Novel compound, NO production inhibitor, anti-inflammatory agent, and method for producing a compound

Abstract

To provide a novel compound having inhibitory activity of nitrogen monoxide (NO) production in a living body, an NO production inhibitor, an antiinflammatory agent and a method for producing a compound having an NO production inhibitory activity.SOLUTION: There is provided an NO production inhibitor in a living body which contains a compound represented by the chemical formula 1, a compound represented by the chemical formula 2 or a salt thereof as an active ingredient. Since NO production can be suppressed as an inflammation mediator, the NO production inhibitor can suppress an inflammatory reaction and thus can contribute to suppression of tissue damage due to excessive inflammatory reactions and chronic inflammation or inhibition of disease onset and disease aggravation.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a novel compound, an NO production inhibitor, an anti-inflammatory agent, and a method for producing a compound. More specifically, it relates to a novel compound shown in Chemical Formula 2 or a salt thereof. Further, it relates to an inhibitor for suppressing the production of nitric oxide (NO) in a living body, which contains as an active ingredient a compound shown in Chemical Formula 1, a compound shown in Chemical Formula 2, or a salt thereof, or an extract of the genus Carthamus containing them. Further, it relates to an anti-inflammatory agent containing as an active ingredient a compound shown in Chemical Formula 1, a compound shown in Chemical Formula 2, or a salt thereof, or an extract of the genus Carthamus containing them. Further, it relates to a method for producing the compound shown in Chemical Formula 1 and the compound shown in Chemical Formula 2.

Background Art

[0002] NO is generated in the atmosphere due to high-temperature environments such as lightning discharges or the combustion of fossil fuels, but it is also produced in vivo. In vivo, it is produced by NO synthase in immune system cells such as vascular endothelial cells, nerve cells, and macrophages. NO produced from vascular endothelial cells is involved in blood pressure regulation through the relaxation of vascular smooth muscle, and NO produced from nerve cells is involved in information transmission between neurons. On the other hand, NO produced from immune system cells is known to enhance inflammatory reactions such as the killing of pathogenic microorganisms (Non-Patent Document 1, Non-Patent Document 2).

[0003] Here, inflammation is a biological defense reaction against harmful stimuli applied to the living body and is an essential physiological reaction for maintaining homeostasis. However, when this is excessive or lasts for a long time, it is known to cause tissue damage and diseases, and technologies for suppressing inflammation are required. In this regard, controlling the production of NO in immune system cells, which produce a large amount in particular, is considered effective for controlling inflammation.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention has been made to solve such problems, and an object thereof is to provide a novel compound having NO production inhibitory activity in a living body, a NO production inhibitor, an anti-inflammatory agent, and a method for producing a compound having NO production inhibitory activity.

Means for Solving the Problems

[0006] As a result of intensive studies, the present inventors have found that the genus Calendula contains the compounds shown in Chemical Formula 1 and the novel compounds shown in Chemical Formula 2. Further, it has been found that the compounds can inhibit NO production in a living body. Therefore, based on these findings, the following inventions have been completed.

Chemical Formula

Chemical Formula

[0007] (1) The novel compound according to the present invention is the compound shown in Chemical Formula 2 or a salt thereof.

[0008] (2) The first aspect of the NO production inhibitor in a living body according to the present invention contains the compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof as an active ingredient.

[0009] (3) The second aspect of the NO production inhibitor in a living body according to the present invention uses an extract of the genus Calendula containing the compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof as an active ingredient.

[0010] (4) The first aspect of the anti-inflammatory agent according to the present invention uses the compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof as an active ingredient.

[0011] (5) The second aspect of the anti-inflammatory agent according to the present invention uses an extract of the genus Calendula containing the compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof as an active ingredient.

[0012] (6) In the present invention, the plant of the genus Calendula may be Calendula officinalis.

[0013] (7) The method for producing the compound shown in Chemical Formula 1 or a salt thereof according to the present invention includes a step of extracting the compound shown in Chemical Formula 1 or a salt thereof from the genus Calendula.

[0014] (8) The method for producing the compound shown in Chemical Formula 2 or a salt thereof according to the present invention includes a step of extracting the compound shown in Chemical Formula 2 or a salt thereof from the genus Calendula.

