQ peptide-supplemented high-fat diet group (HFD + 1xYQ or HFD + 3xYQ) group compared with the high-fat diet (HFD) group; # indicates p < 0.05, ## indicates p < 0.01.

[0046] Figure 30Results were obtained in an animal model of acute renal failure, using tyrosine-glutamine (YQ) peptide treatment to assess renal damage (part (A)), and to assess the expression of IL-1β (part (B)), IL-6 (part (C)), and MCP-1 (part (D)) in renal tissue inflammatory responses. Sham represents the sham-operated group, renal IR represents the acute renal failure-induced group, and veh represents the water-administered group. ** indicates a statistically significant increase in creatinine concentration, IL-1β, IL-6, or MCP-1 expression in the water-administered acute renal failure-induced group (veh + renal IR) compared to the sham-operated group (p < 0.01). #, ## indicate that the creatinine concentration, IL-1β, IL-6, or MCP-1 expression levels in the acute renal failure-induced group administered with YQ peptide (YQ+renal IR) were statistically significantly reduced compared with the acute renal failure-induced group administered with water (veh+renal IR). # indicates p < 0.05, and ## indicates p < 0.01.

[0047] Figure 31 Results were obtained by examining the expression of nitrotyrosine and lipid peroxide (4-HNE) in renal tissue following treatment with a tyrosine-glutamine (YQ) peptide in an animal model of acute renal failure. * indicates a statistically significant increase in nitrotyrosine and lipid peroxide (4-HNE) expression in the acute renal failure-inducing group (veh + renal IR) administered with water compared to the sham group (p < 0.05). # indicates a statistically significant decrease in nitrotyrosine and lipid peroxide (4-HNE) expression in the acute renal failure-inducing group (YQ + renal IR) administered with YQ peptide compared to the acute renal failure-inducing group (veh + renal IR) administered with water (p < 0.05).

[0048] Figure 32This study examined the effects of tyrosine-glutamine (YQ) peptide treatment on blood ammonia concentrations (Part (A)) and nitrotyrosine expression in liver tissue in an animal model of hyperammonemia. The control group is a normal control group, and the AOM group is an azoxymethane-induced hyperammonemia group. * and ** indicate statistically significant increases in blood ammonia concentrations and nitrotyrosine expression in liver tissue in the water-treated hyperammonemia group (veh+AOM) compared to the normal control group (Control). * indicates p < 0.05, ** indicates p < 0.01. # indicates statistically significant decreases in blood ammonia concentrations and nitrotyrosine expression in liver tissue in the YQ peptide-induced hyperammonemia group (YQ200+AOM) compared to the water-treated hyperammonemia group (veh+AOM). DETAILED DESCRIPTION

[0049] To achieve the object of the present invention, the present invention provides a health functional food composition for preventing or improving diseases caused by protein nitration, wherein the composition comprises a peptide having a terminal tyrosine or a food-acceptable salt thereof as an active ingredient.

[0050] The tyrosine of the peptide having a tyrosine at the end can be located at both ends or at one end, and the amino acid linked to the tyrosine can be polar / hydrophilic (for example, tyrosine, glutamine, threonine), non-polar / hydrophobic (for example, tryptophan, valine), or charged (for example, arginine), and the number of linked amino acids can be 1 to 29, but is not limited thereto.

[0051] In the health functional food composition for preventing or ameliorating diseases caused by protein nitration of the present invention, the protein is preferably selected from any one of glutamine synthetase, insulin receptor β subunit, manganese superoxide dismutase, heat shock protein 60, copper / zinc superoxide dismutase, and catalase, but is not limited thereto.

[0052] Furthermore, preferably, the disease caused by protein nitration is any one selected from, but not limited to, depression, anxiety disorder, stroke, epilepsy, seizure, cognitive impairment, Alzheimer's disease, dementia, type 2 diabetes, diabetic nephropathy, sarcopenia, dyslipidemia, obesity, non-alcoholic fatty liver disease, acute kidney injury, hyperammonemia, and hepatic encephalopathy.

[0053] The above-mentioned health functional food composition for preventing or improving diseases caused by protein nitration can be prepared into any one selected from pills, tablets, capsules, powders, granules, candies, syrups and beverages, or prepared by adding it as a food ingredient, and can be appropriately prepared by conventional methods.

[0054] Examples of foods to which the active ingredients of the present invention can be added include any form selected from meat, sausage, bread, chocolate, candies, snacks, biscuits, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and multivitamins, including all health functional foods in the general sense.

[0055] These functional health foods may contain various nutritional supplements, vitamins, minerals (electrolytes), synthetic and natural flavorings, colorants, and flavor enhancers (e.g., cheese, chocolate), pectin and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents for carbonated beverages. Furthermore, they may contain fruit pulp used to prepare natural fruit juices and vegetable beverages. These ingredients may be used alone or in combination.

[0056] The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. These natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame, may be used as sweeteners.

[0057] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by protein nitration, wherein the composition comprises a peptide having a terminal tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient.

[0058] In the pharmaceutical composition for preventing or treating diseases caused by protein nitration of the present invention, the above-mentioned protein and the disease caused by protein nitration are as described above.

[0059] In addition to the aforementioned peptides having a terminal tyrosine, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent. The pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, the pharmaceutical composition may be administered by topical application to the skin or by intraperitoneal, rectal, intravenous, intramuscular, or subcutaneous injection, but is not limited thereto.

[0060] The pharmaceutical compositions of the present invention can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations are prepared by mixing one or more compounds with one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, internal liquids, emulsions, and syrups. In addition to commonly used simple diluents such as water or liquid paraffin, they may contain various excipients, such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used as non-aqueous solvents and suspending solvents. Semi-synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, glycerin, gelatin, etc. can be used as suppository bases.

[0061] The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may depend on factors including the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration, and excretion rate, treatment period, drugs used simultaneously, and other factors well known in the medical field. The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered in a single or multiple doses. Taking all of the above factors into account, it is important to administer the drug in a minimum amount to obtain the maximum effect without side effects, which can be easily determined by those skilled in the art.

