Screening method for nitrotyrosine reducing agents

Abstract

To provide an efficient screening method of nitrotyrosine reducing agents capable of directly degrading already-produced nitrated proteins.SOLUTION: The present invention includes the following preferred embodiments. [1] A screening method of a nitrotyrosine reducing agent uses a content of an aromatic hydroxycarboxylic acid and / or an aromatic hydroxyaldehyde in a material, as an index.EFFECT: There is provided is an efficient screening method of new agents that can effectively reduce nitrated proteins by directly degrading the already-produced nitrotyrosine. The screening method makes it possible to select an ameliorating agent for diseases such as arteriosclerosis and cerebral ischemic diseases, in which the nitrated proteins may be involved, an ameliorating agent for skin property change due to aging, such as decrease in skin elasticity and skin dullness, or candidate agents thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening for a nitrotyrosine-reducing agent. [Background technology]

[0002] Protein nitration is a type of post-translational modification of proteins caused by reactive nitrogen species generated in vivo. It is a reaction in which a nitro group is added to the benzene ring in the aromatic amino acid residues of tyrosine and tryptophan that make up proteins. The nitration reaction occurs when the benzene ring in the amino acid is converted to a nitronium ion (NO2 + ) or nitrogen dioxide radicals (Non-Patent Document 1). Because the content of tryptophan in many proteins present in living organisms is much lower than that of tyrosine, nitration of proteins is thought to occur mainly at tyrosine residues, resulting in the formation of nitrotyrosine in proteins (Non-Patent Document 2).

[0003] It is known that protein nitration affects cellular function by causing a decrease in the function of enzymes and tyrosine kinase receptors (Non-Patent Document 3). It has also been reported that nitrotyrosine in proteins accumulates in a variety of age-related diseases, such as arteriosclerosis and cerebral ischemic disease, and is involved in these diseases (Non-Patent Document 4). It has also been reported that nitrated proteins may be involved in neurodegenerative diseases, hypertension, asthma, rheumatoid arthritis, diabetic nephropathy, and inflammatory bowel disease (Non-Patent Documents 5-10). Furthermore, the present inventors have discovered that nitrated proteins are also involved in age-related changes in skin properties, such as loss of elasticity and dullness (Non-Patent Documents 11-12, Patent Documents 1-2).

[0004] To improve various diseases associated with the formation of nitrated proteins, methods have been disclosed for selecting active ingredients that eliminate peroxynitrite, which nitrates tyrosine residues in proteins (Patent Documents 3 and 4). However, these screening methods are solely for the purpose of identifying active ingredients that inhibit the formation of nitrated proteins, and are not for screening ingredients that are effective against nitrated proteins that have already formed. Furthermore, Goishicha (registered trademark) extract and hibiscus extract have been found to be effective as decomposers of nitrated proteins (Patent Documents 1 and 5). However, the active ingredients and commonalities among these materials that have the effect of decomposing nitrated proteins were unknown. Therefore, conventional screening for nitrated protein decomposers has required random screening, which required considerable effort, time, and expense for the preparation and testing of candidate materials. Therefore, an effective method for discovering agents that can decompose nitrated proteins was needed to fundamentally resolve various diseases associated with the formation of nitrated proteins. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Chem Res Toxicol.,22(5):894-898,2009 [Non-patent document 2] Front Chem.,3:70,2016 [Non-patent document 3] Diabetes,57(4):889-98,2008 [Non-patent document 4] Science,290(5493):985-9,2000 [Non-Patent Document 5] Int J Neurosci.,130(10):1047-1062,2020 [Non-patent document 6] JAMA.,289(13):1675-80,2003 [Non-Patent Document 7] Free Radic Res.,38(1):49-57,2004 [Non-patent document 8] Osteoarthritis Cartilage.,21(1):151-6,2013 [Non-Patent Document 9] Kidney Int.,57(5):1968-72,2000 [Non-Patent Document 10] J Pathol.,186(4):416-21,1998 [Non-Patent Document 11] The 31st IFSCC Congress 2020 Yokohama [Non-Patent Document 12] FRAGRANCE JOURNAL,vol.45 / No.8,2017 [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-178899 [Patent Document 2] Patent Publication No. 2021-124313 [Patent Document 3] Japanese Patent Application Laid-Open No. 2002-326922 [Patent Document 4] Patent Publication No. 2021-155362 [Patent Document 5] Patent Publication No. 2021-147335 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an efficient screening method for an excellent nitrotyrosine-reducing agent that can directly decompose an already produced nitrated protein. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above problems, the present inventors have found that aromatic hydroxycarboxylic acids and aromatic hydroxyaldehydes have a high nitrotyrosine-reducing effect. Therefore, it was determined that materials having a nitrotyrosine-reducing effect can be screened by using the content of aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes as an index, and this led to the completion of the present invention.

