Composition for preventing, ameliorating or treating diseases caused by tyrosine nitration in proteins, containing tyrosine as active ingredient

Abstract

The present invention relates to a composition for preventing, ameliorating or treating diseases caused by nitration of tyrosine in a protein, comprising tyrosine as an active ingredient. The tyrosine as an active ingredient of the present invention not only enhances reduced GS activity, but also can restore the content (proportion) of glutamine and glutamic acid in the brain and the amount of ammonia to a normal state. The composition has an insulin sensitivity enhancing effect in a type 2 diabetes mellitus model, an excitatory toxicity and oxidative stress inhibiting effect in an epileptic seizure model, a cerebral infarction reducing and GS activity enhancing effect in a stroke model, a tyrosine denitration effect using human recombinant MnSOD, an acute renal failure effect, and a hyperammonemia effect. Therefore, the compound can be effectively used as a health-care functional food composition for preventing or alleviating diseases caused by tyrosine nitrification in proteins or as a pharmaceutical composition for preventing or treating diseases caused by tyrosine nitrification in proteins.

Description

[0001] Cross - reference to related applications

[0002] This application is a divisional application of a Chinese patent application with an application date of June 30, 2021, a Chinese patent application number of 202180047217.6, and an invention title of "Composition for preventing, improving or treating diseases caused by tyrosine nitration in proteins, containing tyrosine as an active ingredient", and this application claims the priority of a Korean application with an application number of 10 - 2020 - 0080308. Technical field

[0003] The present invention relates to a composition for preventing, improving or treating diseases caused by tyrosine nitration in proteins, containing tyrosine as an active ingredient. Background art

[0004] In the presence of •NO or •NO - derived metabolites, excessive levels of reactive oxygen species in the body can lead to the formation of nitrate substances such as peroxynitrite. Therefore, when tyrosine in the amino acids contained in proteins is nitrated, the structure and function of the proteins may change. Thus, tyrosine nitration may be an inducer of various diseases.

[0005] For example, glutamine synthetase (GS) is an enzyme that synthesizes glutamine from glutamate and ammonia in the body, is produced in various organs of the body, and plays a role in the nitrogen balance of organs and the whole body. When glutamine synthetase is exposed to reactive nitrogen due to reasons such as chronic stress, tyrosine is nitrated and the activity of glutamine synthetase also decreases, thereby causing various diseases.

[0006] Recently, a gene therapy based on overexpression of glutamine synthetase in skeletal muscle has been proposed for the treatment of acute hyperammonemia. The basic principle of this therapy is to increase the removal of ammonia from muscle by glutamine synthetase by replacing or enhancing glutamine synthetase that is usually lacking in the muscles of liver disease patients.

[0007] On the other hand, Korean Patent Publication No. 2020 - 0018488 discloses the use of glutamine synthetase in the treatment of hyperammonemia, and Korean Patent Publication No. 2020 - 0038481 discloses a composition for treating stress - related diseases, but the composition of the present invention for preventing, improving or treating diseases caused by tyrosine nitration in proteins, containing tyrosine as an active ingredient, has not been disclosed. Summary of the invention

[0008] Technical problem

[0009] The present invention is proposed in view of the above-mentioned needs, and provides a composition containing tyrosine as an active ingredient for preventing, improving or treating diseases caused by tyrosine nitration in proteins. It has been confirmed that tyrosine as an active ingredient of the present invention not only reduces the N-tyr content in glutamine synthetase produced by stress, but also enhances the activity of glutamine synthetase with reduced activity, and restores the contents of glutamine, glutamate and ammonia in the brain to the normal state. Moreover, it has been confirmed for the insulin sensitivity enhancement effect in the type 2 diabetes model, the excitotoxicity and oxidative stress inhibition effects in the seizure model, the reduction of cerebral infarction and the enhancement of GS activity in the stroke model, the denitrification effect of tyrosine using human recombinant MnSOD, the effect in acute renal failure, and the effect on hyperammonemia, thus completing the present invention.

[0010] Solution to the problem

[0011] In order to achieve the above object, the present invention provides a health functional food composition containing tyrosine or a food pharmaceutically acceptable salt thereof as an active ingredient for preventing or improving diseases caused by tyrosine nitration in proteins.

[0012] Moreover, the present invention provides a pharmaceutical composition containing tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating diseases caused by tyrosine nitration in proteins.

[0013] Moreover, the present invention provides a method for removing nitro groups from nitrated tyrosine by treating a protein containing nitrated tyrosine with tyrosine.

[0014] Effects of the invention

[0015] The present invention relates to a composition containing tyrosine as an active ingredient for preventing, improving or treating diseases caused by tyrosine nitration in proteins. It has been confirmed that the expression level of glutamine synthetase does not change due to stress, but the N-tyr content in GS significantly increases statistically, thus reducing the activity of GS. And in cell-based experiments, it can not only enhance the GS activity reduced by free tyrosine, but also has the effect of restoring the contents (ratios) of glutamine and glutamate and the ammonia amount in the brain to the normal state, and has the insulin sensitivity enhancement effect in the type 2 diabetes model, the excitotoxicity and oxidative stress inhibition effects in the seizure model, the reduction of cerebral infarction and the enhancement of GS activity in the stroke model, the denitrification effect of tyrosine using human recombinant MnSOD, the effect in acute renal failure, and the effect on hyperammonemia. Description of the drawings

[0016] Figure 1Figure showing the effects produced by chronic immobilization stress (CIS). Part (A) shows the results of confirming the changes in body weight and food intake during the experiment. Part (B) shows the results of confirming the changes in corticosterone levels in plasma and the levels of ROS / RNS in plasma and the prefrontal cortex (PFC). Part (C) shows that the GS level did not change, but the N-Tyr level in the stress group increased significantly statistically, and the GS activity decreased significantly statistically. Part (D) shows the results of confirming the GS activity in vitro after treating the PFC tissue lysate with peroxynitrite and tyrosine. *, **, *** indicate significant statistical differences compared with CTL, * for p < 0.05, ** for p < 0.01, *** for p < 0.001. # indicates that the GS activity in the group treated with tyrosine increased significantly statistically compared with the group not treated with tyrosine, p < 0.05.

[0017] Figure 2 Results of confirming the body weight and feed intake of the chronic immobilization stress (CIS) induction group (STR) on a tyrosine diet (TD). Part A shows the schedules of the normal diet group (ND) and the tyrosine diet group (TD). Part B shows the results of confirming the body weight changes. Part C shows the results of confirming the feed intake.

[0018] Figure 3 Figure showing the depressive behaviors of the chronic immobilization stress (CIS) induction group (STR) on a tyrosine diet (TD). Part A shows the results of the open field test. Part B shows the results of the tail suspension test. Part C shows the results of the sucrose preference test.

[0019] Figure 4 Results of confirming the corticosterone levels in plasma (A) and the changes in ROS / RNS in plasma and the PFC of the chronic immobilization stress (CIS) induction group (STR) on a tyrosine diet (TD) by dichlorofluorescin (DCF), showing that the stress hormone and oxidative stress were reduced by TD (B, C).

[0020] Figure 5 Results of confirming the GS activity (A), the expression level of GS (B), and the nitration level of tyrosine contained in GS (C) in the PFC of the chronic immobilization stress (CIS) induction group (STR) on a tyrosine diet (TD).