Effects of the Invention

[0015] The compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof has an activity capable of suppressing the production of NO in a living body. According to the present invention, such a useful compound can be obtained.

[0016] The NO production inhibitor and anti-inflammatory agent of the present invention can suppress the production of NO in a living body. That is, since it can suppress the production of NO, which is an inflammatory mediator, it can suppress the inflammatory reaction, and thus can contribute to the suppression of tissue damage, onset of diseases, and deterioration of diseases caused by excessive inflammatory reactions and chronic inflammation.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0018] Hereinafter, the present invention will be described in detail.

[0019] The NO production inhibitor according to the present invention is an agent that suppresses the production of NO in a living body. Here, suppressing the production of NO in a living body means reducing the amount of NO produced by any cell in the living body. Since NO is extremely reactive and disappears in a short time, it is difficult to directly confirm whether the production of NO in the living body is suppressed. Therefore, whether the production of NO is suppressed can be confirmed using cultured cells as shown in the examples described later. That is, after administering a test substance to cultured cells having NO-producing ability, the amount of NO (amount of nitrous acid) is measured by a colorimetric method using Griess reagent. If the amount of NO decreases due to the administration of the test substance, it can be determined that the test substance has an activity of suppressing the production of NO in the living body.

[0020] The NO production inhibitor according to the present invention has the following first and second aspects. First aspect: It contains, as an active ingredient, the compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof. Second aspect: It contains, as an active ingredient, an extract of the genus *Carthamus* containing the compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof.

[0021] As described above, NO is an inflammatory mediator that enhances inflammatory responses such as tissue damage and sterilization. Therefore, it can be said that the inflammatory response can be suppressed by suppressing the production of NO in the living body. From this, the present invention also provides an anti-inflammatory agent containing the same substance as the NO production inhibitor as an active ingredient. That is, the anti-inflammatory agent according to the present invention has the following first and second aspects. First aspect; The compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof is used as an active ingredient. Second aspect; An extract of the genus Calendula containing the compound shown in Chemical Formula 1, the compound shown in Chemical Formula 2, or a salt thereof is used as an active ingredient.

[0022] The genus Calendula refers to plants belonging to the genus Calendula of the Asteraceae family. Examples of the genus Calendula include Calendula officinalis (Pot Marigold, Calendula), Calendula arvensis (Honkinsenka, Fuyushirazu, Fuyuzakikinsenka), and the like.

[0023] The compound shown in Chemical Formula 1 (Compound 1) and its salt, and the compound shown in Chemical Formula 2 (Compound 2) and its salt (hereinafter, Compound 1, the salt of Compound 1, Compound 2, and the salt of Compound 2 may be collectively referred to as, or any one of them may be referred to as, "the present compound") have high NO production inhibitory activity as shown in the examples described later. Therefore, it is a useful substance at least in this respect, and the present compound can be used as a NO production inhibitor or an anti-inflammatory agent in the living body.

[0024] Among the present compounds, Compound 2 is a novel compound that has not been reported so far. Compound 2 can be used for all meaningful applications, not limited to the suppression of NO production and inflammation suppression in the living body.

[0025] The present compound is contained in the genus Calendula. Therefore, an extract of the genus Calendula containing the present compound can also be used as a NO production inhibitor or an anti-inflammatory agent in the living body.

[0026] Compound 1 and Compound 2 can form salts with their hydroxyl groups, and in the present invention, these salts may also be used. Here, the salts include "pharmaceutically acceptable salts" and are interpreted broadly. For example, they may be various salts such as metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, etc. Examples of metal salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, and zinc salts. Examples of ammonium salts include salts such as ammonium and tetramethylammonium. Examples of organic amine addition salts include morpholine addition salts and piperidine addition salts. Examples of amino acid addition salts include glycine addition salts, phenylalanine addition salts, lysine addition salts, aspartic acid addition salts, and glutamic acid addition salts.

[0027] This compound may be a synthetic product or a commercially available reagent. Alternatively, it can be extracted and purified from natural products such as animals and plants containing this compound and used, or it can be chemically synthesized and used.

