JP2024041560A - Inhibitor of phenol generation by 4-hydroxybenzoic acid decarboxylase - Google Patents

Abstract

To provide a low molecular weight compound that is highly safe when ingested, has a relatively inexpensive production cost, and has action of inhibiting phenol generation by Had.SOLUTION: A compound represented by the following formula (I) and one or more kinds of compounds selected from a physiologically acceptable salt thereof are used as an inhibitor of phenol generation by 4-hydroxybenzoic acid decarboxylase. (in the formula, R1 denotes OH and an oxygen heterocyclic compound, R2 denotes H, OH, monosaccharide, (1→2) disaccharide, or trisaccharide). It is preferable that a compound represented by the formula (I) be one or more kinds of compounds selected from sesamol, sesaminol, sesamolin, and sesaminol monoglucoside.SELECTED DRAWING: None

Description

The present invention is directed to the production of phenol by 4-hydroxybenzoic acid decarboxylase (hereinafter sometimes referred to as "Had"), which contains a specific lignan compound or its derivative contained in sesame seeds as an active ingredient. Concerning inhibitors of.

Diabetic nephropathy is one of the complications that develop as type 2 diabetes progresses, which is the most prevalent type of diabetes, and is a disease in which kidney function declines. Treating diabetic nephropathy costs as much as 1.5 trillion yen per year, the largest factor being dialysis treatment. To date, many causative substances for diabetic nephropathy have been identified, but no therapeutic drugs have yet been developed.

Recently, phenyl sulfate has been identified as a biomarker that can determine the risk of onset and stage of diabetic nephropathy (Patent Document 1), and phenyl sulfate has been implicated in renal damage caused by diabetic nephropathy. It has also been reported that there are some (non-patent document 1).

On the other hand, phenol is a precursor of phenyl sulfate, and is produced from L-tyrosine by tyrosine phenol lyase (TPL) possessed by intestinal bacteria resident in the intestines. Since the generated phenol is thought to be converted to phenyl sulfate through internal metabolism, it is predicted that inhibiting phenol production by TPL will improve renal damage caused by diabetic nephropathy. In fact, it has been reported that when 2-aza-DL-tyrosine, an inhibitor of TPL, is administered to renal failure model mice, blood phenyl sulfate concentration decreases and renal function improves (Patent Document 2). , Non-Patent Document 1). Furthermore, the present inventors have discovered that a specific lignan compound contained in sesame seeds has the effect of inhibiting phenol production by TPL (Japanese Patent Application No. 2022-26918).

Furthermore, in addition to TPL, it is presumed that there is a pathway in which 4-hydroxybenzoic acid, which is generated from L-tyrosine through internal metabolism, is generated by Had (non-TPL). Patent Document 2). However, it was not known that specific lignan compounds contained in sesame seeds have the effect of inhibiting phenol production by Had.

International Publication No. 2017/056498 pamphlet International Publication No. 2018/079832 pamphlet

Nat Commun. 2019 Apr 23;10(1):1835.doi:10.1038/s41467-019-09735-4. FEMS Microbiol Ecol. 2018 Sep 1;94(9):fiy125.

An object of the present invention is to provide a low-molecular compound that is highly safe when ingested, is relatively inexpensive to produce, and has the effect of inhibiting phenol production by Had.

Sesame seeds contain a large amount of antioxidant components, so they are widely used in foods, drinks, and medicines. Metabolic products called lignans (ie, sesame lignan compounds) are known as representative antioxidant components derived from sesame seeds, and are collectively referred to as plant-specific metabolites. Sesame lignan compounds have a structure in which two molecules of phenylpropanoid are bonded to each other through carbon atoms at the center of their side chains, and many of them have a basic skeleton of phenol, which serves as a substrate for the Had enzymatic reaction. Therefore, we focused on sesame lignan compounds and continued intensive research to solve the above problems, and as a result, we discovered that a specific sesame lignan compound has the effect of inhibiting phenol production by Had enzyme using 4-hydroxybenzoic acid as a substrate. Ta. Furthermore, it was confirmed that the above-mentioned specific sesame lignan compound has an effect of inhibiting Had-induced phenol production in various Had-producing bacteria. The present invention has been completed based on these findings.

That is, the present invention is as follows.
[1] Phenol produced by 4-hydroxybenzoic acid decarboxylase containing one or more compounds selected from the group consisting of the compound represented by the following formula (I) and its physiologically acceptable salts inhibitor of production.

