Composition for preventing, ameliorating, or treating disease caused by nitration of tyrosine in proteins comprising tyrosine as effective component

Abstract

To provide a composition for preventing, ameliorating, or treating a disease caused by nitration of tyrosine in proteins comprising tyrosine as an effective component.SOLUTION: Tyrosine as an effective component of the present invention not only can enhance the activity of GS having reduced activity and but also has an effect of restoring the amount (i.e., ratio) of glutamine and glutamic acid in brain and the amount of ammonia to normal level, an effect of enhancing the insulin sensitivity in a model of type 2 diabetes, an effect of suppressing the excitotoxicity and oxidative stress in a model of epileptic seizure, an effect of reducing cerebral infarction and enhancing the activity of GS in a model of brain stroke, an effect of eliminating the nitration of tyrosine using human recombinant MnSOD, and an effect for acute renal failure and hyperammonemia. Therefore, the tyrosine as an effective component of the present invention can be efficiently used as a functional health food composition for preventing or ameliorating a disease caused by nitration of tyrosine in proteins or a pharmaceutical composition for preventing or treating a disease caused by nitration of tyrosine in proteins.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition containing tyrosine as an active ingredient for preventing, ameliorating or treating diseases caused by the nitration of tyrosine in proteins. [Background technology]

[0002] In vivo, excessive levels of reactive oxygen species in the presence of ·NO or ·NO-derived metabolites lead to the formation of nitrifying species such as nitrogen peroxide. Therefore, tyrosine nitration, which is one of the amino acids contained in proteins, can lead to structural and functional changes in the protein. Therefore, tyrosine nitration can also be a cause of various diseases. For example, glutamine synthetase (GS) is an enzyme that synthesizes glutamine using glutamic acid and ammonia in the body, and is produced in many organs of the body to play a role in the nitrogen balance of the organs and the whole body. If glutamine synthetase is exposed to reactive nitrogen due to late-growing stress or other reasons, tyrosine is nitrated, which reduces the activity of glutamine synthetase and can cause various diseases.

[0003] Recently, gene therapy based on overexpression of glutamine synthetase in skeletal muscle has been proposed for the treatment of acute hyperammonemia, with the aim of replacing or enhancing glutamine synthetase, which is commonly deficient in the muscles of patients with liver disease, thereby increasing the removal of ammonia by glutamine synthetase in the muscles.

[0004] Meanwhile, Korean Patent Publication No. 2020-0018488 discloses the use of glutamine synthetase for treating hyperammonemia, and Korean Patent Publication No. 2020-0038481 discloses a composition for treating stress-related disorders, but does not disclose a composition for preventing, improving or treating diseases caused by the nitration of tyrosine in proteins, which contains tyrosine of the present invention as an active ingredient. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been derived in response to the above-mentioned requirements, and provides a composition for preventing, improving or treating diseases caused by the nitration of tyrosine in proteins, which contains tyrosine as an active ingredient. It has been confirmed that tyrosine, the 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 whose activity has been reduced, thereby restoring the contents of glutamine, glutamic acid and ammonia in the brain to a steady state. The present invention has been completed by confirming the following: enhancement of insulin sensitivity in a type 2 diabetes model; suppression of excitotoxicity and oxidative stress in an epilepsy seizure model; reduction of cerebral infarction and enhancement of GS activity in a stroke model; elimination of tyrosine nitration using human recombinant MnSOD; effect on acute renal failure and effect on hyperammonemia. [Means for solving the problem]

[0006] In order to achieve the above object, the present invention provides a functional health food composition for preventing or improving diseases caused by nitration of tyrosine in proteins, comprising tyrosine or a food-based acceptable salt thereof as an active ingredient.

[0007] The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by nitration of tyrosine in a protein, comprising tyrosine or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0008] The present invention also provides a method for treating a protein containing tyrosine nitration with tyrosine to remove the nitro group from the nitrated tyrosine. Effect of the Invention

[0009] The present invention relates to a composition for preventing, improving or treating diseases caused by the nitration of tyrosine in proteins, containing tyrosine as an active ingredient. It has been confirmed that the expression level of glutamine synthetase does not change due to stress, but the content of N-tyr in GS increases statistically significantly, thereby decreasing GS activity. In cell-based experiments, it not only increases the GS activity decreased by free tyrosine, but also has the effect of restoring the content (ratio) of glutamine and glutamic acid and the amount of ammonia in the brain to a steady state, increases insulin sensitivity in a type 2 diabetes model, inhibits excitotoxicity and oxidative stress in an epilepsy seizure model, reduces cerebral infarction and increases GS activity in a stroke model, has the effect of removing tyrosine by nitration using human recombinant MnSOD, has an effect on acute renal failure and hyperammonemia. [Brief description of the drawings]