[0021] Figure 6To confirm the results of the contents of glutamate, glutamine, tyrosine, and GABA in the tyrosine diet (TD) chronic immobilization stress (CIS) induction group (STR) in the prefrontal cortex (PFC).

[0022] Figure 7 To confirm the results of the ammonia content in the tyrosine diet (TD) chronic immobilization stress (CIS) induction group (STR) in the prefrontal cortex (PFC).

[0023] Figure 8 To induce protein nitration by treating mouse PFC proteins with ONOO - and to confirm the results of the nitration inhibition effect produced by tyrosine.

[0024] Figure 9 Figure for confirming the nitration inhibition effect of the peptide of the present invention. Part A shows the Western blot results for nitrotyrosine protein in the prefrontal cortex (PFC) tissue, Part B shows the Western blot results for nitrotyrosine protein in the liver tissue, Part C shows the Western blot results for glutamine synthetase protein in the prefrontal cortex (PFC) tissue, and Part D shows the Western blot results for glutamine synthetase, insulin receptor β, and phosphorylated insulin receptor β proteins in the liver tissue.

[0025] Figure 10 Results for confirming the body weight change (A), blood glucose (B), and feed intake (C) caused by L-tyrosine diet in type 2 diabetic model mice induced by high-fat diet over time. *** indicates that the blood glucose content in the high-fat diet group (HFD) is significantly increased statistically compared with the normal diet group (ND), p < 0.001, #, ## indicate that the blood glucose content in the L-tyrosine diet-containing group (HFD + L-Tyr) is significantly decreased statistically compared with the high-fat diet group (HFD), # is p < 0.05, and ## is p < 0.01.

[0026] Figure 11 Results for confirming the glucose tolerance of mice caused by L-tyrosine diet in type 2 diabetic model mice induced by high-fat diet. Part (A) shows the blood glucose content changing over time, and Part (B) shows the area under the curve (AUC) in Part (A). **, *** indicate that the blood glucose content in the high-fat diet group (HFD) is significantly increased statistically compared with the normal diet group (ND), ** is p < 0.01, *** is p < 0.001, and # indicates that the blood glucose content in the L-tyrosine diet-containing group (HFD + L-Tyr) is significantly decreased statistically compared with the high-fat diet group (HFD), p < 0.05.

[0027] Figure 12To confirm the results of insulin resistance induced by L-tyrosine diet in type 2 diabetes model mice induced by high-fat diet. Part (A) shows the blood glucose content over time, and part (B) shows the area under the curve (AUC) in part (A). *, **, *** indicate that compared with the normal diet group (ND), the blood glucose content in the high-fat diet group (HFD) is significantly increased statistically, * is p < 0.05, ** is p < 0.01, *** is p < 0.001. # indicates that compared with the high-fat diet group (HFD), the blood glucose content in the L-tyrosine diet group (HFD + L-Tyr) is significantly decreased statistically, p < 0.05.

[0028] Figure 13 To confirm the results of changes in fat accumulation in tissues (A) and changes in ROS / RNS content in plasma (B). *** indicates that compared with ND, the tissue weight and ROS / RNS content in plasma in HFD or HFD + Tyr are significantly increased statistically, p < 0.001, #, ## indicate that compared with HFD, the tissue weight and ROS / RNS content in plasma in HFD + Tyr are significantly decreased statistically, # is p < 0.05, ## is p < 0.01.

[0029] Figure 14 To confirm the results of changes in seizure severity (A), body weight changes (C), and survival rate (D) induced by tyrosine diet. Part (B) shows the area under the curve (AUC) in part (A). **, *** indicate that compared with ND, the seizure level in 5x L-Tyr is significantly decreased statistically, ** is p < 0.01, *** is p < 0.001. # indicates that compared with before seizure in CTL, the body weight increases after seizure, p < 0.05.

[0030] Figure 15 To confirm the results of changes in ROS / RNS quantity (A) and GS activity in the hippocampal tissue of surviving mice after kainic acid injection. **, *** in part (A) indicate that compared with CTL, the content of ROS / RNS increases, ** is p < 0.01, *** is p < 0.001. ##, indicate that compared with the ND group fed a normal diet after KA injection, the content of ROS / RNS in the tyrosine diet group is significantly decreased statistically, ## is p < 0.01, is p < 0.001. * in part (B) indicates that compared with the ND group fed a normal diet after KA injection, the GS activity in the tyrosine diet group is significantly increased statistically, p < 0.05.

[0031] Figure 16Immunohistochemical staining results for analyzing the number of nerve cells in the hippocampal region (CA1 (A), CA3 (B)) and the activity of microglia in the same region by kainic acid. * indicates a decrease in the number of nerve cells or an increase in the activity of microglia inducing inflammation compared with CTL, p < 0.05, and # indicates a significant decrease in microglia activity in the tyrosine diet group, p < 0.05.

[0032] Figure 17 Results for confirming the preventive effect of D-tyrosine on cognitive impairment in a chronic restraint stress model mouse.

[0033] Figure 18 Results for confirming the reduction of brain infarct and the enhancement of GS activity by L-tyrosine in a stroke model mouse using endothelin-1.

[0034] Figure 19 Results for confirming the denitration effect of tyrosine using human recombinant MnSOD. *** indicates an increase in MnSOD activity in the tyrosine administration group (L-Tyr, D-Tyr) compared with the peroxynitrite (2.1 mM PN) group, p < 0.001.

[0035] Figure 20 Results for confirming the effect of tyrosine on acute renal failure. Part (A) shows the gene expression level of the antioxidant factor NQO-1, part (B) shows CuZnSOD activity, part (C) shows MnSOD activity, part (D) shows the ROS / RNS content, and part (E) shows the expression level of nitrotyrosine protein in the renal tissue 24 hours after renal ischemia and reperfusion.

[0036] Figure 21 Results for measuring plasma creatinine as an index for evaluating kidney injury. *, ** indicate that the expression levels of creatine (A), IL-1β (B), and IL-6 (C) are significantly increased statistically compared with the Sham group, * is p < 0.05, and ** is p < 0.01. #, ## indicate that the expression levels of creatine, IL-1β, and IL-6 are decreased compared with the CMC + IR group, # is p < 0.05, and ## is p < 0.01.

[0037] Figure 22 Results for confirming the effect of tyrosine on hepatic encephalopathy (hyperammonemia). Part (A) shows the ammonia content in the blood, part (B) shows ALT in the plasma, and part (C) shows the Western blot result for confirming the nitrated protein content. Detailed implementation mode

[0038] The present invention relates to a health functional food composition for preventing or improving diseases caused by tyrosine nitration in proteins, which contains tyrosine or a food-grade acceptable salt thereof as an active ingredient.

[0039] Preferably, the tyrosine nitration in the above-mentioned protein is tyrosine nitration in any one of proteins selected from tyrosine hydroxylase, Mn superoxide dismutase, Cu / Zn superoxide dismutase, insulin receptor β subunit, annexin IV, glutamate dehydrogenase, 3-α-hydroxy steroid dehydrogenase, glutathione S-transferase, 3-ketoacyl CoA thiolase, catalase, Tau protein, mitochondria complex 1, α-synuclein, apolipoprotein-A1, amyloid-β, and NMDA receptor, but is not limited thereto.