[0062] The dosage of the composition of the present invention can be used within various ranges depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of the disease.

[0063] Furthermore, the present invention provides a composition for inhibiting nitration of tyrosine in a protein, the composition comprising a peptide having a terminal tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient.

[0064] In the composition for inhibiting nitration of tyrosine in a protein of the present invention, the protein is as described above.

[0065] Furthermore, the present invention provides a method for removing a nitro group from nitrated tyrosine by treating a protein containing nitrated tyrosine with a peptide having a tyrosine residue at the terminal end.

[0066] Furthermore, the present invention provides a health functional food composition for preventing or ameliorating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, wherein the composition comprises a peptide having a terminal tyrosine or a food-acceptable salt thereof as an active ingredient.

[0067] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, the composition comprising a peptide having a terminal tyrosine or a food-acceptable salt thereof as an active ingredient.

[0068] In the pharmaceutical composition for preventing or treating a disease caused by an increase in reactive oxygen species / reactive nitrogen species of the present invention, preferably, the disease caused by an increase in reactive oxygen species / reactive nitrogen species is any one selected from the group consisting of depressive disorder, anxiety disorder, stroke, epilepsy, seizure, cognitive impairment, Alzheimer's disease, dementia, type 2 diabetes, diabetic nephropathy, sarcopenia, dyslipidemia, obesity, non-alcoholic fatty liver disease, acute kidney injury, hyperammonemia and hepatic encephalopathy, but is not limited thereto.

[0069] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only used to more specifically illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0070] Materials and methods

[0071] 1. Synthetic peptides

[0072] In order to analyze the effect of the peptide of the present invention, glutamine (Q), L -Tyrosine ( L -Y) or D -Tyrosine ( D-Y) as individual amino acids, tyrosine-glutamine (YQ), glutamine-tyrosine (QY), tyrosine-tryptophan (YW), tryptophan-tyrosine (WY), tyrosine-threonine (YT), threonine-tyrosine (TY), tyrosine-arginine (YR), arginine-tyrosine (RY), tyrosine-valine (YV), valine-tyrosine (VY), tyrosine-tyrosine-glutamine (YYQ), glutamine-tyrosine-tyrosine (QYY), tyrosine-threonine-glutamine (YTQ), glutamine-threonine-tyrosine (QTY), tyrosine Acid-tryptophan-glutamine (YWQ), glutamine-tryptophan-tyrosine (QWY), tyrosine-glutamine-glutamine (YQQ), glutamine-glutamine-tyrosine (QQY), tyrosine-tyrosine-tyrosine-glutamine (YYYQ), glutamine-tyrosine-tyrosine-tyrosine (QYYY), tyrosine-tryptophan-threonine-glutamine (YWTQ), glutamine-tryptophan-threonine-tyrosine (QWTY), tyrosine-8 random amino acids-tyrosine (Y(A)8Y), tyrosine-18 random amino acids-tyrosine (Y(A) 18 Y) or Tyrosine-28 random amino acids-Tyrosine (Y(A) 28 Y) as a peptide with a terminal tyrosine. Glutamine was a 100% pure product from Nutricost, L-tyrosine was a product from Sigma-Aldrich (#93829, >99.0% BioUltra), and D-tyrosine was a product from Sigma-Aldrich (#855456, >99.0% BioUltra). Peptides used for in vitro experiments were synthesized by Peptron (98% or higher purity), and peptides used for in vivo experiments were synthesized by GL Biochem (95% or higher purity).

[0073] 2. Animal experiments

[0074] In the present invention, 7-week-old C57BL / 6 male mice, 3xTG-AD mice (a model of Alzheimer's disease), 4-week-old ICR male mice, or Mongolian gerbils (65-75 g) were housed at 22-24°C and 50-70% humidity with a 12-hour light-dark cycle, with free access to diet and water. Experiments on animals used in the present invention were conducted according to protocols (GNU-161128-M0068) approved by the Gyeongsang National University Institutional Animal Care and Use Committee (GNU IACUC) in accordance with the guidelines of the National Institutes of Health (NIH, Bethesda, Maryland, USA).

[0075] 3. Preparation of mouse brain and liver tissue samples

[0076] The prefrontal cortex (PFC) and liver tissues were removed from CO2-anesthetized mice and weighed. The tissues were then crushed using a tissue crusher and centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was collected to obtain PFC and liver tissue lysates.

[0077] 4. Prepare feed supplemented with tyrosine peptides

[0078] For animal experiments using the peptide diet according to the present invention, mouse feed supplemented with tyrosine-containing peptides was prepared. Tyrosine-glutamine (YQ), glutamine-tyrosine (QY), or tyrosine-tryptophan (YW) peptides with molecular weights of 309.32 g / mol or 367.40 g / mol, respectively, were added to a standard feed (AIN-93G) (Table 1).

[0079] Table 1

[0080]

[0081] 5. Cognitive function test

[0082] The object recognition test (ORT) and the object location recognition test (OLT) were performed to analyze the long-term memory ability of the animals fed with the peptide diet according to the present invention. Figure 1The experiment consists of three phases: habituation, familiarization, and testing. Mice are habituated to the test box for 10 minutes each day for two days. During the third day's familiarization phase, mice are allowed to explore two identical objects for 10 minutes. Then, during the fourth day's testing phase, one of the two objects is replaced with a novel object, and the mice are allowed to explore for 10 minutes. The first 5 minutes of the experiment are used as data. The object recognition function (ORT) index is calculated using the following formula.

[0083] Discrimination index = (NF) / (N + F)

[0084] N: New object exploration time

[0085] F: Familiar object exploration time

[0086] After 24 hours of ORT, the location of the familiar object was changed and the mice were allowed to explore for 5 minutes. The object location recognition function (OLT) index was calculated by calculating the proportion of the time spent exploring the moved object to the total object exploration time.