[0009] In addition, the present inventors have found that derivatives of aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes also have a high nitrotyrosine-reducing effect. However, because the structures of these derivatives are diverse, there are many compound structures that could serve as screening indicators, making it difficult to carry out measurements and compare the effects of different materials. However, the present inventors have solved this problem by subjecting the material to a process of hydrolyzing the material. Specifically, the present inventors determined that by converting the aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde derivatives contained in the material into aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes by hydrolysis, it would be possible to screen for nitrotyrosine-reducing agents using the aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes as indicators, and have thus completed the present invention.

[0010] That is, the present invention includes the following preferred embodiments. [1] The first invention is: A method for screening for nitrotyrosine reducers using the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in a material as an index. [2] The second invention is: A method for screening a nitrotyrosine reducing agent, comprising: A) A process of hydrolyzing the material, B) measuring aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes in the hydrolysate; C) a step of selecting a material having a high content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde; The method comprising: [3] The third invention is: The screening method according to [1] or [2], wherein the aromatic hydroxycarboxylic acid is dihydroxybenzoic acid and / or trihydroxybenzoic acid. [4] The fourth invention is: The screening method according to any one of [1] to [3], wherein the aromatic hydroxyaldehyde is dihydroxybenzaldehyde and / or trihydroxybenzaldehyde. [Effects of the Invention]

[0011] According to the present invention, by using the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in a material as an indicator, an efficient screening method for a new agent that can effectively reduce nitrated proteins by directly decomposing already produced nitrotyrosine is provided. The present invention makes it possible to select agents for improving diseases in which nitrated proteins may be involved, such as arteriosclerosis and cerebral ischemic disease, and agents for improving age-related changes in skin properties, such as loss of skin elasticity and dullness, or candidate agents for these. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. Note that the scope of the present invention is not limited to the embodiments described herein, and various modifications can be made without departing from the spirit of the present invention.

[0013] The present invention provides a method for evaluating and selecting new substances that can effectively reduce nitrated proteins by directly decomposing nitrotyrosine that has already been produced.

[0014] In the present invention, the use of the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde as an index means that the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde measured by any method is used as a criterion for assessing the effectiveness of the nitrotyrosine-reducing action.

[0015] The aromatic hydroxycarboxylic acid used as an indicator in the present invention is not particularly limited as long as it is a compound having at least one carboxy group and at least one hydroxy group bonded to a benzene ring, but is preferably a compound having one carboxy group and 1 to 5 hydroxy groups bonded to the benzene ring, and more preferably a compound having one carboxy group and 2 to 3 hydroxy groups bonded to the benzene ring.

[0016] Specific examples of aromatic hydroxycarboxylic acids include monohydroxybenzoic acids such as 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid, dihydroxybenzoic acids such as 2,3-dihydroxybenzoic acid (2-pyrocatechuic acid), 2,4-dihydroxybenzoic acid (β-resorcylic acid), 2,5-dihydroxybenzoic acid (gentisic acid), 2,6-dihydroxybenzoic acid (γ-resorcylic acid), 3,4-dihydroxybenzoic acid (protocatechuic acid), and 3,5-dihydroxybenzoic acid (α-resorcylic acid), and trihydroxybenzoic acids such as 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid (phloroglucinolcarboxylic acid), and 3,4,5-trihydroxybenzoic acid (gallic acid). Among these, one or more compounds selected from the group consisting of 2,5-dihydroxybenzoic acid (gentisic acid), 3,5-dihydroxybenzoic acid (α-resorcylic acid), and 3,4,5-trihydroxybenzoic acid (gallic acid) are preferred.