[0010] [Figure 1] The effect of late immobility stress (CIS) was confirmed. (A) shows the change in body weight and food intake during the experiment. (B) shows the change in plasma corticosterone content and ROS / RNS content in plasma and PFC (prefrontal cortex). (C) shows that GS content did not change, but N-tyr content in the stress group increased significantly and GS activity decreased significantly. (D) shows the in vitro GS activity after treating PFC tissue homogenate with nitric oxide and tyrosine. *, **, *** show statistically significant difference compared to CTL. * shows p<0.05, ** shows p<0.01, and *** shows p<0.001. # shows that GS activity in the tyrosine-treated group increased significantly compared to the non-tyrosine-treated group, p<0.05. [Diagram 2]The results of checking the body weight and food intake of the late immobility stress (CIS) induced group (STR) on a tyrosine diet (TD). (A) shows the schedule of the normal diet group (ND) and the tyrosine diet group (TD), (B) shows the results of checking the body weight change, and (C) shows the results of checking the food intake. [Diagram 3] The depressive behavior of the late immobility stress (CIS) induced group (STR) fed a tyrosine diet (TD) was confirmed. (A) shows the results of an open field test, (B) shows the results of a tail suspension test, and (C) shows the results of a sucrose preference test. [Figure 4] The results show that the tyrosine diet (TD) reduced stress hormones and oxidative stress by measuring the corticosterone content in plasma (A) and the changes in ROS / RNS in plasma and PFC in the late immobility stress (CIS) induced rats (STR) fed a tyrosine diet (TD) using dichlorofluorescin (DCF) (B,C). [Diagram 5] The results show that GS activity (A), GS expression level (B), and tyrosine nitration level (C) in GS were confirmed in the PFC of a late-developing immobility stress (CIS)-induced group (STR) fed a tyrosine diet (TD). [Figure 6] The results were obtained by examining the glutamate, glutamine, tyrosine and GABA contents in the PFC of late immobility stress (CIS)-induced rats (STR) fed a tyrosine diet (TD). [Figure 7] This is the result of examining ammonia content in the PFC of late immobility stress (CIS) induced rats (STR) fed a tyrosine diet (TD). [Figure 8] Mouse PFC protein was treated with ONOO- to induce protein nitration, and the inhibitory effect of tyrosine on nitration was confirmed. [Figure 9]The nitration inhibitory effect of the peptide according to the present invention was confirmed. A is a Western blot result for nitrotyrosine protein in PFC (prefrontal cortex) tissue, B is a Western blot result for nitrotyrosine protein in liver tissue, C is a Western blot result for glutamine synthetase protein in PFC (prefrontal cortex) tissue, and D is a Western blot result for glutamine synthetase, insulin receptor β, and phosphorylated insulin receptor β proteins in liver tissue. [Figure 10] The results are as follows: body weight change (A), blood glucose (B), and food intake (C) for each period in a high-fat diet-induced type 2 diabetes model mouse model. *** indicates that blood glucose content in the high-fat diet group (HFD) was statistically significantly increased compared to the normal diet group (ND), p<0.001; # and ## indicate that blood glucose content in the L-tyrosine-containing diet group (HFD+L-Tyr) was statistically significantly decreased compared to the high-fat diet group (HFD), # indicates P<0.05, and ## indicates P<0.01. [Figure 11] The results of confirming glucose tolerance in mice fed an L-tyrosine diet in a type 2 diabetes model mouse induced by a high-fat diet. (A) shows the blood glucose content over time, and (B) shows the area under the curve (AUC) in (A). **, *** indicate that the blood glucose content of the high-fat diet group (HFD) was statistically significantly increased compared to the normal diet group (ND), where ** is P<0.01, *** is P<0.001, and # indicates that the blood glucose content of the L-tyrosine-containing diet group (HFD+L-Tyr) was statistically significantly decreased compared to the high-fat diet group (HFD), where p<0.05. [Figure 12]The results of confirming insulin resistance in mice fed an L-tyrosine diet in a type 2 diabetes model mouse induced by a high-fat diet. (A) shows blood glucose content over time, and (B) shows the area under the curve (AUC) in (A). *, **, *** indicate a statistically significant increase in blood glucose content in the high-fat diet group (HFD) compared to the general diet group (ND), with * being P<0.05, ** being P<0.01, and *** being P<0.001. # indicates a statistically significant decrease in blood glucose content in the L-tyrosine-containing diet group (HFD+L-Tyr) compared to the high-fat diet group (HFD), with p<0.05. [Figure 13] The results show changes in fat accumulation in tissues (A) and changes in ROS / RNS content in plasma (B). *** indicates that tissue weight and plasma ROS / RNS content in HFD or HFD+Tyr were significantly increased compared to ND, p<0.001, # and ## indicate that tissue weight and plasma ROS / RNS content in HFD+Tyr were significantly decreased compared to HFD, # indicates P<0.05, ## indicates P<0.01. [Figure 14] The results show that the change in seizure severity (A), body weight change (C) and survival rate (D) due to the tyrosine diet are confirmed, and (B) shows the area under the curve (AUC) in (A). **, *** indicate that the seizure severity of 5xL-Tyr was statistically significantly reduced compared to ND, ** is P<0.01, *** is P<0.001. # indicates that the body weight after seizure was increased compared to before seizure in CTL, p<0.05. [Figure 15]The results of analyzing the quantitative change in ROS / RNS (A) and GS activity in hippocampal tissues of mice that survived after injection of kainic acid. In (A), ** and *** indicate an increase in ROS / RNS content compared to CTL, with ** being P<0.01 and *** being P<0.001. ## and ### indicate a statistically significant decrease in ROS / RNS content in the tyrosine diet group compared to the ND group that received KA injection and a general diet, with ## being P<0.01 and ### being P<0.001. In (B), * indicates a statistically significant increase in GS activity in the tyrosine diet group compared to the ND group that received KA injection and a general diet, with p<0.05. [Figure 16] These are the results of histoimmunochemical staining of the number of neurons in the hippocampus (CA1(A), CA3(B)) and the activity of microglia in the same area after kainic acid. * indicates a decrease in the number of neurons or an increase in inflammation-inducing microglia activity compared to CTL, p<0.05, and # indicates a significant decrease in microglia activity in the tyrosine diet group, p<0.05. [Figure 17] This result confirmed the preventive effect of D-tyrosine on cognitive impairment in a late-developing physical restraint stress model mouse. [Figure 18] The results confirmed the effect of L-tyrosine in reducing brain infarct and enhancing GS activity in a stroke model mouse using endothelin-1. [Figure 19] The results confirm the denitration effect of tyrosine using human recombinant MnSOD. *** indicates that MnSOD activity was increased in the tyrosine administration group (L-Tyr, D-Tyr) compared to the nitric oxide (2.1 mM PN) group, p<0.001. [Figure 20] The results confirm the effect of tyrosine on acute renal failure, and show (A) gene expression level of NQO-1, an antioxidant factor, (B) CuZnSOD activity, (C) MnSOD activity, (D) ROS / RNS content, and (E) nitrotyrosine protein expression level in kidney tissue 24 hours after renal ischemia and reperfusion. [Figure 21] The results show that serum creatinine was measured as an index of kidney damage. * and ** indicate that the expression levels of creatine (A), IL-1β (B) and IL-6 (C) were significantly increased compared to the sham group, with * indicating P<0.05 and ** indicating P<0.01. # and ## indicate that the expression levels of creatine, IL-1β and IL-6 were decreased compared to the CMC+IR group, with # indicating P<0.05 and ## indicating P<0.01. [Figure 22] The effect of tyrosine on hepatic encephalopathy (hyperammonemia) was confirmed. (A) Western blot analysis confirmed blood ammonia content, (B) plasma ALT, and (C) nitrated protein content. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to a functional health food composition for preventing or improving diseases caused by nitration of tyrosine in proteins, which contains tyrosine or a food-logically acceptable salt thereof as an active ingredient.

[0012] Nitration of tyrosine in the protein is mediated by tyrosine hydroxylase, Mn superoxide dismutase, Cu / Zn superoxide dismutase, insulin receptor β subunit, annexin IV, glutamate dehydrogenase, 3-α-OH steroid dehydrogenase, glutathione S-transferase, 3-ketoacyl CoA thiolase, catalase, Tau protein, mitochondria complex 1, and other enzymes. The nitration of tyrosine in any one of proteins selected from the group consisting of α-synuclein, apolipoprotein-A1, amyloid-β, and NMDA receptor is preferred, but is not limited thereto.