In formula (I), R ¹ represents OH, a group represented by the following formula (II) or (III), and R ² represents H, OH, a monosaccharide, a (1→2) disaccharide, or a trisaccharide. Represents sugars.

〔２〕Ｒ^２が、Ｈ、ＯＨ、又は単糖類である、上記〔１〕に記載の阻害剤。
〔３〕式（Ｉ）で表される化合物が、セサモール、セサミノール、セサモリン、及びセサミノールモノグルコシドから選択される１種又は２種以上の化合物である、上記〔１〕又は〔２〕に記載の阻害剤。
〔４〕式（Ｉ）で表される化合物が、４－ヒドロキシ安息香酸デカルボキシラーゼによるフェノール生成を混合阻害する、上記〔１〕又は〔２〕に記載の阻害剤。

[2] The inhibitor according to [1] above, wherein R ² is H, OH, or a monosaccharide.
[3] In the above [1] or [2], the compound represented by formula (I) is one or more compounds selected from sesamol, sesaminol, sesamolin, and sesaminol monoglucoside. Inhibitors as described.
[4] The inhibitor according to [1] or [2] above, wherein the compound represented by formula (I) mixedly inhibits phenol production by 4-hydroxybenzoic acid decarboxylase.

Further, as another embodiment of the present invention,
One or more compounds selected from the compounds represented by the following formula (I) and physiologically acceptable salts thereof (hereinafter sometimes referred to as "the sesame lignan compound") are prepared using phenol by Had. A method of inhibiting the production, including the step of ingesting it into a subject in need of inhibition of the production;
the subject sesame lignan compounds for use in inhibiting phenol production by Had;
Use of the subject sesame lignan compounds to produce an inhibitor of phenol production by Had;
can be mentioned.

In formula (I), R ¹ represents OH, a group represented by the following formula (II) or (III), and R ² represents H, OH, a monosaccharide, a (1→2) disaccharide, or a trisaccharide. Represents sugars.

Further, as another embodiment of the present invention,
The present sesame lignan compound is ingested by a subject in need of prevention or treatment of symptoms or diseases caused by uremic substances (e.g., phenyl sulfate; the same shall apply hereinafter) whose precursor is phenol produced by Had in Had-producing bacteria. A method of preventing or treating the condition or disease, comprising:
The present sesame lignan compound for use in the prevention or treatment of symptoms or diseases caused by uremic substances having phenol precursors produced by Had in Had-producing bacteria;
Use of the present sesame lignan compound for producing a preventive or therapeutic agent for symptoms or diseases caused by uremic substances having phenol as a precursor produced by Had in Had-producing bacteria;
can be mentioned.

The present sesame lignan compound has the effect of inhibiting phenol production by Had derived from Had-producing bacteria. Furthermore, the present sesame lignan compound is a low-molecular compound related to components derived from sesame seeds used as foods, so it is highly safe when ingested, and the manufacturing cost is relatively low. Therefore, when the sesame lignan compound of the present invention is ingested, it is possible to obtain preventive and therapeutic effects on symptoms and diseases caused by uremic substances whose precursors are phenols produced by Had in Had-producing bacteria in living organisms such as the intestines.