[0017] The aromatic hydroxyaldehyde used as an indicator in the present invention is not particularly limited as long as it is a compound having at least one aldehyde group and at least one hydroxy group bonded to a benzene ring, but is preferably a compound having one aldehyde group and 1 to 5 hydroxy groups bonded to the benzene ring, and more preferably a compound having one aldehyde group and 2 to 3 hydroxy groups bonded to the benzene ring.

[0018] Specific examples of aromatic hydroxyaldehydes include monohydroxybenzaldehydes such as 2-hydroxybenzaldehyde (salicylaldehyde), 3-hydroxybenzaldehyde, and 4-hydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde (β-resorcylaldehyde), 2,5-dihydroxybenzaldehyde (gentisinaldehyde), and 2,6-dihydroxybenzaldehyde. Examples of the dihydroxybenzaldehyde include dihydroxybenzaldehydes such as 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde) and 3,5-dihydroxybenzaldehyde (α-resorcyl aldehyde), and trihydroxybenzaldehydes such as 2,3,4-trihydroxybenzaldehyde, 2,4,5-trihydroxybenzaldehyde, 2,4,6-trihydroxybenzaldehyde and 3,4,5-trihydroxybenzaldehyde. Among these, one or more compounds selected from the group consisting of 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde) and 2,3,4-trihydroxybenzaldehyde are preferred.

[0019] In a first embodiment of the screening method of the present invention, in a system for measuring the amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde present in a material, a material in which the amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde present in the material is high can be selected as a nitrotyrosine reducer.

[0020] When measuring the amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in a material, the state of the material to be tested does not matter. For example, if the material is a solid, it is desirable to extract or solubilize the aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in the material by any method and then subject it to measurement.

[0021] In a second embodiment of the screening method of the present invention, a material is mixed with a hydrolysis reagent and subjected to a hydrolysis treatment step, and then the amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde present in the hydrolysis product is measured in a system in which a material present in a large amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in the material is selected as a nitrotyrosine reducer.

[0022] In the present invention, the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in a material is measured and used as an index for assessing effectiveness, but it has been confirmed that aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde also exist in the material in the form of derivatives, and that these derivatives also have the effect of reducing nitrotyrosine. Therefore, by hydrolyzing a candidate material to convert the aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde derivatives into aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde, it becomes possible to measure the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde present in the candidate material.

[0023] The amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde present in a material can be determined based on the desired level of effect, and it is sufficient that either one is present to an extent that can be detected using known detection equipment. As a rough guide, a significant effect can be expected if the amount is 0.000001 g, preferably 0.00001 g or more, and 0.0001 g or more per gram of dry weight. For example, although this varies slightly depending on the individual, it has been confirmed that eucalyptus leaves contain approximately 0.00003 g or more of gallic acid as an aromatic hydroxycarboxylic acid per gram of dry weight, and clove flower buds contain approximately 0.0002 g or more of gallic acid as an aromatic hydroxycarboxylic acid per gram of dry weight, confirming their high effectiveness in reducing nitrotyrosine.

[0024] The step of hydrolyzing the material in the present invention is not particularly limited, and can be carried out using known means such as hydrothermal treatment, acid or alkali treatment, or enzyme treatment.