[0040] Preferably, the diseases caused by tyrosine nitration in the above-mentioned protein are any one of diseases selected from depressive disorder, anxiety disorder, stroke, epilepsy, glaucoma, diabetes, diabetic retinopathy, seizure, hepatic encephalopathy, cognitive impairment, brain development impairment, cancer, Alzheimer's disease, acute kidney injury, and hyperammonemia, but are not limited thereto.

[0041] Tyrosine nitration in the above-mentioned protein can occur in the brain, liver, muscle, adipose tissue, kidney tissue, pancreas or lungs, but is not limited thereto.

[0042] Preferably, tyrosine as the active ingredient of the present invention is administered in the doses shown in Table 1 below, but is not limited thereto.

[0043] Table 1

[0044] Doses and dietary periods for each disease

[0045]

[0046] The above-mentioned health functional food composition can be prepared from any one selected from pills, tablets, capsules, powders, granules, candies, syrups and beverages, or can be prepared by adding as a food ingredient, and can be appropriately prepared according to conventional methods.

[0047] An example of a food to which the active ingredient of the present invention can be added can be any form selected from meats, sausages, breads, chocolates, candies, pastries, cookies, pizzas, ramen, other noodles, chewing gums, dairy products including ice creams, various soups, beverages, teas, rehydration fluids, alcoholic beverages and vitamin complexes, including all health functional foods in the general sense.

[0048] The above-mentioned health functional food may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, coloring agents and enhancers (such as cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonating agents for carbonated beverages, etc. In addition, it may also contain the pulp for producing natural fruit juices and vegetable beverages. These ingredients can be used alone or in combination.

[0049] The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates, etc. as additional ingredients. The above-mentioned carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and 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, etc. can be used as sweeteners.

[0050] Furthermore, the present invention relates to a pharmaceutical composition for preventing or treating diseases caused by tyrosine nitration in proteins, which contains tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient.

[0051] In addition to the above-mentioned tyrosine, it may also contain pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, skin topical application or intraperitoneal, rectal, intravenous, intramuscular or subcutaneous injection is preferably selected, but it is not limited thereto.

[0052] The pharmaceutical composition of the present invention can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations are formulated by mixing one or more compounds with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. And, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspending agents, internal liquids, emulsions, syrups, etc. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. can also be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspending agents, emulsions, freeze-dried preparations, suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the matrix of suppositories, witepsol, polyethylene glycol, Tween 61, cocoa butter, laurel fat, glycerin, gelatin, etc. can be used.

[0053] The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level may depend on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, 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 conventional therapeutic agents, and can be administered once or multiple times. Considering all the above factors, it is important to administer in an amount that can achieve the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0054] Depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate and severity of the disease, the dose of the composition of the present invention can be used in a variety of ranges.

[0055] Moreover, the present invention relates to a method for treating a protein containing nitrated tyrosine with tyrosine to remove the nitro group from the nitrated tyrosine.

[0056] Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those skilled in the art that these examples are only for more specifically illustrating the present invention, and the scope of the present invention is not limited to these examples.

[0057] 1. Animal model

[0058] In the present invention, 28 male C57BL / 6 mice at 7 weeks of age (KOATECH, Korea) were housed under standard conditions (temperature 22 - 24 °C, humidity 50 - 70%, lights on at 6:00 am, 12-hour light-dark cycle), and at this time, the animals were allowed to freely access food and water. According to the guidelines of the National Institutes of Health (NIH, Bechesda, MD, USA), experiments on the animals used in the present invention were conducted according to the protocol (GNU-161128-M0068) approved by the Gyeongsang National University Institutional Animal Care and Use Committee (GNU IACUC).

[0059] 2. Preparation of plasma and prefrontal cortex (PFC) samples

[0060] Both brain blood and PFC samples were taken from mice anesthetized with CO 2 gas. Mouse blood was collected between 9:00 am and 11:00 am and collected in a vacuum container coated with K 3 EDTA. Plasma was obtained by centrifuging at 500 × g for 10 minutes at 4 °C. The PFC sample was weighed, and the tissue was lysed using a Bullet tissue lyser and centrifuged at 12,000 rpm for 20 minutes at 4 °C. The above-obtained plasma and tissue lysate were stored at -80 °C until use.

[0061] 3. Confirmation of corticosterone and ROS / RNS (reactive oxygen species / reactive nitrogen species)

[0062] 10 μg of the lysate was mixed in 50 ml of GS analysis buffer (50 mM imidazole-HCl, pH 6.8, 50 mM L-glutamine, 25 mM hydroxylamine, 25 mM sodium arsenate, 2 mM MnCl 2 and 0.16 mM ADP) and incubated at 37 °C for 1 hour.

[0063] After mixing, 50 μl of termination buffer (90 mM FeCl 3, The reaction was terminated with 1.8 N HCl and 1.45% trichloroacetic acid, and the γ-glutamylhydroxamate synthesized from the reaction of glutamine and hydroxylamine using GS was measured at 560 nm using a microplate reader.

[0064] The levels of GS and nitrated tyrosine GS were measured using Western blotting and immunoprecipitation (IP)-WB, respectively. Immunoprecipitation analysis of nitrated tyrosine GS was performed using anti-3-nitrotyrosine (ab61392, Abcam, Cambridge, UK) and protein A / G+ agarose (Santa Cruz, Dallas, TX, USA) according to the manufacturer's protocol, and Western blotting of GS was performed using anti-GS (1:5000, Abcam).

[0065] Example 1. Analyze behavior according to chronic immobilization stress (CIS), confirm changes in glutamine synthetase (GS) activity, and the content of nitrated tyrosine in GS

[0066] The animal models were divided into two groups (normal group, stress group). The stress group was forced to move alone in the restraint device for 2 hours every day (from 2 to 4 pm) and given chronic immobilization stress for 15 days. The body weight changes and feed intake were measured every other day.

[0067] After that, the animals were sacrificed to confirm the changes in corticosterone content in the plasma and to confirm ROS / RNS in the plasma and PFC. The corticosterone content was measured using an ELISA kit (Cayman) according to the manufacturer's method, and ROS / RNS was analyzed using a ROS / RNS analysis kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's method.

[0068] The results are as Figure 1 shown. Compared with the control group (CTL) without stress, the body weight of the stress group (STR) with chronic stress tended to decrease between 7 and 15 days, but there was little difference in feed intake, and the corticosterone content and ROS / RNS also increased significantly statistically.

[0069] On the other hand, it was confirmed that compared with the control group, the expression level of glutamine synthetase (GS) in the stress group did not decrease, the level of tyrosine nitration in GS increased significantly statistically, and the GS activity decreased significantly statistically.

[0070] Furthermore, 0.0031 - 0.45 mg / ml of tyrosine was added to 10 μg of PFC (prefrontal cortex) tissue lysate and mixed, incubated on ice for 10 minutes, and peroxynitrite (PN) was mixed to make the volume 10 times that of the above tissue lysate, then incubated for 10 minutes. After that, GS activity was confirmed in vitro, and it was confirmed that GS activity increased with the increase in tyrosine concentration.

[0071] Example 2. Confirmation of changes in body weight and feed intake in the tyrosine diet (TD) chronic immobilization stress (CIS) - induced group (STR)

[0072] The animal models were divided into two groups (normal group, stress group). In the stress group, the animals were individually forced to move in a restraint device for 2 hours (from 2 to 4 pm) every day and given chronic immobilization stress for 15 days. The changes in body weight and feed intake were measured every other day.