Had from Escherichia coli strain BL21 (hereinafter sometimes referred to as "recombinant Escherichia coli") into which Had gene derived from Bacillus subtilis strain JCM1465 was introduced and its substrate 4-hydroxybenzoic acid were combined with eight types of sesame lignan compounds (sesamol). [SML], sesaminol monoglucoside [SMG], sesaminol [1→2] diglucoside [1-2 SDG], sesaminol [1→6] diglucoside [1-6 SDG], sesaminol triglucoside [STG], FIG. 2 is a diagram showing the results of incubation in the presence of sesamin, sesamolin, and sesamol) and measuring the amount of phenol produced. “*” and “**” in the figure indicate statistically significant differences (p< 0.05 and p<0.01). Had from Escherichia coli BL21 strain into which the Had gene from Bacillus subtilis JCM1465 strain was introduced was grown in the presence of its substrate 4-hydroxybenzoic acid ("substrate" in the figure) and the sesame lignan compound (sesamol) ("+" in the figure). ) or in the absence ("-" in the figure) for 30 minutes (Figure 2A), 1 hour (Figure 2B), 3 hours (Figure 2C), and 24 hours (Figure 2D), and the amount of phenol produced was determined. It is a figure showing the result of measuring. "**" in the figure indicates that there is a statistically significant difference (p<0.01) (the same applies hereinafter). Had-producing bacteria derived from Citrobacter coseri strain JCM1658 were grown in the presence of Had substrate 4-hydroxybenzoic acid (“substrate” in the figure) and the sesame lignan compound (sesamol) (“+” in the figure) or in the absence of Had. Results of measuring the amount of phenol produced after incubation for 30 minutes (Figure 3A), 1 hour (Figure 3B), 3 hours (Figure 3C), and 24 hours (Figure 3D) in the presence of FIG. Had-producing bacteria derived from Clybella intermedia strain JCM1238 were incubated in the presence (“+” in the figure) or in the absence of 4-hydroxybenzoic acid (“substrate” in the figure), which is a substrate for Had, and the sesame lignan compound (sesamol). FIG. 4 is a diagram showing the results of measuring the amount of phenol produced after incubation for 30 minutes (FIG. 4A) and 1 hour (FIG. 4B) in the presence (“-” in the diagram). Had-producing bacteria derived from Enterobacter strain ALC0001 were incubated in the presence ("+" in the diagram) or in the absence of 4-hydroxybenzoic acid ("substrate" in the diagram), which is a substrate for Had, and the sesame lignan compound (sesamol). FIG. 5 is a diagram showing the results of measuring the amount of phenol produced after incubation for 30 minutes (FIG. 5A), 1 hour (FIG. 5B), and 3 hours (FIG. 5C) in the presence (“-” in the diagram). Had-producing bacteria derived from Shigella strain ALC0002 were cultured in the presence (“+” in the figure) or absence of Had substrate 4-hydroxybenzoic acid (“substrate” in the figure) and the subject sesame lignan compound (sesamol). The results of incubating for 30 minutes (Figure 6A), 1 hour (Figure 6B), 3 hours (Figure 6C), and 24 hours (Figure 6D) and measuring the amount of phenol produced are shown below ("-" in the figure). FIG.

The inhibitor of the present invention contains the present sesame lignan compound, that is, one or more compounds selected from the following compounds of formula (I) and physiologically acceptable salts thereof, and It is an agent specified for the purpose of "inhibiting phenol production" (hereinafter sometimes referred to as "the subject inhibitor"). The sesame lignan compound of the present invention may be used alone as feed for non-human mammals, food/beverage products, or pharmaceuticals (preparations), or it may be mixed with additives to form a composition (i.e., a non-human mammal). It may also be used as a feed composition for human mammals, a food/beverage composition, or a pharmaceutical composition. Examples of the above-mentioned foods and drinks include health foods (functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally adjusted foods, supplements, etc.), foods with health claims (foods for specified health uses, nutritionally functional foods, functional foods, etc.) (labeled foods, etc.). In order to distinguish the present inhibitor from the inhibitor of phenol production by TPL in the previous application (Japanese Patent Application No. 2022-26918), it is not used for the purpose of "inhibiting phenol production by TPL."

In formula (I), R ¹ represents OH, a group represented by the following formula (II) or (III), and R ² represents H, OH, a monosaccharide, a (1→2) disaccharide, or a trisaccharide. Represents sugars.

In formula (I), examples of the monosaccharide in R ² include glucose, fructose, galactose, etc., and examples of the (1→2) disaccharide in R ² include (1→2) diglucoside, etc. Examples of the trisaccharide for ^R2 include triglucoside.

In this example, which will be described later, four types of sesame lignan compounds (sesamol, sesaminol, sesamolin, and sesaminol monoglucoside) have been shown to particularly effectively inhibit phenol production by Had. ), R ² is preferably H, OH, or a monosaccharide, and the compound of formula (I) is one or more selected from sesamol, sesaminol, sesamolin, and sesaminol monoglucoside. Compounds are more preferred.

As used herein, "Had" refers to an enzyme that catalyzes reaction 1 (4-hydroxybenzoic acid - carbon dioxide [CO ₂ ] → phenol) and the reverse reaction 2 (phenol + carbon dioxide [CO ₂ ] → 4-hydroxybenzoic acid), as shown in the following reaction formula.

The present sesame lignan compound has the effect of inhibiting phenol production by Had by inhibiting at least reaction 1 in the above reaction formula.