[0025] In the hydrolysis step using hydrothermal treatment, the raw material is immersed in a water-containing solvent and then subjected to a reaction by heating or in a hydrolysis medium such as steam or pressurized steam. The water-containing solvent may contain an organic solvent as long as it does not affect the reaction. While there are no particular limitations on the organic solvent, examples include alcoholic solvents such as methanol, ethanol, ethylene glycol, and butylene glycol; etheric solvents such as tetrahydrofuran, ethyl ether, 1,4-dioxane, and methyl tert-butyl ether; nitrile solvents such as acetonitrile and propionitrile; esteric solvents such as ethyl acetate, n-propyl acetate, and isopropyl acetate; hydrocarbon solvents such as pentane, hexane, benzene, toluene, and xylene; and ketone solvents such as acetone and methyl ethyl ketone. These organic solvents may be used alone or in combination. Preferred are alcoholic solvents such as ethanol and butylene glycol, with ethanol and butylene glycol being more preferred. The mixing ratio of the solvent is not particularly limited as long as the solvent contains water. The heating temperature or hydrolysis medium temperature during hydrolysis by hydrothermal treatment is 50 to 200°C, preferably 100 to 180°C, and the treatment time is 0.5 to 90 hours, preferably 24 to 72 hours.

[0026] In the hydrolysis step using acid or alkali treatment, the material is immersed in one or more acid or alkali solutions to cause a reaction. Suitable acids are not particularly limited, but examples include hydrochloric acid, sulfuric acid, nitric acid, formic acid, trifluoroacetic acid, and hydrobromic acid. Suitable alkalis are not particularly limited, but examples include sodium hydroxide, potassium hydroxide, and lithium hydroxide. The concentration of the acid or alkali is preferably 1 to 25N, and more preferably 1 to 10N. A supplementary solvent may be added to ensure the fluidity of the acid or alkali-mediated hydrolysis reaction and to accelerate the reaction. The auxiliary solvent is not particularly limited as long as it does not affect the reaction. Specific examples include water, alcoholic solvents such as methanol, ethanol, ethylene glycol, and butylene glycol; etheric solvents such as tetrahydrofuran, ethyl ether, 1,4-dioxane, and methyl tert-butyl ether; nitrile solvents such as acetonitrile and propionitrile; esteric solvents such as ethyl acetate, n-propyl acetate, and isopropyl acetate; hydrocarbon solvents such as pentane, hexane, benzene, toluene, and xylene; and ketone solvents such as acetone and methyl ethyl ketone. These solvents may be used alone or in combination. Preferred are water, alcoholic solvents such as ethanol and butylene glycol, and more preferred are water, ethanol, and butylene glycol. When the auxiliary solvent is added to an acid or alkali, the mixing ratio is not particularly limited, and the concentration of the acid or alkali in the system is preferably 1 to 25N, more preferably 1 to 10N. In this step, the order of mixing the raw materials and the acid or alkali and / or auxiliary solvent is not particularly limited, as long as both are present in the test system.

[0027] The treatment time for hydrolysis by acid or alkali treatment is 0.5 to 90 hours, preferably 1 to 72 hours, and the reaction temperature is 20 to 100°C, preferably 50 to 90°C. The hydrolysate obtained by hydrolysis by acid or alkali treatment is preferably neutralized and / or washed before measuring the amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde present. Since neutralization generates salts derived from the acid or alkali, it is preferable to carry out washing and / or desalting and / or dilution following neutralization. For washing, in addition to the solvent of the acid or alkali solution used in the acid or alkali treatment, water or a solvent containing water can be used. Washing is preferably carried out about 1 to 5 times, or until the washed solution becomes nearly neutral. Desalting can be carried out by a commonly used method, such as filtration or membrane treatment.

[0028] In the enzymatic hydrolysis step, the raw material is mixed with one or more hydrolases and reacted. Suitable enzymes are not particularly limited as long as they are glycoside hydrolases, but examples include glycosidase, galactosidase, glucuronidase, and tannase. Extracts from cells or fungi containing these enzymes can also be used. In the enzymatic hydrolysis step, the treatment time is 1 to 144 hours, preferably 3 to 72 hours, and the reaction temperature is 20 to 60°C, preferably 30 to 45°C.