[0087] Example 1. Analysis of protein nitration inhibition effect

[0088] 1-1. Analysis of protein nitration inhibition effects in mouse brain and liver tissues

[0089] Mouse brain and liver tissue lysates were used to analyze the inhibitory effect of the peptides of the present invention on protein nitration. Peroxynitrite (PN), which induces protein nitration, was added to the tissue lysates. The peptides of the present invention were then added at a concentration of 2 mM, mixed by vortexing for 5 seconds, and incubated on ice for 10 minutes. Western blotting was then performed using an anti-nitrotyrosine antibody (1:1000), an anti-insulin receptor β antibody (1:1000), or an anti-phospho-insulin receptor β antibody (1:1000).

[0090] The results confirmed that PN caused an increase in proteins with increased tyrosine nitration in PFC and liver tissues, and Western blotting results of multiple bands confirmed an increase in nitration of various proteins except glutamine synthetase (GS) and insulin receptor β subunit (IRb). It was confirmed that the proteins with increased tyrosine nitration due to PN had reduced nitration by treatment with a peptide having a tyrosine (Y) at the terminal. Figure 2Furthermore, it was confirmed that in liver tissue, there was no difference in the expression of IRb protein after treatment with PN or a peptide having a terminal tyrosine (Y). In contrast, the expression of phosphorylated IRb protein decreased due to nitration by PN, but increased due to treatment with a peptide having a terminal tyrosine (Y). Figure 3 ).

[0091] 1-2. Analysis of the nitrification inhibition effect of Cu / ZnSOD and MnSOD

[0092] The SOD colorimetric reaction activity kit (Thermo Scientific, #EIASODC) was used to analyze the inhibitory effect of the peptides of the present invention on the nitration of copper / zinc superoxide dismutase (SOD-1) and manganese superoxide dismutase (SOD-2). Human recombinant Cu / ZnSOD or MnSOD within a standard range was added to a PCR tube. The peptides of the present invention were then added at a concentration of 1 mM, along with 1 mM peroxynitrite (PN). The mixture was then incubated on ice for 10 minutes. Subsequently, 25 μL of xanthine oxidase provided in the kit was added and the reaction was continued at room temperature for 20 minutes. The absorbance was then measured at 450 nm. The activity of the Cu / ZnSOD or MnSOD was determined by subtracting the absorbance from the absorbance of the first measurement.

[0093] The results confirmed that the activity of Cu / ZnSOD or MnSOD rapidly decreased due to PN treatment, but the activity of Cu / ZnSOD or MnSOD was restored by a peptide having a terminal tyrosine ( Figure 4 ).

[0094] 1-3. Analysis of the nitration inhibition effect of glutamine synthetase

[0095] Mouse brain tissue lysate was used to analyze the inhibitory effect of the peptide of the present invention on glutamine synthetase nitration. The PFC tissue lysate was added to a PCR tube, and the peptide of the present invention was added at a concentration of 1 mM, along with 1 mM peroxynitrite (PN). The mixture was then allowed to react on ice for 10 minutes. 50 μL of the reacted sample was then placed in a 96-well plate, and 50 μL of glutamine synthetase assay buffer (50 mM imidazole-HCl, 25 mM L-glutamine, pH 6.8, 12.5 mM hydroxylamine, 12.5 mM sodium arsenate, 1 mM MnCl₂, 0.08 mM ADP) was added. The reaction was continued at 37°C for approximately 40 minutes, and the absorbance was measured at 560 nm. The activity of glutamine synthetase was calculated based on a standard curve prepared using γ-glutamylhydroxamate.

[0096] The results confirmed that glutamine synthetase activity rapidly decreased due to PN treatment, but that glutamine synthetase activity was restored by a peptide having a terminal tyrosine. In particular, it was confirmed that even a peptide consisting of 30 amino acids had the effect of restoring the activity of glutamine synthetase that had been nitrated and reduced by PN treatment ( Figure 5 ).

[0097] 1-4. Analysis of the nitrification inhibition effect of catalase

[0098] Western blotting was performed using human recombinant catalase to analyze the inhibitory effect of the peptides of the present invention on catalase nitration. 0.2 μg of human recombinant catalase was added to a PCR tube, followed by the addition of the peptides of the present invention at a concentration of 2 mM and 1 mM peroxynitrite (PN). The mixture was then reacted on ice for 10 minutes. SDS sample buffer was then added and the mixture was boiled for 5 minutes. The mixture was then run on an SDS-PAGE gel and transferred to a PVDF membrane. Western blotting was then performed using an anti-nitrotyrosine antibody (1:1000).

[0099] The results confirmed that the expression of catalase that was nitrated increased due to PN treatment, while the expression of catalase that was nitrated by a peptide having a terminal tyrosine decreased ( Figure 6 ).

[0100] 1-5. Analysis of Nitration Inhibition Effects by Assaying HSP60 (Heat Shock Protein 60) Refolding Activity

[0101] It is well known that the activity of HSP60, one of the chaperone proteins, is inhibited by protein nitration, and various diseases are known to be related to this. To this end, the HSP60 / HSP10 Glow-Fold Protein Refolding Kit (R&D Systems, #K-300) was used to analyze the inhibitory effect of the peptide of the present invention on HSP60 nitration. The HSP60 solution provided in the kit was added to a PCR test tube, and then the peptide of the present invention was added at a concentration of 1mM and 1mM peroxynitrite (PN) was added and mixed, and then reacted on ice for 10 minutes. Then, the HSP10 solution provided in the kit, MgCl2, and MgSO4 were added. 2+ -ATP solution and Glow-Fold matrix protein were reacted at room temperature for 15-30 minutes. The reacted samples were then heated at 45°C for 7 minutes and immediately placed on ice. 50 μL of luciferin solution and 4 μL of sample were mixed in a 96-well plate, and luminescence was measured within 1-2 minutes using a microplate reader (Time 0). The remaining sample was reacted at 30°C for 60 minutes. 50 μL of luciferin solution and 4 μL of sample were then mixed in a 96-well plate, and luminescence was measured within 1-2 minutes using a microplate reader (Time 60). HSP60 refolding activity of each sample was analyzed by the difference in absorbance (RLU) measured at Time 0 and Time 60.