[0013] The disease caused by tyrosine nitration in a protein is preferably any one selected from the group consisting of depressive disorder, anxiety disorder, stroke, epilepsy, glaucoma, diabetes, diabetic retinopathy, seizures, hepatic encephalopathy, cognitive impairment, brain development impairment, cancer, Alzheimer's disease, acute kidney injury, and hyperammonemia, but is not limited thereto.

[0014] The nitration of tyrosine in proteins occurs in, but is not limited to, the brain, liver, muscle, adipose tissue, kidney tissue, pancreas, or lung.

[0015] The amount of tyrosine, which is the active ingredient of the present invention, to be administered is preferably the amount shown in Table 1 below, but is not limited thereto.

[0016] [Table 1]

[0017] The health functional food composition may be prepared in the form of any one selected from pills, tablets, capsules, powders, powders, granules, candies, syrups and beverages, or may be added as an ingredient to food, and may be appropriately prepared by conventional methods.

[0018] Examples of foods to which the active ingredient of the present invention can be added include any one of meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages and vitamin complexes, and include any of the health functional foods in the usual sense.

[0019] The health functional foods may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. In addition, fruit pulp for the production of natural fruit juices and vegetable drinks may be included. Such ingredients may be used independently or in combination.

[0020] The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates are 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. As sweeteners, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.

[0021] The present invention also relates to a pharmaceutical composition for preventing or treating a disease caused by the nitration of tyrosine in a protein, which comprises tyrosine or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0022] In addition to the tyrosine, the pharmaceutical composition of the present invention may further include a pharma- ceutical acceptable carrier, excipient, or diluent. The pharmaceutical composition of the present invention may be administered orally or parenterally, and in parenteral administration, it is preferable to select, but is not limited to, topical application on the skin or intraperitoneal, rectal, intravenous, intramuscular, or subcutaneous injection.

[0023] The pharmaceutical composition of the present invention may be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. As bases for suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, gelatin, etc. may be used.

[0024] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is determined by factors including the type and severity of the patient's disease, the activity of the agent, sensitivity to the agent, administration time, administration route and excretion rate, treatment duration, co-administered drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. It is important to administer an amount that provides maximum effect at a minimum dose without side effects, taking into account all of the above factors, which can be easily determined by one of ordinary skill in the art.

[0025] The dosage of the composition of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0026] The present invention also relates to a method for treating a protein containing tyrosine nitration with tyrosine to remove the nitro group from the tyrosine nitration. EXAMPLES

[0027] The present invention will be described in more detail below using examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail and that the scope of the present invention is not limited thereto.

[0028] 1. Animal Model In the present invention, 28 7-week-old C57BL / 6 male mice (Korea Coretech) were raised under standard conditions (temperature 22-24°C, humidity 50-70%, 12-hour light-dark cycle with lights on at 6 a.m.) with free access to food and water. The animals used in the present invention were maintained in accordance with the protocol (GNU-161128-M0068) approved by the Kyungsang National University Animal Care and Use Committee (GNU IACUC) in accordance with the guidelines of the National Institutes of Health (NIH, Bechesda, MD, USA).

[0029] 2. Plasma and prefrontal cortex (PFC) sample preparation Brain blood and PFC samples were sampled from mice anesthetized with CO2 gas. Mouse blood was collected between 9:00 and 11:00 a.m. and collected in a vacuum container coated with K3EDTA. Plasma was obtained by centrifugation at 500 x g for 10 minutes at 4°C. PFC samples were weighed, disrupted using a Bullet tissue disrupter, and centrifuged at 12,000 rpm for 20 minutes at 4°C. The plasma and tissue lysate obtained were stored at -80°C until use.

[0030] 3. Confirmation of corticosterone and ROS / RNS (reactive oxygen species / reactive nitrogen species) 10 μg of the homogenate was mixed in 50 ml of GS assay buffer (50 mM imidazole-HCl, pH 6.8, 50 mM L-glutamine, 25 mM hydroxylamine, 25 mM sodium arsenate, 2 mM MnCl2, and 0.16 mM ADP) and incubated at 37° C. for 1 hour.

[0031] After mixing, 50 μl of stop buffer (90 mM FeCl3, 1.8 N HCl, and 1.45% trichloroacetic acid) was added to stop the reaction, and γ-glutamylhydroxamate synthesized from the glutamine and hydroxylamine reaction by GS was measured at 560 nm using a microplate reader.

[0032] Western blot and immunoprecipitation (IP)-WB were used to measure the levels of GS and Tyr-nitrification GS, respectively. Immunoprecipitation analysis of Tyr-nitrification GS was performed using anti-3-nitrotyrosine antibody (ab61392, Abcam, Cambridge, UK) and Protein A / G Plus Agarose (Santa Cruz, Dallas, TX, USA) according to the manufacturer's protocol, and Western blot of GS was performed using anti-GS (1:5000, Abcam).

[0033] Example 1. Behavioral analysis of chronic immobilization stress (CIS), changes in glutamine synthetase (GS) activity, and confirmation of nitration-tyrosine content in GS The animal model was divided into two groups (normal group and stress group). The stress group was individually forced into a restraining device for two hours each day (2:00-4:00 pm) for 15 days, during which they were subjected to late immobility stress. Body weight changes and food intake were measured every other day.

[0034] The animals were then sacrificed to determine changes in plasma corticosterone levels, and ROS / RNS levels in plasma and PFC were determined. Corticosterone levels were measured using an ELISA kit (Cayman) according to the manufacturer's method, and ROS / RNS levels were measured using a ROS / RNS assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's method.

[0035] As a result, as shown in Figure 1, the body weight of the stressed group (STR) subjected to late-growing stress tended to decrease for 7 to 15 days compared to the control group (CTL) that was not subjected to stress, but there was almost no difference in feed intake, and corticosterone content and ROS / RNS also increased statistically significantly.

[0036] On the other hand, the expression level of glutamine synthetase (GS) in the stress group did not decrease compared to the control group, and not only did the level of tyrosine nitration in GS increase statistically significantly, but GS activity also decreased statistically significantly.

[0037] In addition, 10 μg of PFC (prefrontal cortex) tissue homogenate was mixed with 0.0031 to 0.45 mg / ml of tyrosine and incubated on ice for 10 minutes. Nitric peroxide (PN) was then added to the tissue homogenate in an amount 10 times its volume, and the mixture was incubated for 10 minutes. The in vitro GS activity was then confirmed, and it was found that GS activity increased with increasing tyrosine concentration.

[0038] Example 2. Confirmation of changes in body weight and food intake in the late immobility stress (CIS) induced group (STR) fed a tyrosine diet (TD) The animal model was divided into two groups (normal group and stress group). The stress group was individually confined to a restraining device for two hours each day (2:00-4:00 pm) and subjected to late immobility stress for 15 days. Body weight changes and food intake were measured every other day.