[0029] In the enzymatic hydrolysis step, the preferred pH conditions for the hydrolysis reaction are not particularly limited, but are carried out at a pH of 5 to 9, preferably 6.5 to 8.0. The pH adjustment during hydrolysis may be performed by adjusting the pH of both the material and the hydrolases before adding one or more hydrolases to the material, or by adding one or more hydrolases to the material and then adjusting the pH. An acid, alkali, or pH buffer solution can be used to adjust the pH. The type of acid, alkali, or pH buffer solution is not particularly limited as long as it can adjust the pH to the desired level. Examples of acids or alkalis include those listed above. Preferred acids include hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, and acetic acid, and preferred alkalis include sodium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, and trisodium phosphate. Examples of pH buffer solutions include acetate buffer, citrate buffer, phosphate buffer, and Tris-HCl buffer. From the viewpoint of adjusting the pH to 6.0 to 8.0, phosphate buffer and Tris-HCl buffer are preferred.

[0030] In the enzymatic hydrolysis step, after completion of the hydrolysis reaction, a thermal inactivation step may be carried out to inactivate the enzymes used in the enzymatic hydrolysis. In this step, the order of mixing the raw material and the hydrolytic enzyme and / or pH adjuster is not particularly limited as long as both are present in the test system at the same time.

[0031] In the hydrolysis step of the material of the present invention, the hydrolyzed hydrolyzate may be extracted and / or purified before measuring the amount of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde present. The extraction method is not particularly limited, but for example, a common extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, or hexane may be added to a solution containing the hydrolyzate to perform the extraction operation. The purification method is not particularly limited, but may be performed by, for example, reverse osmosis membrane filtration, dialysis, electrodialysis, ultrafiltration, or separation and fractionation using size exclusion chromatography (e.g., gel filtration chromatography, gel permeation chromatography), reverse phase chromatography, etc. Furthermore, the hydrolyzate may be purified by a combination of these methods.

[0032] In the step of measuring the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde after the hydrolysis step of the material, it is preferable to use the material after the reaction has been completed by sufficiently reacting it. In addition, it is preferable to distill off the reaction solution or replace it with a different solution before measurement, in accordance with the method for measuring the aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in the reaction product.

[0033] The content of the aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde of the present invention can be measured by a known method, such as absorbance, color development, immunostaining, ELISA, liquid chromatography, thin-layer chromatography, gas chromatography, mass spectrometry, capillary electrophoresis, NMR, or infrared absorption spectroscopy.

[0034] The material used in the present invention is not particularly limited. Animals and plants, animal and plant-derived extracts, fungal cultures, or products thereof treated with enzymes or the like can be used as test substances, and they may be in solid, powder, liquid, gel, or other forms. Depending on the material, the hydrolysis method and the method for measuring the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde may be appropriately selected. In the hydrolysis step of the material, processing such as cutting or crushing may be performed before the hydrolysis treatment, if necessary. [Example]

[0035] The present invention will be described in more detail below. Unless otherwise specified, the blending amounts are expressed in mass %. Furthermore, unless otherwise specified, special reagent grade ethanol (99.5%) was used.

[0036] (Test 1) <Preparation of test substance> In Examples 1 to 13, compounds represented by the following formulae (E1) to (E5) and (E6) to (E13) were used as test substances, and in Comparative Examples 1 to 11, compounds represented by the following formulae (x1) to (x11) were used. Each compound was dissolved in ethanol (Fujifilm) or distilled water to prepare a 100 mM solution. Distilled water or ethanol was used as a control substance.