[0102] The results confirmed that the refolding activity of HSP60 decreased rapidly due to PN treatment, but the refolding activity of HSP60 was restored by a peptide having a terminal tyrosine ( Figure 7 Based on the above results, it was determined that peptides having a tyrosine residue at the end can prevent or improve diseases induced by decreased HSP60 activity.

[0103] Example 2. Analysis of GS Nitration Inhibition and Antidepressant Effects in Chronic Physical Restraint Stress Animal Model

[0104] Twenty-eight 7-week-old C57BL / 6 male mice were divided into two groups (normal group and stress group). The stress group was individually restrained in a restraint device for 2 hours daily (2-4 pm) for a total of 15 days to induce chronic physical restraint stress. The normal (CTL) and stress (STR) groups were further divided into a nutritionally balanced normal diet group (ND) and a diet group (PD) supplemented with YQ or QY peptide (330 mg / kg). Mice that completed 15 days of chronic physical restraint stress underwent a sucrose preference test (SPT), and blood and PFC tissue were collected for subsequent experiments.

[0105] (1) Analysis of GS nitrification inhibition effect

[0106] GS activity and GS expression were measured using PFC lysates from mice that had undergone chronic physical restraint stress using the same method as described above.

[0107] The results confirmed that GS expression levels were similar, but GS activity decreased due to stress. It was also confirmed that GS activity in the YQ or QY peptide diet group was statistically significantly increased compared to the normal diet group ( Figure 8 Part (A) Figure 8 Part (B) Figure 9 Part (A) Figure 9 (Part B).

[0108] Furthermore, immunoprecipitation (IP)-WB was used to determine the level of tyrosine nitration in GS. Tyr-nitrated GS was immunoprecipitated using an anti-nitrotyrosine antibody (ab61392, Abcam) and protein A / G agarose (Santa Cruz) according to the manufacturer's protocol.

[0109] The results confirmed that the level of tyrosine nitration in GS was statistically significantly increased due to stress, and the level of tyrosine nitration in GS in the YQ or QY peptide diet group was statistically significantly decreased compared with the normal diet group ( Figure 8 Part (C) Figure 8 Part (D) Figure 9 Part (C) Figure 9 (Part D).

[0110] (2) Analysis of antidepressant effects

[0111] Blood and PFC tissue were collected from mice after chronic physical restraint stress to analyze the antidepressant effects. Blood was collected from mice and centrifuged at 1000 × g for 10 minutes at 4°C. The supernatant was collected to separate plasma, which was then diluted to 1 / 20 with PBS. Plasma corticosterone concentrations were measured using a corticosterone EIA kit (Cayman) according to the manufacturer's protocol.

[0112] The results confirmed that stress caused a statistically significant increase in plasma corticosterone concentrations, and that in the stress group, the YQ or QY peptide diet group had a decreased plasma corticosterone concentration compared to the normal diet group ( Figure 10 Part (A) Figure 11 (Part A).

[0113] Plasma and 50 μg of PFC tissue lysate were diluted to 1 / 3 with PBS and the reactive oxygen species / reactive nitrogen species (ROS / RNS) concentrations in plasma and PFC tissue were measured using a ROS / RNS assay kit (Cell Biolabs) according to the manufacturer's protocol.

[0114] The results confirmed that stress caused a statistically significant increase in the concentrations of ROS / RNS in plasma and PFC tissues. In the stress group, the concentrations of ROS / RNS in plasma and PFC tissues were reduced in the YQ or QY peptide diet group compared with the normal diet group ( Figure 10 Part (C) Figure 10 Part (D) Figure 11 Part (C) Figure 11 (Part D).

[0115] Furthermore, depression was assessed by measuring the sucrose preference of mice after 15 days of chronic physical restraint stress. The sucrose preference test was conducted over four days, with the same amount of 0.1M sucrose and water provided in drinking bottles of identical size and shape. On day one, 0.1M sucrose and water were provided for 24 hours. On day two, the positions of the sucrose and water were swapped and provided for 24 hours. On day three, 0.1M sucrose and water were not provided. On day four, the same amount of 0.1M sucrose and water were provided again for three hours, then the positions were swapped and provided again for three hours. On day four, the amount of sucrose and water consumed over a six-hour period was measured, and sucrose preference (%) was calculated according to the following formula.

[0116] Sucrose preference (%) = sucrose consumption / (water consumption + sucrose consumption) × 100

[0117] The results confirmed that stress caused a decrease in sucrose preference, and in the stress group, sucrose preference increased in the YQ or QY peptide diet group compared with the normal diet group ( Figure 10 Part (B) Figure 11 (Part B).

[0118] Example 3. Analysis of cognitive function improvement in a chronic physical restraint stress animal model

[0119] Twenty-eight 7-week-old C57BL / 6 male mice were divided into two groups (normal group and stress group) and fed a diet supplemented with tyrosine-containing peptides (1xYQ, 2xYQ, or 1xYW) for one week. The stress group was individually restrained in a device for 2 hours daily (2-4 pm) for a total of 2 weeks to induce chronic physical restraint stress. Then, the mice were subjected to an object recognition test (ORT).

[0120] The results confirmed that object recognition function decreased due to stress, and in the stress group, object recognition function increased in the YQ or YW peptide diet group compared with the normal diet group ( Figure 12 ).

[0121] Example 4. Analysis of Cognitive Function Improvement in an Animal Model of Alzheimer's Dementia

[0122] 4-1. Transgenic Mouse Model of Alzheimer's Disease

[0123] Cognitive function was tested in two-month-old 3xTG-AD transgenic mice, a model of Alzheimer's dementia, and two-month-old normal C57BL / 6 mice. The mice were then divided into two groups and fed either a normal diet (ND) or a diet supplemented with a tyrosine-containing peptide (1xYQ). Furthermore, cognitive function was reassessed at eight months of age, when the 3xTG-AD mice are known to exhibit mild cognitive impairment, to test the cognitive protective effects of the tyrosine-containing peptide.