[0073] The above - mentioned normal group and stress group were divided into a general diet group with a balanced diet and a diet group supplemented with 181.2 mg / kg of the tyrosine of the present invention for feeding.

[0074] The results were as Figure 2 shown, and it was confirmed that there was no difference in the change in body weight or feed intake whether tyrosine was in the diet or not.

[0075] After that, behavioral analyses including OFT (open field test), TST (tail suspension test), and SPT (sucrose preference test) were conducted.

[0076] The OFT (open field test) was conducted in a square box (60×60×20 cm). The mice were placed in the central area (30×30 cm) of the box and their movements were tracked for 5 minutes to measure the total moving distance, the duration in the central area, and the frequency of attempting to enter the central area.

[0077] In the TST (tail suspension test), the mice were individually suspended in a box with a horizontal bar, and their movements were tracked for 6 minutes to measure the duration of immobility.

[0078] In the SPT (sucrose preference test), 0.1 M of sucrose and water were provided for 48 hours to develop a habit, then 0.1 M of sucrose and water were not provided for 24 hours. After that, 0.1 M of sucrose and water were provided for 6 hours, and then 0.1 M of sucrose and water were provided after swapping positions for 3 hours.

[0079] After that, the consumption of sucrose and water was measured, and the ratio of the provided sucrose to the total consumption of sucrose preference was calculated.

[0080] The results are as Figure 3 shown. It was confirmed that in the open field test, compared with the normal group (CTL), the time spent in the central area, the moving distance, and the number of times moving to the center of the stress group (STR) decreased. In the stress group, compared with the normal diet group (ND), the time spent in the central area, the moving distance, and the number of times moving to the center of the tyrosine diet group (TD) increased.

[0081] Moreover, in the tail suspension test (TST), compared with the normal group (CTL), the immobility time of the normal diet group (ND) in the stress group increased significantly statistically (p < 0.05). In contrast, the immobility time of the tyrosine diet group decreased significantly statistically (p < 0.05).

[0082] Also, in the sucrose preference test, compared with the normal group, the sucrose preference of the stress group decreased significantly statistically (p < 0.001). Compared with the normal diet group of the stress group, the sucrose preference of the tyrosine diet group of the present invention increased significantly statistically (p < 0.01).

[0083] After that, the content of corticosterone in the plasma was confirmed after sacrificing the animals. The results are as Figure 4 shown. Stress increased the content of corticosterone, but compared with the normal diet group of the stress group, the content of corticosterone in the tyrosine diet group decreased significantly statistically (#, p < 0.05). The results of confirming ROS / RNS in the plasma and PFC tissue showed that compared with the normal diet group of the stress group, ROS / RNS in the tyrosine diet group decreased significantly statistically (**: p < 0.01, *: p < 0.05).

[0084] On the other hand, compared with the control group, the expression levels of glutamine synthetase (GS) in the normal diet group (ND) and the tyrosine diet group (TD) of the stress group were similar, but stress decreased the GS activity. However, in the stress group, compared with the normal diet group, the GS activity of the tyrosine diet group increased significantly statistically. The content of N-Tyr (nitrated tyrosine) in GS increased significantly statistically by stress, but compared with the normal diet stress group, the content of N-Tyr in the tyrosine diet group decreased significantly statistically ( Figure 5 )

[0085] Example 3. Confirm the contents of glutamate, glutamine, tyrosine, and GABA in the prefrontal cortex (PFC) of the chronic immobilization stress (CIS) induction group (STR) with tyrosine diet (TD)

[0086] Quantify the contents of amino acids (glutamate, glutamine, tyrosine) and GABA in PFC by liquid chromatography - mass spectrometry (LC - MS / MS).

[0087] First, lyse the PFC tissue and dilute the supernatant of the tissue with an internal standard substance (L - Glu - d5) and the mobile phase. Divide the diluted sample into 5 portions and inject them into the LC - MS / MS system (Agilent 6460). The chromatographic column used is a SeQuant ZIC ® -HILIC chromatographic column (2.1×100 mm, 3.5 μm, 100 Å), and perform a concentration gradient separation of the mobile phase using acetonitrile containing 0.1% formic acid.

[0088] Detect amino acids (m / z 148→84 for Glu, m / z 147→84 for Gln, m / z 104→87 for GABA, and m / z 153→88 for the internal standard substance) by multiple reaction monitoring detection

[0089] The results are as Figure 6 shown. It was confirmed that, compared with the normal group, the contents of glutamate, glutamine, tyrosine, and GABA in the PFC of the general diet group in the stress group decreased. In contrast, the contents of glutamate, glutamine, tyrosine, and GABA in the tyrosine diet group increased above the normal group level.

[0090] Example 4. Changes in ammonia content in the tyrosine diet (TD) chronic immobilization stress (CIS) - induced group (STR) in PFC

[0091] The weight of the PFC tissue was measured at temperatures below -20 °C to prevent tissue melting. Then, 100 μl of RIPA (proteinase inhibitor cocktail, including PIC) per 10 mg of tissue weight and an appropriate amount of glass beads were placed in a 1.5 ml tube, and the tissue was lysed twice using a bullet blender for 30 seconds each time. The tissue lysate was centrifuged (spin down), transferred to a new 1.5 ml tube, and the tissue was sonicated for 1 minute. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was transferred to a new 1.5 ml tube and stored at -80 °C until use. During the above process, all procedures were carried out at a temperature of 4 °C except for the measurement of the PFC tissue weight. The ammonia standard solution (provided at 10 μg / ml = 588 μM) in the ammonia assay kit (Ammonia assay kit: Sigma-Aldrich, Cat Num. AA0100) was diluted with water to make 0 (blank control), 0.2, 0.4, 0.6, 0.8, 1.0 μg / ml, and the PFC tissue lysate was diluted 2-fold with PBS buffer.

[0092] Add 10 μl of the sample diluted 2-fold with PBS buffer (corresponding to 5 μg of tissue) to 100 μl of the ammonia assay reagent provided in the above ammonia assay kit and incubate at room temperature for 5 minutes. At the same time, add 10 μl of the standard solution to 100 μl of the ammonia assay reagent and incubate at room temperature for 5 minutes. RIPA + PIC diluted 2-fold with PBS buffer was used as a buffer blank control to compensate for the interference of the PFC tissue lysis buffer (RIPA + PIC). After 5 minutes, the absorbance (A initial ) was measured at 340 nm using a microplate reader.

[0093] Add 10 μl of L-glutamate dehydrogenase and measure the absorbance (A final ) at 340 nm after incubating at room temperature for 5 minutes.

[0094] Obtain DA340nm (A final - A initial ). Under standard conditions, subtract the DA340nm of the blank control group, and in the case of the sample, subtract the DA340nm of the buffer blank control group.

[0095] Use the standard result values to obtain linear regression and use it to calculate the ammonia concentration of the sample.

[0096] The results are asFigure 7 As shown, it was confirmed that compared with the normal group, the ammonia content in the general diet group of the stress group increased. In contrast, the ammonia content in the tyrosine diet group of the present invention decreased to a level similar to that of the normal group.

[0097] Example 5. Analysis of in vitro nitration inhibition effect

[0098] Treat mouse PFC protein with ONOO - to induce protein nitration and confirm the nitration inhibition effect produced by tyrosine. The results are as Figure 8 shown. It was confirmed that protein nitration was inhibited in a tyrosine concentration-dependent manner, and both L-tyrosine and D-tyrosine showed similar levels of nitration inhibition effect.