[0039] The normal and stressed groups were again divided into a general diet group with balanced nutrition and a diet group supplemented with tyrosine (181.2 mg / kg) of the present invention, and fed.

[0040] As a result, as shown in FIG. 2, it was confirmed that there was no difference in body weight change or feed intake depending on the tyrosine diet.

[0041] Thereafter, behavioral analyses including the open field test (OFT), tail suspension test (TST) and sucrose preference test (SPT) were performed.

[0042] The OFT (open field test) was performed in a square box (60 × 60 × 20 cm). Mice were placed in the central area of ​​the box (30 × 30 cm). Their movements were tracked for 5 min, and the total distance traveled, duration in the central area, and frequency of attempts to enter the central area were measured.

[0043] In the TST (tail suspension test), mice were individually suspended in a box with a horizontal bar and their movements were tracked for 6 min to measure the duration of immobility.

[0044] In the SPT (sucrose preference test), 0.1 M sucrose and water were provided for 48 hours to induce habituation, after which 0.1 M sucrose and water were not provided for 24 hours, after which 0.1 M sucrose and water were provided for 6 hours, and the positions of 0.1 M sucrose and water were changed for 3 hours.

[0045] Sucrose and water consumption was then measured and calculated as the percentage of sucrose offered relative to total sucrose consumption.

[0046] As a result, as shown in Figure 3, in the open field test, the time spent in the central area, the distance moved, and the number of times moved to the center were reduced in the stressed group (STR) compared to the normal group (CTL), but among the stressed groups, the time spent in the central area, the distance moved, and the number of times moved to the center were increased in the tyrosine diet group (TD) compared to the normal diet group (ND).

[0047] In addition, in the tail suspension test (TST), the stressed group (ND) showed a statistically significant increase in the immobile time compared to the normal group (CTL) (p<0.05), whereas the tyrosine diet group showed a statistically significant decrease in the immobile time (p<0.05).

[0048] In addition, in the sucrose preference test, the sucrose preference of the stressed group was statistically significantly decreased compared to the normal group (p<0.001), but the sucrose preference of the tyrosine diet group of the present invention was statistically significantly increased compared to the general diet group of the stressed group (p<0.01).

[0049] The animals were then sacrificed and the corticosterone content in the plasma was examined. As a result, as shown in FIG. 4, it was confirmed that the corticosterone content increased due to stress, but the corticosterone content in the tyrosine diet group was statistically significantly decreased compared to the general diet group in the stress group (#, p<0.05). The ROS / RNS in the plasma and PFC tissue was confirmed to be statistically significantly decreased in the tyrosine diet group compared to the general diet group in the stress group (**: p<0.01, *: p<0.05).

[0050] On the other hand, compared to the control group, the expression levels of glutamine synthetase (GS) in both the normal diet (ND) and tyrosine diet (TD) stress groups were similar, but GS activity decreased due to stress. In the stress groups, GS activity was statistically significantly increased in the tyrosine diet group compared to the normal diet group, and the N-tyrosine (nitraion tyrosine) content in GS was statistically significantly increased due to stress, but the N-tyr content in the tyrosine diet group was statistically significantly decreased compared to the normal stress group (Figure 5).

[0051] Example 3. Confirmation of glutamate, glutamine, tyrosine and GABA contents in the PFC of late immobility stress (CIS)-induced mice (STR) fed a tyrosine diet (TD) The contents of amino acids (glutamate, glutamine, tyrosine) and GABA in the PFC were quantified by liquid chromatography-mass spectrometry (LC-MS / MS).

[0052] First, the PFC tissue was broken down, and the supernatant was taken and diluted with an internal standard (L-Glu-d5) and mobile phase. The diluted sample was dispensed into five portions and injected into an LC-MS / MS system (Agilent 6460). The column used was a SeQuant ZIC®-HILIC column (2.1×100 mm, 3.5 μm, 100 Å), and the mobile phase was acetonitrile containing 0.1% formic acid to provide a concentration gradient for separation.

[0053] Amino acids were detected using a multiple reaction monitoring detection method (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).

[0054] As a result, as shown in FIG. 6, it was confirmed that the glutamate, glutamine, tyrosine and GABA contents of the general diet group in the stress group in the PFC were decreased compared to the normal group, whereas the glutamate, glutamine, tyrosine and GABA contents of the tyrosine diet group were increased above the normal group levels.

[0055] Example 4. Changes in ammonia content in the PFC of late immobility stress (CIS)-induced rats (STR) fed a tyrosine diet (TD) The PFC tissue weight was measured at -20°C or lower to prevent the tissue from melting, and then 100 μl of RIPA (proteinase inhibitor cocktail, including PIC) and an appropriate number of glass beads per 10 mg of tissue weight were placed in a 1.5 ml tube, and the tissue was disrupted twice for 30 seconds using a bullet blender. The tissue fragments were spun down and transferred to a new 1.5 ml tube, and the tissue was disrupted using ultrasound 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 in the test. In the above process, all steps except for the measurement of the PFC tissue weight were carried out at 4°C. Ammonia standard solution (10 μg / ml = 588 μM provided) provided by the ammonia assay kit (Sigma-Aldrich, Cat Num. AA0100) was diluted with water to prepare 0 (blank control), 0.2, 0.4, 0.6, 0.8, and 1.0 μg / ml, and the PFC tissue homogenate was diluted 2-fold with PBS buffer.

[0056] 10 μl of sample (corresponding to 5 μg of tissue) diluted 2-fold with PBS buffer was added to 100 μl of ammonia assay reagent provided in the ammonia assay kit, and incubated at room temperature for 5 minutes. At the same time, 10 μl of standard solution was added to 100 μl of ammonia assay reagent, and incubated at room temperature for 5 minutes. To correct for interference caused by PFC tissue homogenate buffer (RIPA+PIC), RIPA+PIC diluted 2-fold with PBS buffer was used as a buffer blank control. After 5 minutes, the absorbance was measured at 340 nm using a microreader (A initial ).

[0057] 10 μl of L-glutamate dehydrogenase solution was added, incubated at room temperature for 5 minutes, and the absorbance was measured at 340 nm (A final ).

[0058] DA340nm(A final-A initial ) was determined, and the DA340nm of the blank control group was subtracted in the case of the standard, and the DA340nm of the buffer blank control group was subtracted in the case of the sample.

[0059] A linear regression was performed using the standard results and used to calculate the ammonia concentration of the samples.

[0060] As a result, as shown in FIG. 7, the stress group fed the general diet had an increased ammonia content compared to the normal group, whereas the group fed the tyrosine diet of the present invention had a decreased ammonia content at a similar level to that of the normal group.

