JP7682451B2 - Novel compound, NO production inhibitor, anti-inflammatory agent, and method for producing the compound - Google Patents

Description

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

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

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, if it becomes excessive or continues for an excessively long period of time, it is known to cause tissue damage and disease, and there is a demand for technology to suppress inflammation. In this regard, controlling NO production, which is produced in particularly large amounts by immune system cells, is thought to be effective in controlling inflammation.

Akio Matsumoto, Regulation of physiological functions by nitric oxide (NO) and its breakdown, Basic Aging Research, Vol. 38, No. 3, pp. 11-18, 2014 Reiji Aoki, 4) Simple evaluation of NO production using macrophages, Food Functionality Evaluation Manual Vol. II, FY2007 Ministry of Agriculture, Forestry and Fisheries Subsidized Project (Food Industry Cluster Development Project), Japan Society for Food Science and Technology, pp. 118-123

The present invention has been made to solve these problems, and aims to provide a novel compound having activity of inhibiting NO production in the body, an NO production inhibitor, an anti-inflammatory agent, and a method for producing a compound having activity of inhibiting NO production.

As a result of intensive research, the present inventors have found that Calendula contains a compound shown in Chemical formula 1 and a novel compound shown in Chemical formula 2. They have also found that the compound can suppress NO production in the living body. Based on these findings, the present inventors have completed the following inventions.

(1) The novel compound of the present invention is a compound shown in Chemical formula 2 or a salt thereof.

(2) A first embodiment of the inhibitor of NO production in a living body according to the present invention has as an active ingredient a compound shown in Chemical formula 1, a compound shown in Chemical formula 2, or a salt thereof.

(3) A second embodiment of the inhibitor of NO production in a living body according to the present invention has as an active ingredient an extract of the genus Calendula that contains the compound shown in Chemical formula 1, the compound shown in Chemical formula 2, or a salt thereof.

(4) A first aspect of the anti-inflammatory agent of the present invention contains as an active ingredient a compound shown in Chemical formula 1, a compound shown in Chemical formula 2, or a salt thereof.

(5) A second aspect of the anti-inflammatory agent of the present invention contains, as an active ingredient, an extract of the genus Calendula that contains the compound shown in Chemical formula 1, the compound shown in Chemical formula 2, or a salt thereof.

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

(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 Calendula genus.

(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 Calendula genus.

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

The NO production inhibitor and anti-inflammatory agent of the present invention can suppress the production of NO in the body. In other words, by suppressing the production of NO, an inflammatory mediator, the inflammatory response can be suppressed, thereby contributing to the suppression of tissue damage and disease onset and disease aggravation caused by excessive inflammatory responses and chronic inflammation.

FIG. 1 shows an HPLC chromatogram of a 50% (v/v) ethanol extract of Calendula officinalis roots. 1 is a graph showing NO mass rate and cell viability in macrophage-like cells cultured in the presence of Compound 1. 1 is a graph showing NO mass rate and cell viability in macrophage-like cells cultured in the presence of Compound 2. 1 is a bar graph showing the radical scavenging rates of curcumin, Compound 1 and Compound 2. 1 is a bar graph showing cell viability in human fibroblasts cultured in the presence of Compound 2.

The present invention will be described in detail below.

The NO production inhibitor of the present invention is an agent that inhibits the production of NO in a living body. Here, inhibiting the production of NO in a living body means reducing the amount of NO produced in any cell in the living body. Because NO is highly reactive and disappears in a short time, it is difficult to directly confirm whether the production of NO in a living body has been inhibited. Therefore, whether the production of NO is inhibited can be confirmed using cultured cells, as shown in the examples described below. That is, after administering a test substance to cultured cells having NO production ability, the amount of NO (amount of nitrite) is measured by a colorimetric method using Griess reagent. If the amount of NO decreases due to administration of the test substance, it can be determined that the test substance has activity of inhibiting the production of NO in a living body.

The NO production inhibitor according to the present invention has the following first and second aspects.
First embodiment: 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 embodiment: 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.

As described above, NO is an inflammatory mediator that enhances inflammatory responses such as tissue damage and bactericidal activity. Therefore, it can be said that inflammatory responses can be suppressed by suppressing the production of NO in the living body. For this reason, 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 embodiment: 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 embodiment: 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.