[0028] Since this compound is included in the genus Celosia as described above, this compound can be obtained by extracting it from the genus Celosia. The content of this compound in the genus Celosia may vary depending on the plant species, the part of the plant body, the collection time, the growth location, the treatment method after collection, etc. For example, when Compound 2 was quantitatively analyzed by high performance liquid chromatography (HPLC), it was confirmed by the present inventors that about 0.2 mg was contained per 1 g of the dried root of Celosia cristata. In the examples described later, 2.3 mg of Compound 2 was isolated from 135 g of the dried root of Celosia cristata. The reasons for the smaller isolated amount compared to the quantitative analysis value by HPLC are considered to be (i) loss occurred during the process of isolating and purifying Compound 2 from the crude fraction after extraction, and (ii) the sample scale and extraction time were different between the quantitative analysis and the isolation in the examples.

[0029] The extraction of this compound from the genus Centaurea can be carried out, for example, by immersing the plant body of the genus Centaurea in an extraction solvent. Specifically, the following methods can be exemplified. ≪Plant body of the genus Centaurea≫ For the plant body of the genus Centaurea, any part such as leaves, stems, flowers, roots or all of them (whole herb) containing these can be used, but it is preferably to contain roots. Also, those collected from the growth place can be used as they are, or can be used after drying. Further, those in the form of leaves, stems, flowers, roots, etc. can be used as they are, or can be used after being crushed into pieces or powder. ≪Extraction solvent≫ There is no particular limitation as long as this compound can be extracted from the genus Centaurea, and it can be appropriately set according to the final use of the product. For example, the solvent can be exemplified by lower alcohols (ethanol, propanol, etc.), ethyl acetate, glycols (glycerin, 1,3-butylene glycol, propylene glycol, 1,3-propanediol, etc.), chloroform, a mixture of these or a mixture of these and water, etc., polar solvents, oils and fats (castor oil, camellia oil, olive oil, apricot oil, rice germ oil, soybean oil, linseed oil, rice oil, sesame oil, corn oil, rapeseed oil, etc.), liquid waxes (jojoba oil, sperm whale oil, etc.), esters, higher alcohols, etc. ≪Extraction conditions≫ Immerse the plant body in the extraction solvent and leave it standing or stirring at a temperature of 1 to 30 °C or at room temperature for 2 to 24 hours.

[0030] Since the solvent after performing the extraction operation as described above contains this compound, it can be used as it is, or can be used after purification, concentration, dilution, sterilization, etc. as necessary. Purification can be carried out by removing plant residues by filtration or centrifugation, or by fractional purification by liquid chromatography. Also, it can be used after being solidified by methods such as spray drying or freeze drying.

[0031] For example, if a 50% (v / v) ethanol extract of the roots of *Tagetes erecta* is subjected to HPLC under the following conditions, the chromatogram shown in Figure 1 can be obtained. In Figure 1, compound 1 can be obtained by collecting the fraction with a retention time of 24 minutes, and compound 2 can be obtained by collecting the fraction with a retention time of 27 minutes. 《HPLC Conditions》 Column: SHISEDO CAPCELL PAK C18 UG120 (5 μm, φ4.6 × 250 mm) Solvent: (A) H 2 O (0.1% (v / v) TFA) (B) Acetonitrile (MeCN) (0.1% (v / v) TFA) Gradient: (B) 0% (v / v) (0 min) → (B) 100% (40 min) Flow rate: 1.0 mL / min Detection: Absorbance detector (210 nm) Injection volume: 10 μL

[0032] This compound, NO production inhibitor, and anti-inflammatory agent can be used in any form or for any purpose. For example, it can be used for the purpose of exerting an effect on the skin. In such a case, it can be in the form of pharmaceuticals or quasi-drugs (such as compresses and ointments) used transdermally, cosmetics (such as packs, lotions, milks, gels, creams, lip creams, etc.), or raw materials or additives used in combination therewith. Also, for example, it can be used for the purpose of exerting an effect in the body. In such a case, it can be in the form of pharmaceuticals or quasi-drugs, health foods, foods, beverages, feeds used by oral ingestion, or raw materials or additives used in combination therewith. In any form, after formulating this compound, it can be produced by a conventional method. The compounding amount of the active ingredient in the product or the dosage administered to the living body can also be appropriately set according to the use, safety, and other raw materials of the product.

[0033] Hereinafter, the present invention will be described based on each example, but the technical scope of the present invention is not limited to the features shown by these examples. In this example, M used as a unit represents mol / L.