[0124] The results confirmed that the object recognition function of the dementia model mice was reduced compared to that of normal mice, and the reduction in object recognition function at 8 months of age was greater than that at 2 months of age. In addition, it was confirmed that there was no difference in the normal mouse group due to the peptide diet, but in the dementia model mice, the object recognition function was slightly improved by the YQ peptide diet ( Figure 13 ).

[0125] Furthermore, 100 μL of RIPA buffer (containing proteolytic enzyme and phosphatase inhibitors) was added to the hippocampus of the brain per 10 mg of tissue. The cells were then pulverized using glass beads and a blender for 1 minute. The supernatant was separated by centrifugation at 12,000 × g for 15 minutes at 4°C and diluted 10-fold in PBS for the determination of reactive oxygen species and reactive nitrogen species (ROS / RNS). ROS / RNS concentrations were measured using the OxiSelect ROS / RNS Assay Kit (Cell Biolabs) according to the recommended protocol.

[0126] The results confirmed that the ROS / RNS concentration in the hippocampus of the dementia animal model increased compared to the normal group, but was reduced by YQ peptide ( Figure 14 ).

[0127] 4-2. Alzheimer's disease dementia model using fluorescently labeled glutamatergic neurons

[0128] By crossing vGluT2-IRES-Cre::tdTomato mice, which fluorescently label glutamatergic neurons, with 3xTG-AD mice, a transgenic mouse model of Alzheimer's disease, 3xTG-vGluT2-Cre::tdTomato, a dementia model in which glutamatergic neurons are fluorescently labeled, the animal model was confirmed to be able to label glutamatergic neurons with red color. Figure 15 (Part A).

[0129] 3xTG-vGluT2-Cre::tdTomato mice were fed a diet supplemented with a tyrosine-containing peptide (1xYQ) at 2 months of age. Spontaneous excitatory postsynaptic currents (sEPSCs) were measured at 6 to 8 months of age to assess glutamatergic neuronal activity. To record membrane currents, 200 μm thick cross-sectional brain slices were placed in a recording chamber perfused with artificial cerebrospinal fluid at 1.5 to 2 mL / min. Whole-cell voltage-clamp recordings were then obtained from visualized glutamatergic neurons in the medial prefrontal cortex (mPFC) at a holding potential of -70 mV. Glutamate currents were isolated by the addition of picrotoxin (100 μM). All recordings were performed at 30 ± 2°C using a pipette solution consisting of 130 mM KCl, 5 mM CaCl2, 10 mM EGTA, 10 mM HEPES, 2 mM MgATP, 0.5 mM Na2GTP, and 5 mM creatine phosphate.

[0130] The results confirmed that the activity of glutamatergic neurons in the dementia animal model was reduced compared to the normal group, but was increased by YQ peptide ( Figure 15(Part B).

[0131] Example 5. Analysis of Excitotoxicity and Oxidative Stress Inhibition in an Animal Model of Epileptic Seizures

[0132] Four-week-old ICR male mice were fed a normal diet, a diet supplemented with tyrosine (L-Tyr), or a tyrosine-containing peptide (3xYQ, 1xYW, or 3xYW) for one week and then injected intraperitoneally with 200 mg / kg of YQ (YQ). A 4.5 mg / mL kainic acid (KA) solution (36 mg / kg) was prepared by heating KA in a water bath and dissolving it in normal saline. Seizure activity and symptoms were recorded within two hours. Brain tissue (prefrontal cortex and hippocampus) was removed from surviving mice 16 hours or 5 days after KA injection.

[0133] 5-1. Determining the severity of epileptic seizures

[0134] Seizure level assessment was recorded by applying the criteria in Table 2 below.

[0135] Table 2

[0136] Representative symptoms based on seizure level

[0137]

[0138] The results confirmed that the peptide diet group had significantly reduced levels of epileptic seizures compared to the normal diet group ( Figure 16 ).

[0139] 5-2. Determination of Reactive Oxygen Species / Reactive Nitrogen Species (ROS / RNS)

[0140] For every 10 mg of hippocampal brain tissue, 100 μL of RIPA buffer (containing proteolytic enzyme and phosphatase inhibitors) was added. The cells were then pulverized using glass beads and a blender for 1 minute. The supernatant was separated by centrifugation at 12,000 × g for 15 minutes at 4°C and diluted 10-fold in PBS for ROS / RNS analysis. ROS / RNS concentrations were measured using the OxiSelect ROS / RNS Assay Kit (Cell Biolabs) according to the recommended protocol.

[0141] The results confirmed that the ROS / RNS concentration in the hippocampus of mice that survived the injection of kainic acid was increased by kainic acid but decreased by 3xYQ or 3xYW peptides ( Figure 17 ).

[0142] 5-3. Determination of glutamine synthetase (GS) activity

[0143] 2 μL of hippocampal lysate was placed in a 96-well plate and adjusted to 50 μL with 50 mM imidazole-HCl buffer (pH 6.8). 50 μL of GS activity assay buffer (50 mM imidazole-HCl, pH 6.8, 25 mM L-glutamine, 12.5 mM hydroxylamine, 12.5 mM sodium arsenate, 1 mM MnCl₂, and 0.08 mM ADP) was added and allowed to react at 37°C for approximately 40 minutes. After the reaction, the standard substance γ-glutamylhydroxamate was added to the empty wells at concentrations ranging from 0.391 to 25.0 mM. 100 μL of a reaction stop solution (90 mM FeCl₃, 1.8 N HCl, and 1.45% (w / v) trichloroacetic acid) was added to the sample and standard substances, and the absorbance was measured at 560 nm. The GS activity of each sample was calculated by comparison with the standard curve. The GS activity was expressed as the amount of γ-glutamyl hydroxamate produced as the final product, expressed in μM / min / μg protein.