The genus Calendula refers to plants belonging to the genus Calendula in the family Asteraceae. Examples of Calendula include Calendula officinalis (pot marigold, calendula) and Calendula arvensis (Japanese calendula, winter marigold, winter calendula).

The compound shown in Chemical formula 1 (Compound 1) and its salts, and the compound shown in Chemical formula 2 (Compound 2) and its salts (hereinafter, Compound 1, the salt of Compound 1, Compound 2, and the salt of Compound 2 may be collectively or individually referred to as "the present compound") have high NO production inhibitory activity, as shown in the examples described below. Therefore, they are useful substances at least in this respect, and the present compound can be used as an inhibitor of NO production in the living body or as an anti-inflammatory agent.

Of these compounds, compound 2 is a novel compound that has not been reported before. Compound 2 can be used for all meaningful purposes, not just for suppressing NO production and inflammation in the body.

This compound is included in the Calendula genus. Therefore, an extract of the Calendula genus containing this compound can also be used as an agent for suppressing NO production in the body or as an anti-inflammatory agent.

Compound 1 and Compound 2 can form salts at their hydroxyl groups, and these salts may be used in the present invention. Here, salts include "pharmacologically acceptable salts" and are interpreted in a broad sense. For example, various salts such as metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts may be used. 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 ammonium and tetramethylammonium salts. 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.

The compound may be a synthetic product or a commercially available reagent. Alternatively, the compound may be extracted and purified from natural products such as plants and animals that contain the compound, or the compound may be chemically synthesized and used.

As described above, this compound is included in the Calendula genus, and therefore this compound can be obtained by extraction from the Calendula genus. The content of this compound in the Calendula genus may vary depending on the plant species, the part of the plant body, the time of collection, the place of growth, the processing method after collection, and the like. For example, the present inventors have confirmed that Compound 2 was contained in an amount of about 0.2 mg per 1 g of dried Calendula officinalis roots when quantitatively analyzed by high performance liquid chromatography (HPLC). In the Examples described below, 2.3 mg of Compound 2 was isolated from 135 g of dried Calendula officinalis roots. The reason why this isolated amount is smaller than the quantitative analysis value by HPLC is thought to be (i) due to losses in the process of isolating and purifying Compound 2 from the crude fraction after extraction, and (ii) due to differences in the scale of the sample and extraction time between the quantitative analysis and the isolation in the Examples.

The present compound can be extracted from Calendula by, for example, immersing the Calendula plant in an extraction solvent. Specifically, the following method can be exemplified.
<Calendula plant>
The plant body of the Calendula genus may be used in any part, such as leaves, stems, flowers, roots, or the whole plant including these (whole plant), but preferably includes the roots. Also, the plant collected from the growing area may be used as it is, or may be used after drying. Also, the plant body in the form of leaves, stems, flowers, roots, etc. may be used as it is, or may be used after crushing into pieces or powder.
<Extraction solvent>
As long as the present compound can be extracted from the Calendula genus, there is no particular limitation and the solvent can be appropriately selected depending on the final use of the product. For example, examples of the solvent include polar solvents such as lower alcohols (ethanol, propanol, etc.), ethyl acetate, glycols (glycerin, 1,3-butylene glycol, propylene glycol, 1,3-propanediol, etc.), chloroform, mixtures thereof, or mixtures thereof with water, 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, and higher alcohols.
≪Extraction conditions≫
The plant body is immersed in the extraction solvent and allowed to stand or be stirred at a temperature of 1 to 30° C. or at room temperature for 2 to 24 hours.

The solvent after the extraction procedure described above contains the present compound, and may be used as is, or may be purified, concentrated, diluted, sterilized, or the like as necessary before use. Purification may be performed by removing plant residues through filtration or centrifugation, or by fractional purification through liquid chromatography. The compound may also be solidified by methods such as spray drying or freeze drying before use.