Example

[0034] <Identification of the compound in Example 1> (1) Preparation of ethanol extract from the roots of *Cynanchum atratum* Bunge The roots of *Cynanchum atratum* Bunge collected in Shiraoi-cho, Hokkaido were washed with water and dried by placing them in a thermostat at 45 °C for 2 days. After cutting them into small pieces with scissors, they were powdered using a mixer. 1 L of 50% (v / v) ethanol was added to 135 g of the obtained dried root powder, and the mixture was stirred at 500 revolutions per minute (rpm) at room temperature for 72 hours using a stirrer to obtain an extract, and the supernatant (first time) was collected. 1 L of 50% (v / v) ethanol was added to the residue, and the mixture was stirred at 500 revolutions per minute (rpm) at room temperature for 72 hours using a stirrer to obtain an extract, and the supernatant (second time) was collected. The supernatants of the first and second times were each suction-filtered to collect the filtrates, which were concentrated under reduced pressure using an evaporator to obtain a concentrated solution. Subsequently, the concentrated solutions of the first and second times were combined and freeze-dried for 24 hours to obtain 24.0 g of a solid ethanol extract from the roots of *Cynanchum atratum* Bunge.

[0035] (2) Isolation of the compound 24.0 g of the ethanol extract from the roots of *Cynanchum atratum* Bunge was dissolved in ultrapure water (MilliQ water) to make the total volume 500 mL. 500 mL of hexane was added, and the mixture was fractionated into an aqueous layer and a hexane layer, and 500 mL of the aqueous layer was collected. 2000 mL of ethyl acetate was added thereto, and the mixture was fractionated into an aqueous layer and an ethyl acetate layer, and the ethyl acetate layer was collected and concentrated to dryness to obtain 0.82 g of an ethyl acetate fraction. This was subjected to silica gel chromatography under the following conditions. The fractions eluted with solvent [4] of hexane:ethyl acetate = 3:2 were fractionated and dried to obtain 27.9 mg of a crude purified product. 《Conditions for silica gel chromatography》 Column: Glass column (φ50×280 mm) Stationary phase: Silica gel 60 N Gradient: [1] Hexane:ethyl acetate = 9:1 (300 mL) [2] Hexane:ethyl acetate = 4:1 (400 mL) [3] Hexane:ethyl acetate = 7:3 (500 mL) [4] Hexane:Ethyl acetate = 3:2 (500 mL) [5] Hexane:Ethyl acetate = 1:1 (500 mL) [6] Hexane:Ethyl acetate = 0:1 (600 mL) [7] Methanol (600 mL)

[0036] The crude purified product (dry weight 27.9 mg) was subjected to HPLC under the following conditions, and the first fraction (dry weight 0.2 mg) with a retention time of 34 minutes and the second fraction (dry weight 2.3 mg) with a retention time of 55 minutes were collected. 《HPLC Conditions》 Column: SHISEIDO CAPCELLPAK UG120 (5 μm, φ20×250 mm) Solvent: H 2 O : Acetonitrile (MeCN) = 65 : 35 (v:v) (0.1% (v / v) TFA) Flow rate: 9.6 mL / min Detection: Absorbance photodetector (210 nm)

[0037] (3) Identification of the compound in the first fraction Regarding the first fraction (retention time 34 minutes, dry weight 0.2 mg), as a result of performing structural analysis using a Nuclear Magnetic Resonance (NMR) apparatus and a Liquid Chromatography-Mass Spectrometer (LC-MS), it was revealed that the compound shown in Chemical Formula 1 ((E)-4-(3-acetyl-2,6-dihydroxyphenyl)-2-methylbut-2-enal, molecular formula C 13 H 14 O 4 , molecular weight 234.25, which may be referred to as "Compound 1" in the present invention.) It became clear that it is. The NMR and LC-MS data are shown below.