[0099] Furthermore, the nitration inhibition effect of the tyrosine of the present invention was analyzed using mouse brain and liver tissue lysates. After collecting prefrontal cortex (PFC) and liver tissues from mice anesthetized with CO 2 gas, weighing them respectively, lysing the tissues using a tissue lyser, centrifuging at 12,000 rpm for 20 minutes at 4°C, and taking the supernatant, PFC and liver tissue lysates were obtained. Add peroxynitrite (PN) that induces protein nitration to the above tissue lysates, and add glutamine (Q), L-tyrosine ( L -Y), and D-tyrosine ( D -Y) at a concentration of 2 mM respectively, then vortex mix for 5 seconds, and then react on ice for 10 minutes. After that, Western blotting was performed using anti-nitrotyrosine antibody (1:1000), anti-glutamine synthetase antibody (1:5000), anti-insulin receptor β antibody (1:1000), or anti-phosphorylated insulin receptor β antibody (1:1000).

[0100] The results confirmed that the proteins with increased tyrosine nitration in PFC and liver tissues increased due to PN, and the nitration of multiple proteins other than glutamine synthetase (GS) and insulin receptor β subunit (IRb) was confirmed through the Western blotting results of multiple bands. It was confirmed that the nitration of the proteins with increased tyrosine nitration due to PN decreased due to treatment with peptides with tyrosine (Y) at the end ( Figure 9 part A of Figure 9 and Figure 9Part C of Figure 9 Part D of

[0101] Example 6. Confirmation of the effect of enhancing insulin sensitivity of L-tyrosine in type 2 diabetic model mice with a high-fat diet

[0102] (1)Construction of a type 2 diabetic model with a high-fat diet

[0103] After acclimating 27 3-week-old male C57BL / 6 mice to the breeding environment for 1 week, they were divided into 3 groups and provided with a normal diet (ND, normal diet; 10 kcal% fat, calorie 3.85 kcal / g), a high-fat diet (HFD, high fat diet; 60 kcal% fat, calorie 5.24 kcal / g), and a high-fat diet supplemented with L-tyrosine (L-Tyr) (HFD + L-Tyr; containing 181 mg of L-tyrosine / kg), allowing the animals to freely intake water and feed. The body weight and food intake were measured every 2 days for 3 months, and the blood glucose was measured every 1 week.

[0104] The results were as Figure 10 shown. It was confirmed that the body weight gradually increased, and on the same day, the body weights of the HFD and HFD + L-Tyr groups were higher than that of the ND, and the body weight changes of the HFD and HFD + L-Tyr groups showed similar levels. There was little difference in the feed intake among the groups, and there was no difference at different times. Compared with the normal diet group (ND), the blood glucose of the HFD group increased significantly statistically, and compared with the HFD group, the blood glucose of the HFD + L-Tyr group decreased significantly statistically.

[0105] (2)Glucose tolerance test

[0106] A 40% D-glucose solution was prepared by dissolving D-glucose in 1×PBS. After measuring the blood glucose of the mice fasted for 16 hours, the glucose solution was intraperitoneally injected according to formula (1), with 2 g of glucose intraperitoneally injected per 1 kg of body weight.

[0107] Formula (1):

[0108] Glucose injection volume (μl) = body weight (g) × 5

[0109] As an example, a 45 g mouse was injected with 225 μl of 40% glucose solution. After intraperitoneal injection of the glucose solution, the blood glucose was measured at 30, 60, 90, and 120 minutes respectively.

[0110] The results were as Figure 11As shown, compared with the normal diet group (ND), the blood glucose level in the high-fat diet group (HFD) increased significantly statistically. In contrast, the blood glucose level in the high-fat diet group supplemented with L-tyrosine (L-Tyr) of the present invention (HFD+L-Tyr) decreased.

[0111] (3) Insulin resistance test

[0112] A 0.15 U / ml solution was prepared by diluting insulin in physiological saline. After measuring the blood glucose of mice fasted for 6 hours, insulin (0.75 U / body weight (kg)) was injected intraperitoneally. After intraperitoneal injection of insulin, the blood glucose was measured at 15, 30, 60, and 90 minutes respectively.

[0113] Equation (2):

[0114] Insulin injection volume (μl) = body weight (g) × 5

[0115] The results are as Figure 12 As shown, compared with the normal diet group (ND), the blood glucose level in the high-fat diet group (HFD) increased significantly statistically. In contrast, the blood glucose level in the high-fat diet group supplemented with L-tyrosine (L-Tyr) of the present invention (HFD+L-Tyr) decreased.

[0116] (4) Confirm the changes in fat accumulation in tissues and the changes in the content of ROS / RNS in plasma

[0117] After anesthetizing the mice with Avertin, blood was collected from the heart, reacted with EDTA in a vacutainer, and then centrifuged at 1500×g and 4 °C for 15 minutes to separate the plasma. PBS and 4% paraformaldehyde (PFA) were perfused through the heart to fix the tissues, and each tissue was excised and stored in PFA.

[0118] After diluting the plasma 3-fold in PBS, the Oxiselect ROS / RNS assay kit (Cell Biolabs) was used and the concentration of reactive oxygen / nitrogen (ROS / RNS) in the plasma was measured according to the recommended experimental method.

[0119] The results confirmed that in the HFD animal model, tyrosine reduced the total accumulation of liver fat and decreased the amount of adipose tissue around the kidneys at a statistically different level. Compared with the normal diet group (ND), the ROS / RNS level in the plasma of the high-fat diet group (HFD) increased significantly statistically. In contrast, the ROS / RNS level in the plasma of the high-fat diet group supplemented with L-tyrosine (L-Tyr) of the present invention (HFD+L-Tyr) decreased significantly statistically ( Figure 13).

[0120] Example 7. Confirmation of the excitotoxicity and oxidative stress inhibitory effects of L-tyrosine in a mouse model of kainic acid-induced seizures

[0121] The inhibitory effect of tyrosine on the excitotoxicity of glutamate was confirmed using an animal model of kainic acid (KA)-induced seizures. Equal amounts of kainic acid were intraperitoneally injected into mice, and animals fed different amounts of tyrosine were continuously observed for 7 days to confirm the degree of seizures.

[0122] (1) Seizures

[0123] Four-week-old male ICR mice were provided with a normal diet or a diet containing L-tyrosine, allowing the animals to freely consume water and feed.

[0124] Normal diet (ND): AIN-93G

[0125] 1×L-Tyr: AIN-93G containing 181 mg / kg of L-Tyr

[0126] 3×L-Tyr: AIN-93G containing 543 mg / kg of L-Tyr

[0127] 5×L-Tyr: AIN-93G containing 905 mg / kg of L-Tyr

[0128] Kainic acid (KA, Abcam) was dissolved in physiological saline while heating in a water bath to prepare a 9 mg / ml kainic acid solution. The kainic acid solution (30 mg / kg) was intraperitoneally injected, and the seizure level and symptoms were measured for 2 hours and recorded. The seizure level was judged according to the criteria in Table 2 below, and the body weight was measured and compared 24 hours before and after the injection of kainic acid.

[0129] Furthermore, 24 hours after the injection of kainic acid, the live brain tissue was removed from the surviving mice, or the brain was removed after perfusion fixation of the tissue with PFA.