For example, when a 50% (v/v) ethanol extract from the roots of Calendula officinalis is subjected to HPLC under the following conditions, the chromatogram shown in Figure 1 is 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 (5μm,φ4.6×250 mm)
Solvent: (A) _H2O (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: spectrophotometric detector (210 nm)
Injection volume: 10μL

The compound, NO production inhibitor, and anti-inflammatory agent can be used in any form or for any purpose. For example, they can be used for the purpose of exerting an effect on the skin. In such a case, they can be in the form of a medicine or quasi-drug (such as a compress or ointment), or a cosmetic (such as a face pack, lotion, emulsion, gel, cream, or lip balm) that is used percutaneously, or as a raw material or additive that is mixed with these. They can also be used for the purpose of exerting an effect inside the body. In such a case, they can be in the form of a medicine or quasi-drug, health food, food, beverage, or feed that is taken orally, or as a raw material or additive that is mixed with these. In any form, they can be manufactured by a conventional method after blending the compound. The amount of active ingredient in the product or the amount administered to the living body can also be appropriately set depending on the purpose and safety of the product, other raw materials, etc.

The present invention will be described below based on each example, but the technical scope of the present invention is not limited to the characteristics shown in these examples. In the examples, the unit M represents mol/L.

Example 1: Identification of Compounds (1) Preparation of Calendula officinalis root ethanol extract Calendula officinalis roots collected in Shiraoi-cho, Hokkaido, were washed with water and dried by placing them in a thermostatic bath at 45°C for 2 days. The roots were finely cut with scissors and then powdered using a mixer. 1L of 50% (v/v) ethanol was added to 135g of the obtained dried root powder, and the mixture was stirred at 500 revolutions/minute (rpm) and room temperature for 72 hours using a stirrer to obtain an extract and recover the supernatant (first time). 1L of 50% (v/v) ethanol was added to the residue and stirred at 500 revolutions/minute (rpm) and room temperature for 72 hours using a stirrer to obtain an extract and recover the supernatant (second time). The first and second supernatants were each filtered by suction to recover the filtrate, and the filtrate was concentrated under reduced pressure using an evaporator to obtain a concentrated solution. Subsequently, the first and second concentrated solutions were combined and freeze-dried for 24 hours to obtain 24.0 g of a solid ethanol extract of Calendula officinalis root.

(2) Isolation of Compounds 24.0 g of ethanol extract of Calendula officinalis root was dissolved in ultrapure water (MilliQ water) to a total volume of 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 ethyl acetate fraction. This was subjected to silica gel chromatography under the following conditions. The fraction eluted with the solvent [4], hexane:ethyl acetate = 3:2, was separated and dried to obtain 27.9 mg of crude product.
<Conditions for silica gel chromatography>
Column: Glass column (φ50×280 mm)
Stationary phase: Silica gel 60N
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)

The crude product (dry weight 27.9 mg) was subjected to HPLC under the conditions below, and the first fraction (dry weight 0.2 mg) having a retention time of 34 minutes and the second fraction (dry weight 2.3 mg) having a retention time of 55 minutes were collected.
HPLC conditions
Column: SHISEIDO CAPCELLPAK UG120 (5 μm, φ20 × 250 mm)
Solvent: _H2O :acetonitrile (MeCN) = 65:35 (v:v) (0.1% (v/v) TFA)
Flow rate: 9.6 mL/min Detection: spectrophotometric detector (210 nm)

(3) Identification of the compound in the first fraction The structure of the first fraction (retention time 34 minutes, dry weight 0.2 mg) was analyzed using a nuclear magnetic resonance (NMR) device and a liquid chromatography mass spectrometer (LC-MS), and it was found to be 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, sometimes referred to as "compound 1" in the present invention). The NMR and LC-MS data are shown below.

<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([MH] ^- )

(4) Isolation and identification of the compound in the second fraction Structural analysis of the second fraction (retention time 55 minutes, dry weight 2.3 mg) was performed using an NMR device and LC-MS, and it was found to be the compound shown in Chemical formula 2 (methyl (E)-4-(3-acetyl-2,6-dihydroxyphenyl)-2- _methylbut -2 _- enoate, molecular formula _C14H16O5 , molecular weight 264.28, sometimes referred to as "Compound 2" in the present invention). Compound 2 is a novel substance that has not been reported before. The NMR and LC-MS data are shown below.

<NMR and LC-MS data of Chemical formula 2>
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 = 1.4 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 C 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] ⁺ )

Example 2: Evaluation of NO content (1) Preparation of test substances The medium used was RPMI1640 medium supplemented with 0.1% (v/v) E. coli-derived lipopolysaccharide (LPS) as an NO production stimulant. Compound 1 and compound 2 were each dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. These were added to the medium to give compound concentrations (final concentrations during cell culture) of 12.5 μM, 25 μM, 50 μM, and 100 μM to prepare test substances.