[0144] The results confirmed that the activity of glutamine synthetase in the hippocampus of mice that survived the injection of kainic acid was decreased by kainic acid, but increased by 3xYQ, 1xYW, 3xYW, or 3xYQ peptides ( Figure 18 ).

[0145] Example 6. Analysis of the inhibitory effects on neuronal cell death and antioxidant effects in an ischemic stroke animal model

[0146] Three-month-old male Mongolian gerbils were induced under general anesthesia with 3% isoflurane and maintained with 2.5% isoflurane gas. After disinfection, the neck was incised to expose both common carotid arteries. Blood flow was then blocked with microvessel clips for 5 minutes to induce ischemia, inducing stroke. Body temperature was maintained at 37.0 ± 0.5°C during the application of the clips. Animals undergoing a sham operation served as a control group.

[0147] 6-1. Analysis of the effect of inhibiting neuronal cell death

[0148] After induction of ischemia, reperfusion was performed and YQ peptide was intraperitoneally administered three times every 24 hours at a dose of 200 mg / kg. On the fourth day, the animals were sacrificed and the brain tissue was removed to determine the number of dead neurons.

[0149] The results confirmed that the number of nerve cells in the ischemic stroke animal model decreased compared with the sham operation group, but increased due to YQ peptide ( Figure 19 (Part A).

[0150] 6-2. Measurement of SOD and CAT activities

[0151] After induction of ischemia, reperfusion was performed and YQ peptide was administered intraperitoneally once at a dose of 200 mg / kg. Two or 24 hours after reperfusion, general anesthesia was administered with avertin. The cranial cavity was opened, the brain was quickly removed, and the hippocampus tissue was excised, rapidly frozen in liquid nitrogen, and stored in an ultra-low temperature freezer. SOD and CAT activities were then measured using a SOD colorimetric activity kit and a catalase colorimetric activity kit.

[0152] The results confirmed that the SOD and CAT activities of the ischemic stroke animal model decreased 24 hours after reperfusion compared with the sham operation group, but increased due to YQ peptide ( Figure 19 Part (B) Figure 19 (Part C).

[0153] 6-3. Immunohistochemical staining for confirming neuronal cell death

[0154] Immunohistochemical staining was performed to examine changes in neurons and glial cells, as well as changes in related factors, in the hippocampus of an animal model of ischemic brain injury. After induction of ischemia, reperfusion was performed and YQ peptide was administered intraperitoneally three times every 24 hours at a dose of 200 mg / kg. A control group was administered vehicle (0.9% saline) instead of peptide. On the fourth day, the animals were sacrificed and brain tissue was removed and fixed in 4% paraformaldehyde at 4°C. The tissue was then immersed in 10%, 20%, or 30% sucrose solutions and frozen in liquid nitrogen. Serial sections of 30 μm thickness were prepared, placed in cryopreservative solution, and stored at -20°C. The brain tissue was washed three times with 0.01 M PBS for 10 minutes each time and then incubated with 0.3% H₂O₂ for 30 minutes to remove endogenous peroxidase. To prevent nonspecific immune reactions, sections were reacted with 5% normal serum corresponding to the respective antibodies for 30 minutes. Antibodies against pyramidal neurons (NeuN), astrocytes (Iba-1), and microglia (GFAP) were then diluted to the desired dilution and incubated overnight at room temperature. Following this reaction, the tissues were reacted with a biotin-conjugated secondary antibody for 2 hours, followed by an ABC solution for 1 hour. The tissues were then developed using a 3,3'-DAB kit, smeared on slides, dried at room temperature for 12 hours, and then subjected to standard dehydration and clearing procedures. The slides were then mounted with DPX mounting solution and observed under a microscope (Olympus BX53).

[0155] The results confirmed that in the control group (0.9% saline administration group), almost no neurons were observed in the CA1 region of the hippocampus, but in the YQ peptide administration group, approximately 60.7% of the cells survived compared to the sham operation group. In addition, it was confirmed that in an ischemic stroke animal model, glial cells with expanded cytoplasm and expanded cell processes were observed in the control group (0.9% saline administration group), but the glial cell morphology in the YQ peptide administration group did not change significantly, showing a morphology almost similar to that of the sham operation group ( Figure 20 ).

[0156] Based on the above results, it was found that peptides having a terminal tyrosine inhibit oxidative damage and immune response in brain tissue caused by ischemia, thereby preventing neuronal cell death.

[0157] Example 7. Analysis of the Effect of a High-Fat Diet on Insulin Sensitivity in a Type 2 Diabetes Animal Model

[0158] Three-week-old C57BL / 6 male mice were purchased from Koatech and housed two per cage in an animal housing room maintained at a constant temperature (22±2°C), constant humidity (50±5%), and a 12-hour light cycle. After one week of acclimation, the mice were divided into four groups: a normal diet (ND), a high-fat diet (HFD; 60% kcal fat), and a YQ peptide-supplemented high-fat diet (HFD+1xYQ or HFD+3xYQ). They were housed for a total of 17 weeks.

[0159] 7-1. Measurement of body weight, food intake, and fasting blood glucose

[0160] The body weight and feed intake of each animal were measured weekly, and fasting blood glucose was measured using a blood glucose meter (Accu-Check) after fasting for 12 hours on the first, fifth, or eighth week after the start of feeding.

[0161] The results confirmed that the body weight of all experimental groups gradually increased. On the same day, the body weight of the HFD group was higher than that of the ND group, and the body weight of the HFD+3xYQ group was lower than that of the HFD group ( Figure 21 (Part A)) It was confirmed that the feed intake of the HFD group, HFD+1xYQ group, or HFD+3xYQ group was lower than that of the ND group on the same day, and there was almost no difference between the HFD group, HFD+1xYQ group, or HFD+3xYQ group ( Figure 21 (Part B).

[0162] Furthermore, it was confirmed that the blood glucose level in the HFD group increased compared with the ND group, and that the blood glucose level in the HFD+1xYQ group or the HFD+3xYQ group decreased over time compared with the HFD group ( Figure 22).