[0061] Example 5. In vitro nitration inhibition effect analysis

[0062] ONOO in mouse PFC proteins - The protein nitration was induced by treatment with tyrosine, and the inhibitory effect of tyrosine on the nitration was confirmed. As a result, as shown in Figure 8, it was confirmed that the protein nitration was inhibited in a tyrosine concentration-dependent manner, and the inhibitory effect of nitration was shown to be at a similar level for both L-tyrosine and D-tyrosine.

[0063] In addition, the inhibitory effect of tyrosine on nitration according to the present invention was analyzed using mouse brain and liver tissue lysates. Prefrontal cortex (PFC) and liver tissues were collected from mice anesthetized with CO2 gas, weighed, and then disrupted using a tissue disrupter. After centrifugation at 12,000 rpm at 4°C for 20 minutes, the supernatant was removed to obtain PFC and liver tissue lysates. Peroxynitrite (PN), which induces protein nitration, was added to the tissue lysates, and glutamine (Q), L-tyrosine (LY), and D-tyrosine (DY) were added at a concentration of 2 mM each, and the mixture was mixed by vortexing for 5 seconds and incubated on ice for 10 minutes. Thereafter, Western blotting was performed using anti-nitrotyrosine antibody (1:1,000), anti-glutamine synthetase antibody (1:5,000), anti-insulin receptor β antibody (1:1,000) or anti-phosphorylated insulin receptor β antibody (1:1,000).

[0064] As a result, we confirmed that PN increased the number of proteins with increased tyrosine nitration in the PFC and liver tissues, and confirmed that the nitration of several proteins other than glutamine synthetase (GS) and insulin receptor β subunit (IRb) was increased by Western blot of several bands. We confirmed that the nitration of proteins with increased tyrosine nitration by PN was decreased by treatment with a peptide with a terminal tyrosine (Y) (Figures 9A and 9B). We confirmed that there was no difference in protein amount between each treatment group in the PFC and liver tissues by expression of GS and IRb. However, IRb was nitrated by PN and the phosphorylated form was decreased, as confirmed by Western blot of phosphorylated-IRb (IRb-p) (Figures 9C and 9D).

[0065] Example 6. Confirmation of the insulin sensitivity enhancing effect of L-tyrosine in high-fat diet type 2 diabetes model mice (1) Establishment of a high-fat diet-induced type 2 diabetes model Twenty-seven male C57BL / 6 mice aged 3 weeks were acclimated to the breeding environment for 1 week, and then divided into three groups and provided with a normal diet (ND, normal diet; 10 kcal% fat, 3.85 kcal / g), a high fat diet (HFD, high fat diet; 60 kcal% fat, 5.24 kcal / g), or a high fat diet supplemented with L-tyrosine (HFD+L-Tyr; containing 181 mg L-tyrosine / kg). Water and food were available ad libitum. Body weight and food intake were measured every 2 days, and blood glucose was measured every week for 3 months.

[0066] As a result, as shown in Figure 10, body weight gradually increased, and it was confirmed that the body weights of the HFD and HFD+L-Tyr groups were heavier than that of the ND on the same date, and the body weight changes of the HFD and HFD+L-Tyr groups were at similar levels. There was almost no difference in feed intake between the groups, and no difference occurred depending on the period. Regarding blood glucose, blood glucose of the HFD group was statistically significantly increased compared to the normal diet group (ND), and blood glucose of the HFD+L-Tyr group was statistically significantly decreased compared to the HFD group.

[0067] (2) Glucose tolerance test D-glucose was dissolved in 1x PBS to prepare a 40% D-glucose solution. After measuring the blood glucose of mice that had been fasting for 16 hours, the glucose solution was intraperitoneally injected according to Equation 1 to intraperitoneally inject 2 g of glucose per kg of body weight. (Formula 1) Glucose injection volume (μl) = body weight (g) x 5

[0068] As an example, a mouse weighing 45 g was injected with 225 μl of 40% glucose solution. After intraperitoneal injection of the glucose solution, blood glucose was measured at 30, 60, 90 and 120 minutes.

[0069] As a result, as shown in FIG. 11, the blood glucose content of the high-fat diet group (HFD) was statistically significantly increased compared to the normal diet group (ND), whereas the blood glucose content of the L-tyrosine (L-Tyr)-supplemented high-fat diet group (HFD+L-Tyr) of the present invention was decreased.

[0070] (3) Insulin resistance test Insulin was diluted with saline to prepare a 0.15 U / ml solution. After fasting for 6 hours, the blood glucose of the mice was measured, and insulin was intraperitoneally injected (0.75 U / body weight (kg)). After the intraperitoneal injection of insulin, blood glucose was measured at 15, 30, 60, and 90 minutes. (Formula 2) Amount of insulin injected (μl) = Body weight (g) x 5

[0071] As a result, as shown in FIG. 12, the blood glucose content of the high-fat diet group (HFD) was statistically significantly increased compared to the normal diet group (ND), whereas the blood glucose content of the L-tyrosine (L-Tyr)-supplemented high-fat diet group (HFD+L-Tyr) of the present invention was decreased.

[0072] (4) Confirmation of changes in fat accumulation in tissues and ROS / RNS content in plasma Mice were anesthetized with Avertin, and blood was collected via the heart and reacted with EDTA in a vacutainer, then centrifuged at 1,500 x g for 15 minutes at 4°C to separate plasma. Tissues were fixed by perfusion with PBS and 4% paraformaldehyde (PFA) via the heart, and each tissue was excised and stored in PFA.

[0073] Plasma reactive oxygen / nitrogen (ROS / RNS) concentrations were measured after diluting plasma 3-fold with PBS using the Oxiselect ROS / RNS Assay Kit (Cell Biolabs) according to the recommended experimental procedure.

[0074] As a result, it was confirmed that tyrosine reduced overall hepatic fat accumulation in the HFD animal model, and the amount of perirenal adipose tissue was statistically significantly reduced. Compared to the normal diet group (ND), the high-fat diet group (HFD) had a statistically significant increase in plasma ROS / RNS levels, whereas the L-tyrosine (L-Tyr)-supplemented high-fat diet group (HFD+L-Tyr) of the present invention had a statistically significant decrease in plasma ROS / RNS levels (Figure 13).

[0075] Example 7. Confirmation of the inhibitory effect of L-tyrosine on excitotoxicity and oxidative stress in epilepsy model mice in which seizures were induced with kainic acid The effect of tyrosine on the suppression of excitotoxicity in epilepsy animals was confirmed using a kainic acid (KA)-induced seizure model. Mice were given different amounts of tyrosine for 7 days, and the same amount of kainic acid was injected intraperitoneally to observe the severity of seizures.