[0037] <Nitrotyrosine decomposition activity test> Nitrotyrosine (SIGMA) was dissolved in 1 M phosphate buffer (pH 7.4) to a concentration of 400 μM, and the test substance was added to a concentration of 10 mM. The solution was incubated at 37°C for 3 days, and the amount of nitrotyrosine in the solution was measured by HPLC under the following analytical conditions. (Analysis conditions) Analytical column: Chemcobond 5-ODS-W (150 x 6 mm) Detection wavelength: 355 nm Mobile phase: 10 mM phosphate buffer (pH 2.8) - methanol (9:1 (v / v)) Column temperature: 40℃ Flow rate: 1.0mL / min

[0038] The nitrotyrosine decomposition rate (%) was calculated from the amount of nitrotyrosine in the obtained solution according to the following formula. The results are shown in Tables 1 and 2. Note that if the nitrotyrosine decomposition rate was 5% or higher, it was determined that the nitrotyrosine reduction effect was achieved, and if it was less than 5%, it was determined that the nitrotyrosine reduction effect was not achieved. Furthermore, it can be said that the higher the nitrotyrosine decomposition rate, the greater the nitrotyrosine reduction effect.

[0039]

number

[0040] <Test substance> The following compounds represented by Chemical Formulas 1 to 3 were used as test substances.

[0041] [ka]

[0042] [ka]

[0043] [ka]

[0044] [Table 1]

[0045] [Table 2]

[0046] In Examples 1 to 5, which used a nitrotyrosine reducer comprising at least one compound selected from the group consisting of aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes, a high nitrotyrosine reduction effect was obtained. In addition, in Examples 6 to 13, which used a nitrotyrosine reducer comprising at least one compound selected from the group consisting of derivatives of aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes, a high nitrotyrosine reduction effect was also obtained. In contrast, the compounds of Comparative Examples 1 to 5 and 10, which consist of hydroxycarboxylic acids and / or hydroxyaldehydes that do not have a benzene ring moiety, the compounds of Comparative Examples 6 to 9, which have a benzene ring moiety but the carboxy group is not directly bonded to the benzene ring, and the compound of Comparative Example 11, which does not have a hydroxy group bonded to the benzene ring, did not have a nitrotyrosine-reducing effect.

[0047] From the above results, it was confirmed that an agent comprising at least one compound selected from the group consisting of aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes, and the group consisting of derivatives of aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes, has a nitrotyrosine-reducing effect, and it was found that the agent is useful as a nitrotyrosine-reducing agent.

[0048] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the blending amounts are expressed in mass %.

[0049] <Preparation of test substance> Dried plant materials were mixed with 10 times their mass of distilled water and heated at 60°C for 8 hours to obtain an extract of 10-20% of the mass of the dried plant material. The dried extract residue was diluted with distilled water at a mass ratio of 1:99, and the resulting diluted extract was used as the test substance. The plant materials used were eucalyptus leaves, comfrey leaves, yarrow flowers, clove flower buds, and staghorn fern leaves.

[0050] <Hydrolyzed sample> 600 μL each of the test substance and 8N hydrochloric acid solution were dispensed into glass vials and reacted at 90°C for 4 hours. After cooling, the solution was neutralized with 5N sodium hydroxide solution (Fujifilm), followed by the addition of 8 mL of 100 mM phosphate buffer (pH 6.8). The solution was filtered and used as a sample for HPLC analysis. <Sample without hydrolysis treatment> 600 μL each of the test substance and distilled water were dispensed into glass vials, and 8 mL of 100 mM phosphate buffer (pH 6.8) was added. The solution was filtered to prepare a sample for HPLC analysis.

[0051] <Gallic acid content quantitative test> The content of gallic acid, an aromatic hydroxybenzoic acid, was measured by HPLC, and the content was calculated from a calibration curve of an aqueous solution of gallic acid (Fijifilm). The gallic acid content in the plant material was also calculated. <Analysis conditions> Analytical column: Chemcobond 5-ODS-W (150 x 6 mm) Detection wavelength: 270 nm Mobile phase A: 0.05% trifluoroacetic acid Mobile phase B: MeOH Mobile phase composition: Gradient conditions 0 minutes Mobile phase A 90%: Mobile phase B 10% 0~15 minutes Mobile phase A 80%: Mobile phase B 20% 15~30 minutes Mobile phase A 60%: Mobile phase B 40% 30~40 minutes Mobile phase A 90%: Mobile phase B 10% 40~45 minutes Mobile phase A 90%: Mobile phase B 10% Column temperature: 40℃ Flow rate: 1.0mL / min