Chemical Formula

[0038] <NMR and LC-MS data of Chemical Formula 1> 1 H NMR: 400 MHz,13 C NMR: 100 MHz (measurement solvent CD 3 OD) 1 H NMR: δ(ppm) 1.97 [d, 3H, J = 1.3 Hz], 2.53 [s, 3H], 3.50 [d, 2H, J = 7.4 Hz], 6.43 [d, 1H, J = 8.8 Hz], 6.63 [tq, 1H, J = 1.3, 7.4 Hz], 7.66 [d, 1H, J = 8.8 Hz], 9.32 [s, 1H]; 13 C NMR: δ(ppm) 9.1 [CH 3 , 23.5 [CH 2 , 26.2 [CH 3 , 108.2 [CH], 112.8 [C], 114.3 [C], 132.5 [CH], 140.2 [C], 154.8 [CH], 164.0 [C], 197.5 [CH], 204.5 [C]. HR-ESIMS : m / z = 233.0815([M-H] - )

[0039] (4) Isolation and Identification of the Compound in the Second Fraction For the second fraction (retention time 55 minutes, dry weight 2.3 mg), structural analysis was performed using an NMR apparatus and LC-MS. As a result, it was revealed that the compound shown in Chemical Formula 2 (methyl (E)-4-(3-acetyl-2,6-dihydroxyphenyl)-2-methylbut-2-enoate, molecular formula C 14 H 16 O 5 , molecular weight 264.28, which may be referred to as "Compound 2" in the present invention). Compound 2 is a novel substance that has not been reported so far. The NMR and LC-MS data are shown below.

Chemical Formula

[0040] <NMR and LC-MS Data of Chemical Formula 2> 1 1H NMR: 400 MHz, 13 13C NMR: 100 MHz (measurement solvent CD 3 OD) 1 1H NMR: δ (ppm) 1.97 [d, 3H, J = 1.4 Hz], 2.53 [s, 3H], 3.50 [d, 2H, J = 7.5 Hz], 3.87 [s, 3H], 6.42 [d, 1H, J = 8.8 Hz], 6.77 [tq, 1H, J = 1.4, 7.5 Hz], 7.63 [d, 1H, J = 8.8 Hz]; 13 13C NMR: δ (ppm) 12.5 [CH 3 , 23.1 [CH 2 , 26.2 [CH 3 , 52.2 [CH 3 , 108.2 [CH], 113.4 [C], 114.2 [C], 128.2 [C], 132.3 [CH], 142.0 [CH], 163.9 [C], 170.6 [C], 204.5 [C]. HR-ESIMS: m / z = 265.1069 ([M + H] + )

[0041] <Example 2> Evaluation of NO Quantity Rate (1) Preparation of Test Substances The medium used was RPMI 1640 medium supplemented with 0.1% (v / v) of Escherichia coli-derived lipopolysaccharide (LPS) as a NO production stimulant. Compounds 1 and 2 were each dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. This was added to the medium so that the concentration of the compound (final concentration during cell culture) was 12.5 μM, 25 μM, 50 μM, and 100 μM to obtain test substances.

[0042] (2) Measurement of NO Quantity Rate The nitric oxide (NO) quantity rate was measured according to the following procedures a) to c). A) Mouse-derived macrophage-like cells (J774.1 cells, RCB0434, Cell Materials Development Laboratory, RIKEN BioResource Center) were seeded at a density of approximately 1.0×10 5 cells / well, 100 μL per well, in each well of a 96-well plate, and cultured at 37 °C under 5% CO 2 for 1 day. B) 100 μL of the test substance was added to the medium in each well and cultured under the same conditions for 24 hours (evaluation sample). Wells with only the same amount of medium added without the test substance were also set up and cultured in the same manner (control sample). C) 0.5 mL of phosphoric acid was added to 19.5 mL of deionized water, and 200 mg of sulfanilamide and 20 mg of N-1-naphthylethylenediamine dihydrochloride were dissolved to prepare Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylethylenediamine, 2.5% phosphoric acid). D) After transferring 100 μL of the supernatant from each well of the 96-well plate to a new 96-well plate, 100 μL of Griess reagent was added to each well of that plate and left standing at room temperature for 30 minutes. E) The absorbance at 540 nm was measured using a microplate reader. Wells containing 100 μL of medium and 100 μL of Griess reagent were also set up as blank samples and measured in the same manner. F) The NO content rate was calculated according to the following formula (1), and the 50% inhibitory concentration (IC 50 ) was determined. Formula (1): NO content rate (%) = {(absorbance of evaluation sample - absorbance of blank sample) / (absorbance of control sample - absorbance of blank sample)} × 100