[0163] 7-2. Glucose tolerance test (GTT) and insulin sensitivity test (ITT)

[0164] GTT was performed at week 15 after the start of feeding, and ITT was performed at week 16. GTT was performed by intraperitoneal injection of 2 g / kg of glucose solution after a 12-hour fast, followed by venous blood sampling at 0, 20, 60, 90, and 120 minutes. ITT was performed by intraperitoneal injection of 0.75 U / kg of insulin after a 6-hour fast, followed by venous blood sampling at 0, 15, 30, 60, and 120 minutes.

[0165] It was confirmed that the HFD group had an increased blood glucose level compared to the ND group in the glucose tolerance and insulin sensitivity test results, and that the HFD+3xYQ group had a decreased blood glucose level compared to the HFD group ( Figure 23 、 Figure 24 ).

[0166] 7-3. Measurement of urine output and fat mass

[0167] Urine volume was measured by measuring the amount of urine (ml) collected from metabolic cages over 16 hours. Fat mass of each mouse was measured using an EchoMRI™ instrument and expressed as a percentage of body weight.

[0168] The results confirmed that the urine output and fat mass increased in the HFD group compared to the ND group, and decreased in the HFD+3xYQ group compared to the HFD group ( Figure 25 ).

[0169] 7-4. Plasma biochemical analysis

[0170] After anesthesia, blood was collected from the inferior aorta, and plasma was separated by centrifugation at 3000 rpm for 15 minutes. Insulin concentrations were measured using an ELISA kit (Crystal Chem, Ultra Sensitive Mouse Insulin ELISA Kit), alanine aminotransferase (ALT) concentrations were measured using an assay kit (IVD Lab Co., ChemiLab GPT (ALT) assay kit), and ROS / RNS concentrations were measured using an assay kit (Cell Biolabs, Inc., OxiSelect™ In Vitro ROS / RNS assay kit).

[0171] The results confirmed that the plasma levels of insulin, ALT, and ROS / RNS increased in the HFD group compared to the ND group, and decreased in the HFD+3xYQ group compared to the HFD group ( Figure 26 ).

[0172] Based on the above results, peptides with terminal tyrosine can be used to prevent or treat non-alcoholic liver disease and chronic metabolic diseases caused by reactive oxygen / reactive nitrogen species.

[0173] 7-5. Histological analysis

[0174] After anesthesia, liver tissue was removed and rapidly frozen in liquid nitrogen. A portion was then fixed in 10% formalin, and 5-μm tissue sections were prepared in paraffin blocks. The tissue was then stained with hematoxylin and eosin (H&E) and observed and imaged under a microscope (Olympus, CKX41).

[0175] The results confirmed that traces of hepatocyte fat droplets and tissue fibrosis were observed in the liver of the HFD group. In contrast, the HFD+3xYQ group and the ND group maintained similar liver tissue appearance ( Figure 27 ).

[0176] 7-6. Determination of insulin receptor expression

[0177] It is well known that the reduction of insulin receptors in muscle in metabolic diseases leads to reduced insulin signaling sensitivity, limiting the ability to use glucose in muscle, thus causing sarcopenia. Liver tissue was homogenized in RIPA buffer and protein was quantified by the BCA method, followed by Western blotting using an anti-insulin receptor-β (IRβ) antibody.

[0178] The results confirmed that the expression of insulin receptor β was decreased in the HFD group compared with the ND group, and increased in the HFD+3xYQ group compared with the HFD group ( Figure 28 ).

[0179] Based on the above results, it was found that peptides having a terminal tyrosine increase the expression level of insulin receptor β and thus have the effect of preventing sarcopenia caused by metabolic diseases.

[0180] 7-7. Determination of albumin / creatinine content and neutral fat content

[0181] It is well known that when renal function declines, urinary albumin levels increase and creatinine levels decrease. To this end, the levels of albumin and creatinine in urine of each experimental animal were measured using a mouse albumin ELISA kit (Abcam) and a creatinine assay kit (Abcam), and the albumin / creatinine ratio was calculated. Furthermore, 10 μL of plasma or 40 mg of homogenized liver tissue was centrifuged at 10,000 g for 10 minutes, and the supernatant was separated. The amount of neutral fat (TG) in the plasma or liver was measured using a neutral fat (triglyceride, TG) assay kit (Cayman).

[0182] The results confirmed that the albumin / creatinine ratio and the amount of neutral fat increased in the HFD group compared with the ND group, and decreased in the HFD+1xYQ or HFD+3xYQ groups compared with the HFD group ( Figure 29 ).

[0183] Based on the above results, it is known that peptides having terminal tyrosine inhibit the decline in renal function caused by diabetes, thereby having the effect of preventing diabetic nephropathy, one of the complications of diabetes, and having the effect of preventing or treating dyslipidemia, which is a phenomenon of abnormal increase in neutral fat in the blood.

[0184] Example 8. Analysis of the Effect of Inhibiting Protein Nitration in an Animal Model of Acute Renal Failure Induced by Renal Ischemia-Reperfusion

[0185] Male C57BL / 6 mice weighing 23-25 g were housed in a sterile housing room maintained at a constant temperature and humidity with free access to water and feed. They were then divided into three groups: 1) a control group (Sham), 2) a group receiving water and undergoing renal ischemia-reperfusion (Veh+renal IR), and 3) a group receiving YQ peptide and undergoing renal ischemia-reperfusion (YQ+renal IR). YQ (100 mg / kg) was orally administered once daily for four days, with renal ischemia induced 30 minutes after administration on the fourth day. After abdominal incision, renal ischemia was induced by clamping the renal pedicles bilaterally with Müller atraumatic vascular clamps. After 25 minutes of ischemia, the clamps were removed and reperfusion was performed. The control group (Sham) underwent the same surgical procedures, except for the use of clamps to induce ischemia. After 24 hours of reperfusion, the animals were sacrificed, and blood was collected from the heart and renal tissue was removed.