[0052] [Table 3]

[0053] As shown in Table 3, gallic acid was detected in the eucalyptus leaf and clove flower bud samples, confirming that these materials contained gallic acid. However, gallic acid was not detected in comfrey leaves, yarrow flowers, or kohlrabi leaves. Gallic acid was detected in the eucalyptus leaf and clove flower bud samples even without hydrolysis, but the amount of gallic acid increased after hydrolysis. This suggests that the eucalyptus leaf and clove flower bud samples contain numerous derivatives of gallic acid. Therefore, it can be concluded that the content of gallic acid and its derivatives could be efficiently detected by hydrolyzing the materials.

[0054] <Preparation of test substance> Distilled water was added to 10 times the mass of dried plant materials, and the mixture was heated at 60°C for 8 hours for extraction. The dried extract residue was diluted with distilled water at a mass ratio of 1:99, and the resulting solution was used as the test substance. The plant materials used were eucalyptus leaves, clove flower buds, and staghorn fern leaves. The control substance was the same dissolution medium used to prepare the test substance.

[0055] <Nitrotyrosine decomposition activity test> 3-Nitrotyrosine (Sigma), a constituent amino acid of nitrated proteins, was dissolved in 1 M phosphate buffer (pH 7.4) to a concentration of 400 μM, and 1 / 10 the amount of the test substance was added. The solution was incubated at 37°C for 3 days, and the amount of nitrotyrosine in the solution was measured by HPLC. <Analysis conditions> Analytical column: Chemcobond 5-ODS-W (150 x 6 mm) Detection wavelength: 355 nm Mobile phase: 10 mM phosphate buffer (pH 2.8) - methanol (9:1 (v / v)) Column temperature: 40℃ Flow rate: 1.0mL / min

[0056] The nitrotyrosine decomposition rate (%) was calculated from the amount of nitrotyrosine in the obtained solution according to the following formula. The results are shown in Table 4. Note that if the nitrotyrosine decomposition rate was 5% or higher, it was determined that the nitrotyrosine reduction effect was achieved, and if it was less than 5%, it was determined that the nitrotyrosine reduction effect was not achieved. Furthermore, it can be said that the higher the nitrotyrosine decomposition rate, the greater the nitrotyrosine reduction effect.

[0057]

number

[0058] [Table 4]

[0059] In the eucalyptus leaves and clove flower buds, which were confirmed to contain gallic acid in Table 3, the decomposition rate of nitrotyrosine, a constituent amino acid of nitrated protein, was approximately 8.0 to 13.9%, confirming that these materials have the ability to decompose nitrotyrosine. On the other hand, in the eucalyptus leaves, which do not contain gallic acid, the decomposition rate of nitrotyrosine was 3.6%, indicating that they did not have the ability to decompose nitrotyrosine.

[0060] In other words, by using this screening method, aromatic hydroxycarboxylic acids and / or aromatic hydroxyaldehydes that have nitrotyrosine decomposition activity contained in candidate materials can be effectively detected, and highly effective nitrotyrosine reducers can be efficiently selected.

Claims

1. A method for screening for a nitrotyrosine decomposing agent using the content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in a material as an index.

2. A method for screening a nitrotyrosine degrading agent, comprising: A) hydrolyzing the material; B) measuring aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde in the hydrolysate; C) a step of selecting a material having a high content of aromatic hydroxycarboxylic acid and / or aromatic hydroxyaldehyde; The method comprising:

3. 3. The screening method according to claim 1, wherein the aromatic hydroxycarboxylic acid is dihydroxybenzoic acid and / or trihydroxybenzoic acid.

4. 4. The screening method according to claim 1, wherein the aromatic hydroxyaldehyde is dihydroxybenzaldehyde and / or trihydroxybenzaldehyde.

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