[0043] (3) Measurement of cell viability The cell viability was measured according to the following procedures A) to F). A) J774.1 cells were seeded at a density of approximately 1.0×10 5 cells / well, 100 μL per well, in each well of a 96-well plate, and cultured at 37 °C under 5% CO 2 for 1 day. i) 100 μL of the test substance was added to the medium in each well and cultured for 24 hours under the same conditions (evaluation sample). Wells with only the same amount of medium added without the test substance were also set up and cultured in the same manner (control sample). ii) The supernatant was removed from the 96-well plate, 5 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reagent was added, and a color reaction was carried out for 3 hours under the same conditions. iii) The supernatant of each well was removed, 200 μL of DMSO was added, and it was shaken and stirred for 3 minutes. iv) The absorbance at 535 nm was measured with a microplate reader. Wells containing 200 μL of DMSO were also set up as blank samples and measured in the same manner. v) The cell viability was calculated according to the following formula 2. Formula 2: Cell viability (%) = { (Absorbance of evaluation sample - Absorbance of blank sample) / (Absorbance of control sample - Absorbance of blank sample)} × 100

[0044] (4) Results The NO production rate and cell viability when compound 1 was used as the test substance are shown in Figure 2. As shown in Figure 2, the NO production rate decreased as the concentration of compound 1 increased. Also, the IC 50 of compound 1 was 57.4 μM. This value is significantly smaller than the IC G of N 50 -monomethyl-L-arginine (L-NMMA) (116.2 μM), which is known as a NO production enzyme inhibitor. Therefore, it can be said that the NO production inhibitory activity of compound 1 is significantly strong. On the other hand, no decrease in cell viability was observed at any concentration. That is, despite having no cytotoxicity, the NO production rate decreased in a concentration-dependent manner with compound 1. From this result, it became clear that compound 1 can suppress the production or presence of NO in cells.

[0045] Also, the NO production rate and cell viability when compound 2 was used as the test substance are shown in Figure 3. As shown in Figure 3, the NO production rate decreased as the concentration of compound 2 increased. Also, the IC 50was 69.2 μM. Since this value was significantly smaller than that of L-NMMA 50 (116.2 μM), it can be said that the NO amount inhibitory activity of Compound 2 is significantly strong. On the other hand, no decrease in cell viability was observed at any concentration. That is, despite having no cytotoxicity, the NO amount rate decreased in a concentration-dependent manner for Compound 2. From this result, it became clear that Compound 2 can suppress the production or presence of NO from cells.

[0046] <Example 3> Evaluation of NO Radical Scavenging Activity Using nitroprusside (SNP) that generates NO radicals by dissolving in a liquid, the NO radical scavenging activity of the compound was examined. Curcumin, which is known as a NO radical scavenging substance, was used as a positive control. That is, the test substances were Compound 1, Compound 2, and curcumin.

[0047] First, SNP was dissolved in phosphate buffered saline (pH 7.4, PBS) to prepare a 10 mM SNP-PBS solution. Curcumin, Compound 1, and Compound 2 were each dissolved in ethanol so that the concentrations were 25 μM, 50 μM, 100 μM, and 200 μM to prepare test substance-ethanol solutions. After adding 50 μL of each test substance-ethanol solution to a 96-well plate, 50 μL of the SNP-PBS solution was added to each, and the mixture was allowed to stand at room temperature for 150 minutes (the final concentrations of the test substances were 12.5 μM, 25 μM, 50 μM, and 100 μM). Wells containing 50 μL of the SNP-PBS solution and 50 μL of ethanol were also set up and allowed to stand in the same manner (control sample). Subsequently, 100 μL of the Greiss reagent prepared as described in Example 2(2)(v) was added to each well, and the absorbance at 546 nm was measured with a microplate reader. Wells containing 50 μL of PBS and 50 μL of ethanol were also set up as blank samples and measured in the same manner. Based on the measurement results, the NO radical scavenging rate (%) was calculated by the following formula 3. The results are shown in Figure 4. Formula 3: NO radical scavenging rate (%) = [1 - {(absorbance of evaluation sample - absorbance of blank sample) / (absorbance of control sample - absorbance of blank sample)}] × 100

[0048] As shown in Fig. 4, when curcumin was used as the test substance, the NO radical scavenging rate increased in a concentration-dependent manner. In contrast, when Compound 1 was used, the NO radical scavenging rate tended to decrease as the concentration increased. When Compound 2 was used, the NO radical scavenging rate showed almost no change regardless of the concentration. From these results, it was revealed that Compound 1 and Compound 2 do not have the activity of scavenging NO radicals.