[0130] Table 2

[0131] Representative symptoms classified by seizure level

[0132]

[0133] As a result, it was confirmed that the seizure degree decreased differentially with the tyrosine content, and there was no change in body weight among the experimental groups ( Figure 14). Moreover, it was confirmed that, in the case of animals not ingesting tyrosine, there were individuals that experienced strong seizures caused by kainic acid and died within 120 minutes. However, the death phenomenon in mice improved with the intake of tyrosine, and all the mice fed with a diet containing 900 mg / kg (diet weight) of tyrosine survived.

[0134] (2) ROS / RNS measurement

[0135] The hippocampus in the brain tissue was placed in 100 μl of RIPA (containing protease / phosphatase inhibitor) buffer per 10 mg of tissue mass and pulverized for 1 minute using glass beads and a Bullet blender. Centrifugation was performed at 12,000×g and 4 °C for 15 minutes to separate the supernatant, which was diluted 10-fold in PBS for ROS / RNS measurement. The OxiSelect ROS / RNS Assay Kit (Cell Biolabs) was used, and the ROS / RNS concentration was measured according to the recommended experimental method.

[0136] As a result of analyzing the changes in the amount of ROS / RNS and GS activity in the hippocampal tissue of surviving mice after kainic acid injection, the amount of ROS / RNS increased with kainic acid, but decreased to the control group level in mice ingesting tyrosine ( Figure 15 ).

[0137] (3) Glutamine synthetase (GS) activity assay

[0138] 2 μl of hippocampal lysate was placed in a 96-well plate, and 50 mM imidazole-HCl buffer (pH 6.8) was added to adjust the volume to 50 μl. 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 MnCl 2 and 0.08 mM ADP) was added, and the reaction was carried out at 37 °C for 30 - 60 minutes.

[0139] After the reaction, γ-glutamylhydroxamate, used as a standard substance, was placed in an empty well until the concentration reached 0.391 - 25.0 mM. 100 μl of reaction termination solution (90 mM FeCl 3 3, 1.8 N HCl and 1.45% (w / v) trichloroacetic acid) was added to the samples and the standard substance, and the absorbance was measured at a wavelength of 560 nm.

[0140] The GS activity of each sample was obtained by comparison with the standard curve. The GS activity was expressed as the amount of γ-glutamyl hydroxamate produced as the end product, in units of μM / min / μg (μM / min / μg) protein.

[0141] The results are as Figure 15 shown. Kainic acid decreased the GS activity, but the GS activity in the mice that ingested tyrosine increased above the control group level.

[0142] (4) Immunohistochemistry (IHC)

[0143] The brain was removed by cardiac perfusion with PBS and PFA and fixed in PFA for more than 15 hours. It was washed 3 times with PBS for 10 minutes each, and brain tissue sections with a thickness of 40 μm were obtained using a vibratome.

[0144] At room temperature, the obtained brain sections were blocked in IHC buffer (PBS containing 0.3% Triton X-100) dissolved with 3% bovine serum albumin for 1 hour, and then reacted with primary antibodies (anti-NeuN (Millipore, MAB377, 1:500), anti-Iba1 (Wako, 019-19741, 1:200)) at 4 °C for more than 15 hours (IHC buffer containing 3% BSA). They were washed 3 times with IHC buffer for 10 minutes each, and then reacted with fluorescently labeled secondary antibodies (anti-Mouse IgG Alexa Fluor 488 (Invitrogen, 1:1000), anti-Rabbit IgG Alexa Fluor 594 (Invitrogen, 1:1000)) at room temperature for 2 hours (IHC buffer containing 3% BSA). They were washed 3 times with IHC buffer for 10 minutes each, and the sections were fixed onto silane-coated glass slides. An anti-fade solution containing DAPI was placed on the sections and covered with coverslips to dry. The signal density of the images taken with a fluorescence microscope was analyzed using the Image J analysis program.

[0145] The brain tissue of the seizure model induced by kainic acid was examined. The results confirmed that kainic acid decreased the number of neurons in the hippocampal region (CA1, CA3), increased the activity of microglia in the same region, and it was confirmed that when tyrosine was ingested, the number of neurons was maintained and the activity of microglia decreased due to the inflammatory response ( Figure 16 )

[0146] Example 8. Confirmation of the preventive effect of D-tyrosine on cognitive impairment in a chronic restraint stress model mouse

[0147] (1)Preparation of a chronic restraint stress model

[0148] After dividing 28 male C57BL / 6 mice (7 weeks old) into two groups, they were provided with a normal diet (AIN-93G) or a diet containing D-tyrosine (D-Tyr) (181 mg D-Tyr / kg AIN-93G) for 1 week. The animals were allowed free access to water and feed, and their body weights and food intakes were measured twice a week.

[0149] Starting from 8 weeks old, chronic immobilization stress (CIS) was applied for 2 hours every day under 200 lux illumination conditions for 2 weeks. It was divided into a control group (CTL), a D-Tyr intake control group (CTL+D-Tyr), a chronic stress group (STR), and a D-Tyr intake chronic stress group (STR+D-Tyr), with each group consisting of 7 animals.

[0150] (2)Cognitive function test

[0151] Two weeks after the end of CIS, a cognitive function test was conducted. In order to compare the cognitive functions of the D-Tyr intake group and the normal diet intake group during the entire test period including the adaptation period, an object recognition test (ORT) and an object location recognition test (OLT) were conducted to test long-term memory ability.

[0152] ORT consists of three stages: habituation, familiarization, and test. The animals were acclimated to the test chamber for 10 minutes every day for two days. On the third day, the animals were allowed to explore two identical objects for 10 minutes during the familiarization stage. When the mice were placed in the test chamber, they were placed facing the wall without objects. In the test stage on the fourth day, one of the two objects was replaced with a new object, and the animals were allowed to explore for 10 minutes. The records of the first 5 minutes were used as data. The discrimination index (DI) of object cognitive function was calculated by the following formula (3).

[0153] Formula (3):

[0154] DI (Discrimination Index) = [exploration time of the new object - exploration time of the familiar object] / [exploration time of the new object + exploration time of the familiar object]

[0155] OLT: After 24 hours of ORT, the animal was made to change the position of a familiar object and explore it for 5 minutes. Similar to the above object recognition function index (DI), the object position recognition function index was calculated by calculating the ratio of the time spent exploring the moved object to the total object exploration time.

[0156] To confirm the cognitive function protective effect of tyrosine using a chronic stress-induced mild cognitive impairment animal model, while providing mice with a diet supplemented with tyrosine, chronic stress (14 days) was imposed. As a result, it was confirmed that there were no differences in body weight and feed intake with or without the inclusion of tyrosine, no statistically significant differences could be confirmed in ORT, and the cognitive function decreased by stress in OLT was maintained at the control group level due to the intake of tyrosine ( Figure 17 ).

[0157] Example 9. Confirmation of the effect of reducing brain infarct and oxidative stress of L-tyrosine in a stroke model mouse using endothelin-1

[0158] (1) Preparation of a stroke model mouse

[0159] 8-week-old male C57BL / 6 mice were provided with a normal diet or a diet containing L-tyrosine for more than 1 week, and the animals were allowed free access to water and feed.

[0160] Normal diet (ND): AIN-93G

[0161] L-Tyr: AIN-93G containing 905 mg / kg L-Tyr

[0162] (2) Preparation of endothelin-1 and stereotaxic surgery

[0163] Endothelin-1 was added to triple-distilled water (2 μg / μl), vortexed vigorously, and completely dissolved using ultrasound at 4°C for approximately 10 minutes.