(2) Measurement of NO mass rate The nitric oxide (NO) mass rate was measured by the following procedures a) to f).
A) Mouse-derived macrophage-like cells (J774.1 cells, RCB0434, Cell Materials Development Laboratory, RIKEN BioResource Research Center) were seeded in each well of a 96-well plate at 100 μL each to a density of approximately 1.0 × ¹⁰ cells/well, and cultured at 37°C and 5% _CO2 for 1 day.
A) 100 μL of the test substance was added to the medium in each well, and the wells were cultured under the same conditions for 24 hours (evaluation sample). Wells to which the same amount of medium was 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 in the solution to prepare Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylethylenediamine, 2.5% phosphoric acid).
d) 100 μL of the supernatant from each well of the 96-well plate was transferred to a new 96-well plate, and then 100 μL of Griess reagent was added to each well of the plate, followed by allowing to stand at room temperature for 30 minutes.
E) The absorbance at 540 nm was measured using a microplate reader. As a blank sample, wells containing 100 μL of medium and 100 μL of Griess reagent were also set up and similar measurements were performed.
f) The NO amount rate was calculated according to the following formula 1, and the 50% inhibitory concentration (IC ₅₀ ) was determined.
Formula 1: NO content (%)={(absorbance of evaluation sample−absorbance of blank sample)/(absorbance of control sample−absorbance of blank sample)}×100

(3) Measurement of cell viability The cell viability was measured according to the following procedures a) to f).
A) J774.1 cells were seeded in each well of a 96-well plate at 100 μL each to a density of approximately 1.0×10 ⁵ cells/well, and cultured at 37° C. in 5% CO ₂ for 1 day.
A) 100 μL of the test substance was added to the medium in each well, and the wells were cultured under the same conditions for 24 hours (evaluation sample). Wells to which the same amount of medium was added without adding the test substance were also set up, and cultured in the same manner (control sample).
c) The supernatant was removed from the 96-well plate, and 5 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reagent was added, followed by a color reaction for 3 hours under the same conditions.
d) The supernatant from each well was removed, 200 μL of DMSO was added, and the wells were shaken and stirred for 3 minutes.
E) The absorbance at 535 nm was measured using a microplate reader. As a blank sample, a well containing 200 μL of DMSO was also set up and measured in the same manner.
F) The cell survival rate 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

(4) Results The NO amount rate and cell viability rate when compound 1 was used as the test substance are shown in FIG. 2. As shown in FIG. 2, the NO amount rate was smaller as the concentration of compound 1 was higher. In addition, the IC ₅₀ of compound 1 was 57.4 μM. This value is significantly smaller than the IC ₅₀ (116.2 μM) of N ^G -monomethyl-L-arginine (L-NMMA), which is known as an NO-producing enzyme inhibitor, so it can be said that the NO amount inhibitory activity of compound 1 is significantly stronger. On the other hand, no decrease in cell viability was observed at any concentration. That is, despite the lack of cytotoxicity, the NO amount 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.

FIG. 3 shows the NO amount rate and cell viability when compound 2 was used as the test substance. As shown in FIG. 3, the NO amount rate was smaller as the concentration of compound 2 was higher. In addition, the IC ₅₀ of compound 2 was 69.2 μM. Since this value is significantly smaller than the IC ₅₀ of L-NMMA (116.2 μM), it can be said that the NO amount inhibitory activity of compound 2 is significantly stronger. On the other hand, no decrease in cell viability was observed at any concentration. That is, despite the lack of cytotoxicity, the NO amount rate decreased in a concentration-dependent manner with compound 2. From this result, it became clear that compound 2 can suppress the production or presence of NO from cells.

Example 3: Evaluation of NO radical scavenging activity Nitroprusside (SNP), which generates NO radicals when dissolved in liquid, was used to examine the NO radical scavenging activity of the compounds. Curcumin, which is known as an NO radical scavenger, was used as a positive control. That is, the test substances were Compound 1, Compound 2, and curcumin.