In the above reaction formula, the type of inhibition by the present sesame lignan compound is not particularly limited, and examples thereof include competitive inhibition, uncompetitive inhibition, mixed inhibition, and the like.

In this specification, "competitive inhibition" refers to an inhibition form in which an inhibitor (the sesame lignan compound of the present invention) binds to a substrate-binding site in an enzyme (Had) that is not bound to a substrate (4-hydroxybenzoic acid). In competitive inhibition, the inhibitor does not bind to the enzyme-substrate complex.

As used herein, "uncompetitive inhibition" means that the inhibitor (the present sesame lignan compound) does not bind to the enzyme (Had) that is not bound to the substrate (4-hydroxybenzoic acid) and inhibits the enzyme-substrate complex. means an inhibitory form that binds only to the body.

As used herein, "mixed inhibition" means that the inhibitor (the present sesame lignan compound) has different effects on both the enzyme (Had) that is not bound to the substrate (4-hydroxybenzoic acid) and the enzyme-substrate complex. Refers to the type of inhibition that binds with the inhibition constant (Ki).

As the present sesame lignan compound, since its effect has been demonstrated in the present example described below, a compound that inhibits phenol production by Had in the form of mixed inhibition (mixed inhibition) can be suitably exemplified.

The origin of the above-mentioned Had is not particularly limited, but since its effect has been demonstrated in the present embodiment described below, a suitable example is Had produced by Had-producing bacteria (more specifically, Had-producing bacteria in various organs or tissues in the living body of a mammal) (in other words, Had derived from Had-producing bacteria). Therefore, the present inhibitor can be advantageously used as a preventive or therapeutic agent for symptoms or diseases caused by uremic substances whose precursor is phenol produced by Had in Had-producing bacteria in the living body of a mammal.

In this specification, "Had-producing bacteria" may be Had-producing enterobacteria or Had-producing non-intestinal bacteria, and specifically, for example, bacteria of the genus Citrobacter ( For example, bacteria of the genus Kluyvera (e.g., C. koseri, C. freundii), bacteria of the genus Kluyvera (e.g., K. intermedia), spp. Bacteria (e.g., E. cloacae, E. aerogenes), Shigella (e.g., S. Shigella dysenteriae), Klebsiella Bacteria (e.g., Klebsiella pneumoniae), Escherichia (e.g., E. coli), Bacillus (e.g., B. subtilis), etc. can be mentioned.

As used herein, "various organs or tissues" include, for example, large intestine (colon or rectum), stomach, liver, heart, brain, spinal cord, lung, esophagus, duodenum, small intestine, skin, prostate, bladder, uterus, kidney. , pancreas, spleen, trachea, bronchi, gallbladder, bile duct, etc.

In this specification, mammals include humans, non-human mammals (e.g., monkeys, mice, rats, dogs, cats, livestock [e.g., rabbits, pigs, horses, cows, sheep, goats, deer]), etc. can be mentioned.

In this specification, "symptoms or diseases caused by uremic substances whose precursor is phenol produced by Had in Had-producing bacteria" include, for example, anorexia, nausea, vomiting, bad breath, stomatitis, enteritis, etc. Gastrointestinal system abnormalities; nervous system abnormalities such as avolition, apathy, impaired memory, depression, somnolence, coma, polyneuritis, (developmental) coordination disorder, anorexia; arteriosclerosis, anemia, erythropoietic disorders , hypertension, ischemic heart disease, pericarditis, myocarditis, (blood) coagulation abnormalities, heart failure, cardiovascular disorders such as cardiovascular disorders; pigmentation, itching (feeling), subcutaneous bleeding, skin atrophy, infection, atopy. , skin abnormalities (diseases) such as hair loss; albuminuria, acute renal failure, acute tubular necrosis, renal anemia, chronic renal failure, tubulointerstitial disorder, acute nephritis, chronic nephritis, nephrosis, diabetic nephropathy, arteries Renal dysfunction such as sclerosing nephropathy, renal bone abnormalities (e.g., renal osteodystrophy), and effusion; stroke; myocardial infarction; cancer; autism; immunodeficiency; bone abnormalities; hyperparathyroidism ; insulin resistance; malnutrition; inflammation; tremor; etc. In addition, the above-mentioned symptoms or diseases include IgA nephropathy, cystic kidney disease, liver dysfunction (e.g., fulminant hepatitis, fatty liver, NASH, NAFLD), cancer (e.g., biliary carcinogenesis), etc., which involve LPS (Lipopolysaccharide). Symptoms or diseases; inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; arteriosclerosis-related diseases; autoimmune diseases such as lupus, scleroderma, and rheumatism; obesity; metabolic syndrome; diabetes; autism; Parkinson's disease; Alzheimer's disease; also includes sarcopenia. Note that the above symptoms or diseases do not include symptoms or diseases that are not caused by uremic substances whose precursor is phenol produced by Had in Had-producing bacteria, but are caused by factors other than these uremic substances. .