[0049] Considering the results of Example 3 and the results of Example 2, the inhibitory effect of Compound 1 and Compound 2 on the NO production rate was considered to be due to the suppression of NO production in cells, rather than the result of scavenging once-generated NO. That is, it was revealed that Compound 1 and Compound 2 can suppress the production of NO from cells.

[0050] <Example 4> Evaluation of cytotoxicity In Example 2, it was confirmed that Compound 1 and Compound 2 have no cytotoxicity against mouse cancer cell-derived macrophage-like cells (J774.1 cells). Furthermore, in order to confirm the cytotoxicity against normal cells, for Compound 2, the cell viability was measured using normal diploid fibroblasts derived from human skin (ASF4-1, population doubling level (PDL) = 43, provided by Dr. Kazuhiko Kajiwa) according to the following procedures a) to k). MEM medium was used as the medium.

[0051] a) Compound 2 was dissolved in DMSO to a concentration of 10 mM and then diluted with the medium to obtain the test substance. b) The cells were seeded at a density of about 3.5×10 3 cells / well, 100 μL each, into each well of a 96-well plate and cultured at 37 °C and 5% CO 2 for 24 hours to allow the cells to adhere. c) The test substance was added to each well so that the final concentrations of Compound 2 were 12.5 μM, 25 μM, 50 μM, and 100 μM, and the cells were cultured under the same conditions for 24 hours (evaluation samples). Wells without the addition of the test substance were also set and cultured in the same manner (control samples). E) The supernatant was removed from the 96-well plate, 10 μL of MTT reagent was added, and a color reaction was carried out under the same conditions for 4 hours. O) The supernatant of each well was removed, 200 μL of DMSO was added to dissolve formazan. Ka) The absorbance at 535 nm was measured with a microplate reader. A well containing 200 μL of DMSO was also set as a blank sample and measured in the same manner. The cell viability was calculated according to Equation 2 of Example 2(3). The results are shown in Figure 5.

[0052] As shown in Figure 5, the cell viability was almost constant regardless of the concentration of Compound 2. That is, a decrease in cell viability with an increase in the concentration of Compound 2 was not observed. From this result, it was revealed that Compound 2 has no significant cytotoxicity against not only cancer cells but also normal cells.

Claims

1. The compound shown in Chemical Formula 2 below or a salt thereof. [Chemical 2]

2. A nitric oxide (NO) production inhibitor in a living body, comprising, as an active ingredient, the compound shown in Chemical Formula 1 below, the compound shown in Chemical Formula 2 below, or a salt thereof. 【Chemical 1】 【Chemical 2】

3. A nitric oxide (NO) production inhibitor in a living body, comprising, as an active ingredient, an extract of a plant belonging to the genus Calendula containing the compound shown in Chemical Formula 1 below, the compound shown in Chemical Formula 2 below, or a salt thereof. 【Chemical 1】 [Chemical Formula 2]

4. An anti-inflammatory agent, comprising, as an active ingredient, the compound shown in Chemical Formula 1 below, the compound shown in Chemical Formula 2 below, or a salt thereof. 【Chemical 1】 【Chemical Formula 2】

5. An anti-inflammatory agent, comprising, as an active ingredient, an extract of a plant belonging to the genus Calendula containing the compound shown in Chemical Formula 1 below, the compound shown in Chemical Formula 2 below, or a salt thereof. 【Chemical 1】 【Chemical 2】

6. The agent according to Claim 3 or Claim 5, wherein the plant belonging to the genus Calendula is Calendula officinalis.

7. A method for producing the compound shown in Chemical Formula 1 below or a salt thereof, comprising a step of extracting the compound shown in Chemical Formula 1 below or a salt thereof from a plant belonging to the genus Calendula. 【Chemical 1】

8. A method for producing the compound shown in Chemical Formula 2 below or a salt thereof, comprising a step of extracting the compound shown in Chemical Formula 2 below or a salt thereof from a plant belonging to the genus Calendula. 【Chemical 2】

9. The method according to Claim 7 or Claim 8, wherein the plant belonging to the genus Calendula is Calendula officinalis.

Images