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 dissolved in ethanol to give concentrations of 25 μM, 50 μM, 100 μM, and 200 μM, respectively, to prepare test substance-ethanol solutions. 50 μL of the test substance-ethanol solution was added to a 96-well plate, followed by 50 μL of SNP-PBS solution, and the plate was left to stand at room temperature for 150 minutes (final concentrations of the test substance were 12.5 μM, 25 μM, 50 μM, and 100 μM). Wells containing 50 μL of SNP-PBS solution and 50 μL of ethanol were also set and left to stand in the same manner (control sample). Next, 100 μL of Griess reagent prepared as described in Example 2 (2) c) was added to each well, and the absorbance at 546 nm was measured using a microplate reader. As a blank sample, wells containing 50 μL of PBS and 50 μL of ethanol were also set up and measured in the same manner. Based on the measurement results, the NO radical scavenging rate (%) was calculated using the following formula 3. The results are shown in FIG. 4.
Equation 3: NO radical scavenging rate (%)=[1-{(absorbance of evaluation sample-absorbance of blank sample)/(absorbance of control sample-absorbance of blank sample)}]×100

As shown in Figure 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 hardly changed regardless of the concentration. These results demonstrated that compound 1 and compound 2 do not have NO radical scavenging activity.

Considering the results of this Example 3 together with the results of Example 2, it was believed that the inhibitory effect of Compound 1 and Compound 2 on the NO content rate was not the result of capturing NO once it was produced, but was due to the inhibition of NO production in cells. In other words, it was revealed that Compound 1 and Compound 2 can inhibit the production of NO from cells.

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, the cell viability of Compound 2 was measured using human skin-derived normal diploid fibroblasts (ASF4-1, population doubling number (PDL) = 43, provided by Kazuhiko Kaji) according to the following procedures A) to F). MEM medium was used.

A) Compound 2 was dissolved in DMSO to a concentration of 10 mM, and then diluted with culture medium to prepare a test substance.
a) 100 μL of cells were seeded into each well of a 96-well plate to give a density of about 3.5×10 ³ cells/well, 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 wells were cultured under the same conditions for 24 hours (evaluation samples). Wells to which the test substance was not added were also set up and cultured in the same manner (control samples).
d) The supernatant was removed from the 96-well plate, and 10 μL of MTT reagent was added, followed by carrying out a color reaction under the same conditions for 4 hours.
E) The supernatant of each well was removed, and 200 μL of DMSO was added to dissolve the formazan. F) The absorbance at 535 nm was measured using a microplate reader. As a blank sample, a well containing 200 μL of DMSO was also set up and measured in the same manner. The cell viability was calculated using formula 2 in Example 2 (3). The results are shown in FIG. 5.

As shown in Figure 5, the cell viability was almost constant regardless of the concentration of compound 2. In other words, no decrease in cell viability was observed with increasing concentration of compound 2. This result demonstrated that compound 2 does not have any significant cytotoxicity against cancer cells or normal cells.

Claims (9)

A compound represented by the following formula 2 or a salt thereof.
An agent for inhibiting the production of nitric oxide (NO) in a living body, comprising as an active ingredient a compound represented by the following chemical formula 1, a compound represented by the following chemical formula 2, or a salt thereof.
An agent for inhibiting the production of nitric oxide (NO) in a living body, comprising as an active ingredient an extract of a plant belonging to the genus Calendula, which contains a compound shown in Chemical Formula 1 below, a compound shown in Chemical Formula 2 below, or a salt thereof.
An anti-inflammatory agent comprising, as an active ingredient, a compound represented by the following Chemical formula 1, a compound represented by the following Chemical formula 2, or a salt thereof.
An anti-inflammatory agent comprising, as an active ingredient, an extract of a plant belonging to the genus Calendula, which contains a compound represented by Chemical Formula 1 below, a compound represented by Chemical Formula 2 below, or a salt thereof.
The agent according to claim 3 or 5, wherein the plant belonging to the genus Calendula is Calendula officinalis. A method for producing a compound represented by Chemical formula 1 below or a salt thereof, comprising the step of extracting the compound represented by Chemical formula 1 below or a salt thereof from a plant belonging to the genus Calendula.
A method for producing a compound represented by the following formula 2 or a salt thereof, comprising the step of extracting the compound represented by the following formula 2 or a salt thereof from a plant belonging to the genus Calendula.
The method according to claim 7 or 8, wherein the plant belonging to the genus Calendula is Calendula officinalis.