[0076] (1) Epileptic seizures Four-week-old ICR male mice were fed a normal diet or an L-tyrosine-containing diet for one week, with free access to water and food. Normal diet (ND):AIN-93G 1×L-Tyr:AIN-93G containing 181 mg / kg L-Tyr 3×L-Tyr:AIN-93G containing 543 mg / kg L-Tyr 5×L-Tyr:AIN-93G containing 905 mg / kg L-Tyr Kainic acid (KA, Abcam) was dissolved in saline while heating in a water bath to prepare a 9mg / ml kainic acid solution, and the kainic acid solution was intraperitoneally injected (30mg / kg), and the severity of seizures and symptoms were recorded for 2 hours. The severity of seizures was judged according to the criteria in Table 2 below, and the body weight was measured before and 24 hours after the kainic acid injection and compared.

[0077] In addition, kainic acid was injected into the surviving mice, and 24 hours later, the live brain tissue was either removed or fixed by PFA perfusion and then the brain was removed.

[0078] [Table 2]

[0079] As a result, it was confirmed that the severity of seizures was differentially alleviated depending on the tyrosine content, and no change in body weight was observed between the experimental groups (Figure 14).In addition, in the case of animals that did not consume tyrosine, the severe seizure phenomenon caused by kainic acid could not be sustained for more than 120 minutes, and some individuals died, but it was confirmed that the phenomenon of mice dying was also ameliorated depending on the amount of tyrosine consumed, and all mice that consumed food containing 900 mg / kg (food weight) of tyrosine survived.

[0080] (2) ROS / RNS measurement From the brain tissue, the hippocampus was added with 100μl of RIPA buffer (containing proteinase / phosphatase inhibitors) per 10mg of tissue mass, and crushed for 1 minute using glass beads and a Bullet Blender. The tissue was centrifuged at 12,000×g and 4℃ for 15 minutes, and the supernatant was separated and diluted 10-fold with PBS for use in ROS / RNS measurement. ROS / RNS concentrations were measured using the Oxiselect ROS / RNS Assay Kit (Cell Biolabs) according to the recommended experimental method.

[0081] Analysis of the quantitative changes in ROS / RNS and GS activity in hippocampal tissue from surviving mice injected with kainic acid showed that the amount of ROS / RNS was increased by kainic acid, but decreased to the control level in mice fed tyrosine (Figure 15).

[0082] (3) Glutamine synthetase (GS) activity measurement 2 μl of hippocampal lysate was added to a 96-well plate, and 50 mM imidazole-HCl buffer (pH 6.8) was added to make the total volume 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 MnCl2, and 0.08 mM ADP) was added, and the mixture was reacted at 37°C for 30 to 60 minutes.

[0083] After the reaction was completed, the standard substance γ-glutamylhydroxamate was added to the empty wells to a concentration of 0.391 to 25.0 mM. 100 μl of reaction stop solution (90 mM FeCl3, 1.8 N HCl, and 1.45% (w / V) trichloroacetic acid) was added to the samples and standard substance, and the absorbance was measured at a wavelength of 560 nm.

[0084] The GS activity of each sample was determined by comparison with a standard curve. The GS activity was expressed as the amount of the final product, γ-glutamylhydroxamic acid, and the unit was μM / min / μg protein.

[0085] As a result, as shown in FIG. 15, GS activity was decreased by kainic acid, but increased above the control level in mice fed tyrosine.

[0086] (4) Tissue immunostaining (Immunohistochemistry, IHC) After transcardial perfusion with PBS and PFA, the brains were removed and post-fixed in PFA for >15 h, washed three times with PBS for 10 min, and brain tissue sections were obtained at 40 μm thickness using a vibratome.

[0087] The obtained brain sections were blocked in IHC buffer (PBS containing 0.3% Triton X-100) containing 3% bovine serum albumin for 1 hour at room temperature, 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). The sections were washed three times for 10 minutes each with IHC buffer, and reacted with fluorescently labeled secondary antibodies (anti-Mouse IgG Alexa Fluor 488 (Invitrogen, 1:1000), anti-Rabbit IgG AlexaFluor594 (Invitrogen, 1:000)) at room temperature for 2 hours (IHC buffer containing 3% BSA). After washing with IHC buffer three times for 10 min each, the sections were fixed on silane-coated glass slides. Anti-fade solution containing DAPI was placed on the sections, which were then covered with a cover slip and dried. The signal density of the images taken with a fluorescent microscope was analyzed using the Image J analysis program.

[0088] Examination of brain tissue from a kainic acid-induced seizure model revealed that kainic acid reduced the number of neurons in the hippocampus (CA1, CA3) and increased microglial activity in the same areas, whereas administration of tyrosine maintained the number of neurons and reduced microglial activity due to an inflammatory response (Figure 16).

[0089] Example 8. Confirmation of the preventive effect of D-tyrosine on cognitive impairment in late-developing physical restraint stress model mice (1) Preparation of the late-developing physical restraint stress model Twenty-eight male C57BL / 6 mice (7 weeks old) were divided into two groups and fed a normal diet (AIN-93G) or a diet containing D-tyrosine (D-Tyr) (181 mg D-Tyr / kg AIN-93G) for 1 week. Water and food were available ad libitum, and body weight and food intake were measured twice a week.

[0090] From the age of 8 weeks, the rats were subjected to chronic immobilization stress (CIS) for 2 weeks, 2 hours per day under 200 lux lighting conditions. They were divided into a control group (CTL), a D-Tyr-fed control group (CTL+D-Tyr), a chronic stress group (STR), and a D-Tyr-fed chronic stress group (STR+D-Tyr), with 7 animals per group.

[0091] (2) Cognitive function test After the 2-week CIS, cognitive function tests were conducted. To compare the cognitive functions of the group fed the D-Tyr-containing diet and the group fed the normal diet throughout the entire test period, including the adaptation period, an object recognition test (ORT) and an object location recognition test (OLT) were performed to test long-term memory ability.

[0092] ORT consisted of three stages: habituation, familiarization, and test. The mice were allowed to adapt to the test box for 10 minutes each day for two days, and during the familiarization stage on the third day, they were asked to explore the two identical objects for 10 minutes. When placing the mice in the test box, they were asked to look at the wall side where there were no objects. During the test stage on the fourth day, one of the two objects was replaced with a new object and the mice were asked to explore for 10 minutes, with the first 5 minutes of recording being used as data. The object recognition function index (DI) was calculated using the following formula 3. (Formula 3) DI (Description Index) = [time spent exploring novel objects - time spent exploring familiar objects] / [time spent exploring novel objects + time spent exploring familiar objects]

[0093] OLT: 24 hours after the ORT, the location of a familiar object was changed and the subject was asked to explore it for 5 minutes. Using the same method as the object recognition index (DI) above, the proportion of time spent exploring the moved object out of the total object exploration time was calculated to calculate the object location recognition index.