[0186] 8-1. Determination of blood creatinine concentration

[0187] The collected blood was centrifuged at 3000 rpm for 15 minutes to separate the plasma. The blood creatinine concentration was measured using the Jaffe method as an assessment indicator of renal damage. The Jaffe method calculates the creatinine concentration by the difference between the absorbance measured at 510 nm after the plasma sample is reacted with picric acid and the absorbance measured after the reaction is stopped with 60% acetic acid. The concentration is expressed in mg / dL.

[0188] The results confirmed that plasma creatinine increased significantly compared with the control group (sham) 24 hours after renal ischemia and reperfusion, and this increase was statistically significantly inhibited by administration of YQ peptide ( Figure 30 (Part A).

[0189] 8-2. Analysis of real-time qPCR

[0190] Total RNA was extracted from renal tissues using the Trizol method, and cDNA was synthesized using the RevertAid reverse transcription system (Thermofisher). Quantitative PCR (qPCR) of inflammatory cytokines (IL-1β, IL-6, and MCP-1) was performed using iQ SYBR Green Supermix (Bio-Rad) in the CFX Connect Real-Time PCR System (Bio-Rad). -△Ct The relative amount of target mRNA was analyzed by GAPDH calibration. The primers used in the experiment are shown in Table 3 below.

[0191] Table 3

[0192] Primer information used for qPCR analysis

[0193]

[0194] The results confirmed that 24 hours after renal ischemia and reperfusion, the expression levels of IL-1β, IL-6, and MCP-1, which are indicators of inflammatory response, increased significantly compared with the control group (sham). Inflammatory response is the main pathogenesis of acute renal failure, and these increases were statistically significantly inhibited by the administration of YQ peptide ( Figure 30 Part (B) Figure 30 Part (C) Figure 30 (Part D).

[0195] 8-3. Determination of nitrated protein and lipid peroxide expression in renal tissue

[0196] Kidney tissues were homogenized in RIPA buffer and protein was quantified by the BCA method, followed by Western blotting using antibodies against nitrotyrosine and 4-hydroxynonenal (4-HNE), a lipid peroxidation product.

[0197] The results confirmed that the amount of tyrosine-nitrated protein in renal tissue increased compared with the control group (sham) 24 hours after renal ischemia and reperfusion, and this increase was significantly reduced by administration of YQ peptide ( Figure 31 ).

[0198] Example 9. Analysis of the Effect of Blood Ammonia Suppression in a Hyperammonemia Animal Model

[0199] Thirteen-week-old male C57BL / 6 mice were allowed free access to water and feed in a sterile housing maintained at a constant temperature and humidity. They were then divided into four groups: (1) control group (Control), (2) hyperammonemia group (Veh+AOM) administered with water, (3) hyperammonemia group (YQ100+AOM) administered with 100 mg / kg of YQ peptide, and (4) hyperammonemia group (YQ200+AOM) administered with 200 mg / kg of YQ peptide. YQ peptide was orally administered once daily for four days. On the fourth day, hyperammonemia was induced by intraperitoneal injection of azoxymethane (AOM, 100 mg / kg) two hours after oral administration. Twelve hours after AOM administration, 200 μL of saline containing 0.5% glucose was intraperitoneally administered to prevent dehydration. Four hours later, the animals were sacrificed, blood was collected from the heart, and liver tissue was removed.

[0200] 9-1. Analysis of blood ammonia

[0201] Ammonia is a major metabolic product of amino acids and nucleic acids. Since the main enzymes of the urea cycle, which convert ammonia to urea, are found only in hepatocytes, and liver tissue is also rich in enzymes such as GS and SOD, which reduce ammonia concentrations, and catalase, which removes reactive oxygen species, fluctuating blood ammonia levels in the blood vessels that pass from the liver to the brain are important indicators of hepatic encephalopathy. To determine this, a single-wavelength reflectance detection analyzer, the PocketChem BA PA-4140 (Arkray, Japan), was used to measure blood ammonia levels. 20 μL of blood was reacted with a test strip at room temperature for 3 minutes, then inserted into the analyzer. The absorbance was measured at a wavelength of 635 nm (LED) and expressed in μg / dL.

[0202] The results confirmed that the blood ammonia concentration in the hyperammonemia group (Veh+AOM) administered with water increased by approximately 3.6 times compared to the control group (control), and that the increase in ammonia caused by AOM was significantly suppressed by the administration of YQ peptide ( Figure 32 (Part A).

[0203] 9-2. Determination of nitrated protein expression in liver tissue

[0204] Liver tissues were homogenized in RIPA buffer and protein was quantified by the BCA method, followed by Western blotting using an antibody against nitrotyrosine.

[0205] As a result, the nitrotyrosine protein in the liver tissue increased after AOM administration compared to the control group, and this increase was significantly reduced by the administration of YQ peptide. This result shows that YQ peptide inhibits the nitration of proteins present in the liver, thereby removing ammonia flowing into the liver ( Figure 32 ).

[0206] Statistical processing

[0207] All data in the present invention are expressed as mean ± standard deviation, and statistical analysis was performed using analysis of variance (ANOVA) with Dunnett's Multiple Comparison Test or Student's t-test using GraphPad Prism 5 (GraphPad Software).

Claims

1. A pharmaceutical composition for preventing or treating diseases caused by protein nitration, characterized in that: The pharmaceutical composition comprises tyrosine-glutamine (YQ) or a pharmaceutically acceptable salt thereof as an effective ingredient, and the disease caused by protein nitration is selected from type 2 diabetes, diabetic nephropathy, sarcopenia, dyslipidemia, obesity and non-alcoholic fatty liver disease.

2. A food composition for preventing or improving diseases caused by protein nitration, characterized in that: The food composition comprises tyrosine-glutamine (YQ) or a food-technically acceptable salt thereof as an effective ingredient, and the disease caused by protein nitration is selected from type 2 diabetes, diabetic nephropathy, sarcopenia, dyslipidemia, obesity and non-alcoholic fatty liver disease.

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