[0164] After anesthetizing the mouse with the intraperitoneal injection anesthetic avertin (0.5 ml per 25 g of mouse weight), 2 μg of endothelin-1 was injected into the motor cortex region of the brain (centered on the bregma, AP 1.5 mm, ML -1.5 mm, DV 1.8 mm) through stereotaxic surgery. It was injected at a rate of 0.3 μl per minute. After injection, the needle was left in place for about 5 minutes to allow endothelin-1 to be fully absorbed into the tissue. After suturing the skin, the mouse was returned to its original cage for recovery.

[0165] (3)Tissue staining

[0166] Perfuse the heart with PBS and PFA, remove the brain and fix it in PFA for more than 15 hours. Wash it 3 times with PBS for 10 minutes each time, cut the brain tissue into slices with a thickness of 40 μm using a vibratome, and fix four slices each time on a silane-coated glass slide at the position where endothelin-1 is injected. Immerse the dried slices in a 0.1% cresyl violet solution for 5 minutes, then immerse them in triple-distilled water twice to wash away the staining solution. After dehydrating in 100% ethanol for 2 - 3 seconds, wash them twice with xylene for 5 seconds each time. After applying the mounting medium, cover with a coverslip and let it dry. Observe the stained section samples under an optical microscope, and use the Image J program to calculate the area of the infarction site from the stored images.

[0167] (4)Glutamine synthetase (GS) activity assay

[0168] Put 2 μl of brain motor cortex lysate in a 96-well plate, and adjust the volume to 50 μl by adding 50 mM imidazole-HCl buffer (pH 6.8). Add 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 MnCl 2 and 0.08 mM ADP), and react at 37 °C for 30 - 60 minutes.

[0169] After the reaction, put γ-glutamylhydroxamate (0.391 - 25.0 mM) as a standard substance in an empty well. Add 100 μl of reaction termination solution (90 mM FeCl 3 , 1.8 N HCl and 1.45% trichloroacetic acid) to the samples and the standard substance, and measure the absorbance at a wavelength of 560 nm. Obtain the GS activity of each sample by comparing with the standard curve. The GS activity is expressed as the production amount of γ-glutamylhydroxamate as the end product, with the unit of μM / min / μg protein.

[0170] The experimental results of inducing vascular stroke by injecting endothelin-1 into the motor cortex of mice showed that, compared with the control group administered saline, the infarct volume of the experimental group administered endothelin increased sharply, while the infarct volume of the experimental group that ingested tyrosine at a dose of 900 mg / kg (feed weight) decreased. It was also confirmed that the GS activity of the tissue decreased due to endothelin-1 compared with the control group, but the tyr diet maintained the GS activity ( Figure 18 ).

[0171] Example 10. Confirmation of the denitration effect of tyrosine using human recombinant MnSOD

[0172] 1) Quantification of peroxynitrite

[0173] Peroxynitrite was diluted 40-fold in 0.3 M NaOH and the absorbance was measured at 302 nm. The molar absorptivity (ε 302 = 1670 M - 1 cm -1 ) of peroxynitrite was used for quantification.

[0174] 2) Determination of SOD activity

[0175] The SOD activity was determined using a SOD colorimetric reaction activity kit from Thermofisher. Human recombinant MnSOD, peptide, and peroxynitrite within the standard SOD mixing range were reacted on ice for 10 minutes. The standard SOD or 10 μl of the reaction mixture and 50 μl of the substrate solution provided in the kit were added to a 96-well plate, and the absorbance was measured at 450 nm. 25 μl of xanthine oxidase provided in the kit was added, and after reacting at room temperature for 20 minutes, the absorbance was measured at 450 nm. The SOD activity was measured using the data obtained by subtracting the absorbance measured for the first time from the absorbance measured for the second time.

[0176] As a result, the SOD activity was decreased by reactive nitrogen in vivo. It was confirmed that although peroxynitrite decreased the activity of MnSOD, the SOD activity was maintained in the added Tyr (L-, D-form) ( Figure 19 )

[0177] Example 11. Confirmation of the effect of Tyr on acute renal failure

[0178] (1)Animal experiment

[0179] Male C57BL / 6 mice (KOATECH) weighing 23 - 25 g were allowed to freely access water and feed in a sterile breeding room with constant temperature and humidity, and were divided into three experimental groups: 1) control group (Sham), 2) group induced by compounding solvent-treated renal ischemia and reperfusion (CMC + IR), and 3) group induced by L-tyrosine-treated renal ischemia and reperfusion (L-Tyr + IR).

[0180] L-tyrosine (100 mg / kg) was suspended in 0.5% carboxymethyl cellulose (CMC). L-tyrosine or the compounding solvent CMC was orally administered once a day for 4 days. Thirty minutes after oral administration on the 4th day, renal ischemia was induced. Renal ischemia was induced by incising the abdomen and clamping the bilateral renal pedicles with a Muller atraumatic vascular clamp. After 25 minutes of ischemia, the clamp was removed and reperfusion was performed. In the control group (Sham), all surgical procedures were performed in the same manner except for inducing ischemia with forceps. Twenty-four hours after reperfusion, the experimental animals were sacrificed, blood was collected from the heart, and tissues were removed.

[0181] (2)Plasma creatinine

[0182] The collected blood was centrifuged at 3000 rpm for 15 minutes to separate plasma, and plasma creatinine was measured by the Jaffe method. In the Jaffe method, the amount of creatinine was calculated from the difference between the absorbance measured by reacting the plasma sample with picric acid at a wavelength of 510 nm and the absorbance measured after terminating the reaction with 60% acetic acid, and was expressed in mg / dl units.

[0183] (3)Real-time quantitative polymerase chain reaction

[0184] Total RNA was extracted from renal tissues by the Trizol method, and cDNA was synthesized using the Revert Aid reverse transcription system (Thermofisher). Quantitative PCR was performed on a CFX Connect Real-Time PCR system (Bio-Rad) using iQ SYBR Green Super mix (Bio-Rad). GAPDH was used as a housekeeping gene. The primers used in the experiment are shown in Table 3.

[0185] Table 3

[0186]

[0187] (4)Western blot

[0188] Kidney tissues were homogenized in RIPA buffer and centrifuged at 16,000×g for 15 minutes at 4°C. The supernatant was collected and the protein was quantified by the BCA method. After SDS-PAGE electrophoresis, it was transferred onto a PVDF membrane and blocked with 5% skim milk powder. Subsequently, after reacting with primary antibodies against nitrotyrosine and β-actin and the corresponding secondary antibodies, analysis was performed on a ChemiDoc XRS ＋ system (Bio-Rad).

[0189] Oxidative stress caused by ROS (Reactive oxygen species) or RNS (reactive nitrogen species) is the main mechanism by which renal ischemia and reperfusion injury lead to acute renal failure. The increase in antioxidant factors is of great significance for inhibiting renal ischemia and reperfusion injury. The results of measuring the gene expression of the antioxidant factor NQO-1 in kidney tissues 24 hours after renal ischemia and reperfusion showed that compared with the group administered with the vehicle, the administration of L-tyrosine significantly increased the NQO-1 gene expression (#, p < 0.01). It was confirmed that the content of ROS / RNS in mitochondria was also increased by about 6-fold or more by IR, but was inhibited by Tyr. It was confirmed that the activities of MnSOD and Cu / Zn SOD were decreased by IR, but were restored by Tyr. 24 hours after renal ischemia and reperfusion, compared with the sham group, the protein of nitrotyrosine in kidney tissues increased, and this increase was significantly reduced by the administration of L-tyrosine ( Figure 20 ).