[0094] To confirm the efficacy of tyrosine in protecting cognitive function using an animal model of late-developing stress-induced mild cognitive impairment, mice were provided with food supplemented with tyrosine and subjected to late-developing stress (14 days). As a result, no differences in body weight or food intake were observed depending on whether tyrosine was included or not, and no statistically significant differences were observed in the ORT. However, it was confirmed that cognitive function that had been reduced by stress in the OLT was able to be maintained at the control group level by supplementing with tyrosine (Figure 17).

[0095] Example 9: Confirmation of the effect of L-tyrosine in reducing brain infarct and oxidative stress in a stroke model mouse using endothelin-1 (1) Preparation of stroke model mice Eight-week-old C57BL / 6 male mice were fed a normal diet or an L-tyrosine-containing diet for at least one week, with water and food available ad libitum. Normal diet (ND):AIN-93G L-Tyr: AIN-93G containing 905 mg / kg L-Tyr

[0096] (2) Endothelin-1 preparation and stereotaxic surgery Endothelin-1 (2 μg / μl) was added to triple distilled water, vortexed vigorously, and then sonicated at 4° C. for about 10 minutes to completely dissolve.

[0097] Mice were anesthetized by intraperitoneal injection of the anesthetic Avertin (0.5ml per 25g mouse weight), and then 2μg of Endothelin-1 was injected into the motor cortex region of the cerebral cortex (AP 1.5mm, ML -1.5mm, DV 1.8mm, centered on the bregma) through stereotaxic surgery. The injection was performed at a rate of 0.3μl per minute, and the needle was left in place for about 5 minutes after the injection was completed to allow Endothelin-1 to be fully absorbed into the tissue. The skin was sutured and the mice were allowed to recover in their original cages.

[0098] (3) Tissue staining PBS and PFA were perfused through the heart, and the brains were removed and fixed in PFA for more than 15 hours. After washing with PBS three times for 10 minutes, brain tissue sections were cut to a thickness of 40 μm using a vibratome, and four sections were fixed on silane-coated glass slides at the point where endothelin-1 was injected. The dried sections were immersed in 0.1% cresyl violet solution for 5 minutes, and then immersed twice in triple distilled water to wash off the staining solution. The sections were dehydrated by immersing in 100% ethanol for 2 to 3 seconds, and then washed twice in xylene for 5 seconds. Mount solution was applied, and the sections were covered with a cover glass and dried. The stained sections were observed under an optical microscope, and the area of ​​the cerebral infarction was calculated using the Image J program for the saved images.

[0099] (4) Glutamine synthetase (GS) activity measurement 2 μl of cerebral motor cortex lysate was added to a 96-well plate, and 50 mM imidazol-HCl buffer (pH 6.8) was added to make up 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 MnCl2 and 0.08 mM ADP) was added and incubated at 37° for 30-60 minutes.

[0100] After the reaction was completed, the standard substance γ-glutamylhydroxamate (0.391-25.0mM) was added to the empty wells. 100μl of reaction stop solution (90mM FeCl3, 1.8N HCl, and 1.45% trichloroacetic acid) was added to the samples and standard substance, and the absorbance was measured at 560nm wavelength. The GS activity of each sample was calculated by comparing with the standard curve. The GS activity was expressed as the amount of γ-glutamylhydroxamate produced, which is the final product, in μM / min / μg protein.

[0101] In an experiment in which endothelin-1 was injected into the motor cortex of the mouse brain to induce vascular stroke, the infarct volume increased sharply in the experimental group that received endothelin compared to the control group that received saline, and the infarct volume decreased in the experimental group that received tyrosine at a dose of 900mg / kg (food weight).In addition, it was confirmed that the GS activity in the tissue was reduced by endothelin-1 compared to the control group, but that GS activity was maintained by the tyrosine diet (Figure 18).

[0102] Example 10. Confirmation of the denitration effect of tyrosine using human recombinant MnSOD 1) Quantitative analysis of peroxynitrite Peroxynitrite was diluted 40 times with 0.3M NaOH and the absorbance was measured at 302 nm to obtain the molar absorption coefficient (ε 302 = 1670 M -1 cm -1 ) was used for quantification.

[0103] 2) SOD activity measurement SOD activity was measured using Thermofisher's SOD non-color reaction activity kit. Standard SOD was mixed with human recombinant MnSOD, peptides and peroxynitrite within the range and reacted on ice for 10 minutes. 10 μl of standard SOD or the reacted mixture and 50 μl of the substrate solution provided in the kit were placed in a 96-well plate and the absorbance was measured at 450 nm. 25 μl of xanthine oxidase provided in the kit was added and reacted at room temperature for 20 minutes, after which the absorbance was measured at 450 nm. SOD activity was measured using the data obtained by subtracting the primary measurement absorbance from the secondary measurement absorbance.

[0104] As a result, SOD activity was decreased by reactive nitrogen species in the body. MnSOD activity was decreased by peroxynitrite, but SOD activity was maintained in the added Tyr (L-, D-form) (Figure 19).

[0105] Example 11. Confirmation of the effect of Tyr on acute renal failure (1) Animal experiments Male C57BL / 6 mice (Coretec) weighing 23-25g were kept in a sterile breeding room where the temperature and humidity were kept constant and allowed to have free access to water and food. They were divided into three experimental groups: 1) control group (Sham), 2) solvent-treated renal ischemia and reperfusion-induced group (CMC+IR), and 3) L-tyrosine-treated renal ischemia and reperfusion-induced group (L-Tyr+IR).

[0106] L-tyrosine (100mg / kg) was suspended in 0.5% CMC (carboxymethyl cellulose). L-tyrosine or the preparation solvent CMC was orally administered once a day for 4 days, and renal ischemia was induced 30 minutes after oral administration on the 4th day. Renal ischemia was induced by opening the abdomen and clamping both renal pedicles with a Muller atraumatic vascular clamp. After 25 minutes of ischemia, the clamps were removed and reperfusion was performed. The control group (sham) underwent all the same surgical procedures except for the induction of ischemia through clamping. 24 hours after reperfusion, the experimental animals were sacrificed, blood was collected from the heart, and tissues were removed.

[0107] (2) Serum creatinine The collected blood was centrifuged at 3,000 rpm for 15 minutes to separate the plasma, and the blood creatinine was measured using the Jaffe method, which involved calculating the amount of creatinine in mg / dl from the difference in absorbance measured at 510 nm after reacting the plasma sample with picric acid and then terminating the reaction with 160% acetic acid.

[0108] (3) Quantitative real-time polymerase reaction Total RNA was extracted from kidney tissues using the Trizol method, and cDNA was synthesized using the Revert Aid reverse transcription system (Thermofisher). Quantitative PCR was performed using the CFX Connect Real-Time PCR system (Bio-Rad) with iQ SYBR Green Super mix (Bio-Rad). GAPDH was used as the housekeeping gene. The primers used in the experiment are listed in Table 3.