[0190] Moreover, as shown in 21, the results of measuring plasma creatinine, which is an index for evaluating kidney injury, showed that 24 hours after renal ischemia and reperfusion, compared with sham, plasma creatinine increased by about 5.9-fold, and the administration of L-tyrosine significantly inhibited this increase statistically. To confirm that the inflammatory response is the main pathogenesis of acute renal failure after renal ischemia and reperfusion injury, the gene expressions of inflammatory cytokines (IL-1β and IL-6) in kidney tissues were measured. The results showed that 24 hours after renal ischemia and reperfusion, compared with the sham group, the gene expressions of IL-1β and IL-6 increased, and the administration of L-tyrosine significantly inhibited this increase statistically.

[0191] Example 12. Efficacy experiment of Tyr on hepatic encephalopathy (hyperammonemia) using azoxymethane

[0192] (1)Animal experiment

[0193] Thirteen-week-old male C57BL / 6 mice (KOATECH) were freely provided with water and feed in a sterile breeding room with constant temperature and humidity, and were divided into three experimental groups: (1) control group (Control), (2) carboxymethyl cellulose + azoxymethane treatment group (CMC + AOM), and (3) L-tyrosine + AOM treatment group (L-Tyr + AOM). L-tyrosine (100 mg / kg) was suspended in 0.5% carboxymethyl cellulose (CMC). L-tyrosine or the formulated solvent CMC was orally administered once a day for 4 days. Two hours after the oral administration on the 4th day, AOM (100 mg / kg, 100 ml) was intraperitoneally injected. Twelve hours after the AOM administration, 200 ml of physiological saline containing 0.5% glucose was intraperitoneally injected to prevent dehydration. Four hours later, the experimental animals were sacrificed, blood was collected from the heart, and liver tissues were removed.

[0194] (2) Blood ammonia analysis

[0195] The amount of ammonia in the blood was measured using a PocketChem BA PA-4140 analyzer (Arkray, Japan) for single-wavelength reflectance measurement. 20 μl of blood was placed on a test strip, reacted at room temperature for 3 minutes, inserted into the analyzer, and the absorbance was measured at a wavelength of 635 nm (LED). The results were expressed in μg / dl units.

[0196] (3) Plasma ALT analysis

[0197] Alanine aminotransferase (ALT) is a blood index of liver cell damage. The collected blood was centrifuged at 3000 rpm for 15 minutes to separate the plasma, and the ChemiLab GPT assay kit (IVD-LAB, Korea) was used for measurement. The absorbance was measured at a wavelength of 340 nm in a spectrophotometer according to the standard measurement method of the kit. The results were expressed in U / L units.

[0198] (4) Western blot

[0199] The liver tissues were homogenized in RIPA buffer and centrifuged at 16000×g at 4 °C for 15 minutes. The supernatant was collected and the amount of protein was quantified by the BCA method. After SDS-PAGE electrophoresis, it was transferred to a PVDF membrane and blocked with 5% skim milk powder. Then, after reacting with primary antibodies against nitrotyrosine and β-actin and the corresponding secondary antibodies, it was detected by the ECL method on a ChemiDoc XRS＋ Analysis was performed on a system (Bio-Rad).

[0200] Ammonia is a major metabolite of amino acids and nucleic acids. The main enzymes of the urea cycle that convert ammonia into urea are only present in hepatocytes. GS and SOD that reduce ammonia concentration and catalase that removes reactive oxygen species are also abundant in liver tissue. Therefore, the change in blood ammonia concentration in the blood vessels passing through the liver and ascending to the brain is an important indicator of hepatic encephalopathy. As a result of measuring blood ammonia in an acute hepatic encephalopathy model induced by AOM, compared with the normal control group, the blood ammonia concentration in the CMC + AOM treatment group increased by 6.7 times. Administration of L-tyrosine significantly inhibited the ammonia increase caused by AOM statistically. Damage to hepatocytes leads to the release of ALT present in the cytoplasm into the blood, resulting in an increase in its concentration. Therefore, measuring the activity of this enzyme is a direct indicator of the degree of liver damage. As a result of measuring plasma ALT in an acute hepatic encephalopathy model induced by AOM, compared with the control group, plasma ALT in the CMC + AOM treatment group increased by approximately 65 times, and administration of L-tyrosine significantly inhibited the increase in plasma ALT induced by AOM statistically. Sixteen hours after AOM administration, compared with the control group, the nitrotyrosine protein in liver tissue increased, and administration of L-tyrosine significantly reduced this increase. This result demonstrates that Tyr inhibits the nitration of proteins present in the liver to remove ammonia flowing into the liver ( Figure 22 )

[0201] Statistical analysis

[0202] All data of the present invention are expressed as mean ± SEM and were statistically analyzed by one-way ANOVA using Dunnetts Multiple Comparison Test or student t-test using GraphPad Prism5 (GraphPad Software) (p < 0.05).

Claims

1. Use of a composition comprising D-tyrosine or a salt thereof as the sole active ingredient in the preparation of a medicament for preventing or treating a disease caused by tyrosine nitration in a protein, wherein, tyrosine nitration in the above-mentioned protein is tyrosine nitration in any one of the proteins selected from glutamine synthetase, tyrosine hydroxylase, manganese superoxide dismutase, copper / zinc superoxide dismutase, insulin receptor beta subunit, annexin IV, glutamate dehydrogenase, 3-α-hydroxysteroid dehydrogenase, glutathione S-transferase, 3-ketothiolase, catalase, Tau protein, mitochondrial complex 1, α-synuclein, apolipoprotein-A1, amyloid-β protein and NMDA receptor, and the above-mentioned tyrosine or its salt is the removal of a nitro group from nitrated tyrosine.

2. The use according to claim 1, characterized in that, the disease caused by tyrosine nitration in the above-mentioned protein is any one selected from depression, anxiety, stroke, epilepsy, cognitive impairment, glaucoma, diabetes, diabetic retinopathy, convulsion, hepatic encephalopathy, brain development disorder, cancer, Alzheimer's disease, acute kidney injury and hyperammonemia.

3. The use according to claim 1, characterized in that, the disease caused by tyrosine nitration in the above-mentioned protein occurs in the brain, liver, muscle, adipose tissue, kidney tissue, pancreas or lung.

4. The use according to claim 1, characterized in that, the disease caused by tyrosine nitration in the above-mentioned protein is depression, and based on mice, the dose of D-tyrosine is 14 - 140 mg / kg / day.

5. The use according to claim 1, characterized in that, the disease caused by tyrosine nitration in the above-mentioned protein is any one selected from stroke, epilepsy and cognitive impairment. When the above-mentioned disease is stroke, based on mice, the dose of D-tyrosine is 80 - 140 mg / kg / day. When the above-mentioned diseases are epilepsy and cognitive dysfunction, based on mice, the dose of D-tyrosine is 14 - 140 mg / kg / day.

6. The use according to claim 1, characterized in that, the composition further comprises a carrier, excipient or diluent in addition to D-tyrosine.

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