[0109] [Table 3]

[0110] (4) Western blot The kidney tissue was homogenized in RIPA buffer and centrifuged at 16,000×g at 4℃ for 15 minutes, and the supernatant was collected and protein was quantified by BCA method. After electrophoresis by SDS-PAGE, it was transferred to a PVDF membrane and blocked with 5% skim milk powder. Then, nitro-tyrosine and β-actin primary antibodies and corresponding secondary antibodies were reacted, and the protein was analyzed by ECL method using ChemiDoc XRS. + The results were analyzed using a Bio-Rad system.

[0111] Oxidative stress caused by reactive oxygen species (ROS) or reactive nitrogen species (RNS) is the main mechanism of acute renal failure due to renal ischemia and reperfusion injury, and an increase in antioxidant factors is important for suppressing renal ischemia and reperfusion injury. Gene expression of NQO-1, an antioxidant factor, was measured in renal tissue 24 hours after renal ischemia and reperfusion, and L-tyrosine administration statistically significantly increased NQO-1 gene expression compared to the vehicle-treated group (#, p<0.01). The content of ROS / RNS in mitochondria was also increased by IR by approximately 6 times, but was inhibited by Tyr. The activity of MnSOD and Cu / Zn SOD was decreased by IR, but was restored by Tyr. Nitrotyrosine protein was increased in renal tissue 24 hours after renal ischemia and reperfusion compared to the sham group, and such increase was significantly reduced by L-tyrosine administration (Figure 20).

[0112] As shown in FIG. 21, serum creatinine was measured as an index of kidney damage evaluation. Serum creatinine increased by about 5.9 times compared to the sham group 24 hours after renal ischemia and reperfusion, and such increase was statistically significantly suppressed by administration of L-tyrosine. Gene expression of inflammatory cytokines (IL-1b and IL-6) was measured in kidney tissue to confirm the inflammatory response, which is the main pathogenic mechanism of acute renal failure due to renal ischemia and reperfusion injury. Gene expression of IL-1b and IL-6 was increased 24 hours after renal ischemia and reperfusion compared to the sham group, and such increase was statistically significantly suppressed by administration of L-tyrosine.

[0113] Example 12. Efficacy experiment of Tyr against hepatic encephalopathy (hyperammonemia) using azoxymethan (1) Animal experiments Male 13-week-old C57BL / 6 mice (Coretech) were allowed to freely consume water and food in a sterile breeding room where temperature and humidity were kept constant. They were divided into three experimental groups: (1) control group, (2) carboxymethyl cellulose + azoxymethane treatment group (CMC + AOM), and (3) L-tyrosine + AOM treatment group (L-Tyr + AOM). L-tyrosine (100mg / kg) was suspended in 0.5% CMC (carboxymethyl cellulose). L-tyrosine or the preparation solvent CMC was orally administered once a day for four days, and AOM (100mg / kg, 100ml) was injected intraperitoneally two hours after oral administration on the fourth day. 12 hours after AOM administration, 200ml of saline containing 0.5% glucose was injected intraperitoneally to prevent dehydration. Four hours later, the experimental animals were sacrificed, blood was collected from the heart, and liver tissue was excised.

[0114] (2) Blood ammonia analysis Blood ammonia was measured using a PocketChem BA PA-4140 (Arkray, Japan), a single wavelength reflectometry analytical instrument. 20 μl of blood was dropped onto a test strip, reacted at room temperature for 3 minutes, inserted into the instrument, and absorbance was measured at 635 nm (LED) wavelength, and the results were expressed in μg / dl.

[0115] (3) Plasma ALT analysis ALT (alanine aminotransferase), a blood indicator of hepatocellular damage, was measured using a ChemiLab GPT assay kit (IVD-LAB, Korea) after separating plasma from collected blood by centrifugation at 3,000 rpm for 15 minutes. The absorbance was measured at 340 nm wavelength on a spectrophotometer according to the standard measurement method of the kit, and the results were expressed in U / L units.

[0116] (4) Western blot The liver tissue was homogenized in RIPA buffer and centrifuged at 16,000×g for 15 minutes at 4℃, and the supernatant was collected and the protein content was quantified by the BCA method. After electrophoresis on SDS-PAGE, the tissue was transferred to a PVDF membrane and blocked with 5% skim milk powder. After that, the tissue was reacted with nitro-tyrosine and β-actin primary antibodies and the corresponding secondary antibodies, and then the protein content was measured by the ECL method using ChemiDoc XRS. + The results were analyzed using a Bio-Rad system.

[0117] Ammonia is the main metabolite of amino acids and nucleic acids, and the main enzymes of the urea cycle that convert ammonia to urea are only present in hepatocytes. GS and SOD, which reduce ammonia concentration, and catalase, which removes active oxygen, are also abundant in liver tissue, so changes in blood ammonia concentration in blood vessels that go to the brain via the liver are important indicators of hepatic encephalopathy. In an acute hepatic encephalopathy model caused by AOM, blood ammonia concentration was increased 6.7 times in the CMC+AOM treatment group compared to the normal control group, and L-tyrosine administration statistically significantly suppressed the increase in blood ammonia caused by AOM. Damage to hepatic parenchymal cells induces the release of ALT present in the cytoplasm into the blood, increasing its concentration, so measuring the activity of this enzyme is a direct indicator of the extent of liver damage. In the AOM-induced acute hepatic encephalopathy model, plasma ALT was measured, and the CMC+AOM treatment group showed a 65-fold increase in plasma ALT compared to the control group, and L-tyrosine administration statistically significantly suppressed the AOM-induced increase in plasma ALT. In liver tissue 16 hours after AOM administration, nitrotyrosine protein increased compared to the control group, and this increase was significantly reduced by L-tyrosine administration, providing evidence that Tyr inhibits the nitration of proteins in the liver, enabling the removal of ammonia that has entered the liver (Figure 22).

[0118] (Statistical processing) All data herein are presented as mean ± SEM and were statistically analyzed by one-way analysis of variance (ANOVA) with Dunnetts Multiple Comparison Test or Student's t test (p<0.05) using GraphPad Prism 5 (GraphPad Software).

Claims

1. A functional health food composition for preventing or improving depression, anxiety disorder, epilepsy, or cognitive impairment, comprising D-tyrosine or a food-based acceptable salt thereof as an active ingredient.

2. A pharmaceutical composition for preventing or treating depressive disorder, anxiety disorder, epilepsy, or cognitive impairment, comprising D-tyrosine or a pharma- ceutical acceptable salt thereof as an active ingredient.

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