The sesame lignan compound of the present invention can be synthesized by purchasing a commercially available product or by appropriately combining known reactions. The raw material can be synthesized by adding a general protective group to a commercially purchased product or a commercially purchased product. In addition, synthesis, purification, etc. by protection and deprotection, which are commonly used in organic chemistry, can be applied to any of the steps.

As used herein, "physiologically acceptable salts" are salts that, within the scope of reasonable medical, pharmaceutical, or biological judgment, have excessive toxicity when used in contact with mammalian tissue. , salts that are suitable according to a moderate benefit/risk ratio, without irritation, allergic reactions, and other problems and complications. Physiologically acceptable salts include, for example, hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, and succinate. Acid addition salts such as salts, oxalates, lactates, tartrates, glycolates, methanesulfonates, butyrates, valerates, citrates, fumarates, maleates, malates; ammonium Salts; alkali metal salts such as sodium salts, lithium salts, potassium salts; aluminum salts; alkaline earth metal salts such as calcium salts and magnesium salts; salts with organic bases such as dicyclohexylamine salts and N-methyl-D-glucamine. ; Salts with amino acids such as arginine, lysine, ornithine; Salts formed by basic nitrogen-containing groups; and the like.

The present sesame lignan compound also includes various isomers (e.g., optical isomers, positional isomers, tautomers, etc.), solvates such as hydrates, crystal polymorphs, and prodrugs such as esters. be done. Further, compounds in which one or more atoms constituting the compound of formula (I) are isotopes are also included in the present sesame lignan compound. Examples of isotopes contained in the sesame lignan compound include hydrogen, carbon, nitrogen, oxygen, phosphorus, bromine, and chlorine isotopes, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N , ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, and ³⁷ Cl.

The targets for ingestion (administration) of this inhibitor are not particularly limited, but usually include mammals that require inhibition of phenol production by Had, and uremic diseases in which phenol produced by Had in Had-producing bacteria is a precursor. A mammal in need of prevention or treatment of symptoms or diseases caused by the substance.

Examples of methods for ingesting (administering) the subject inhibitor include oral ingestion in the form of powder, granules, tablets, capsules, syrups, suspensions, etc. ); method of injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, local administration) in the form (dosage form) of a solution, emulsion, suspension, etc. (parenteral administration); intranasal administration in the form of a spray. Methods of administration (parenteral administration); and the like.

The subject inhibitor for oral ingestion can be in the form of sustained release or enteric-coated formulations. Such enteric-coated agents, for example, coat granules containing the sesame lignan compound as an active ingredient with an enteric coating (i.e., a base [enteric component] that is resistant to gastric juice and dissolves in the small intestine). Alternatively, tablets obtained by adding a lubricant to granules containing the sesame lignan compound as an active ingredient and compressing the same may be coated with an enteric coating. can.

As a method for obtaining the present inhibitor whose dosage form is a tablet, for example, after mixing the present sesame lignan compound as an active ingredient and optional components (additives) that may be added as necessary, this mixture is compressed. Examples include a method of obtaining a tablet by molding, and a method of coating the tablet obtained after compression molding with an enteric component (a method of forming an enteric agent), with the latter method being preferred.

Examples of the enteric components include shellac, zein, hydroxymethyl cellulose phthalate, carboxymethyl cellulose, carboxymethyl ethyl cellulose, cellulose acetate phthalate, methacrylic acid copolymers, water-insoluble ethyl cellulose, aminoalkyl methacrylate copolymers, beer yeast cell walls (e.g. , trade name Yeast Wrap, etc.), tapioca starch, gelatin, pectin, etc., among which shellac is preferred. In addition, whether or not it is an enteric-coated drug can be confirmed by the disintegration test method of the 14th edition of the Japanese Pharmacopoeia.

The intake amount of the Sesame Lignan Compound contained in the Inhibitor is appropriately determined depending on the age, weight, sex, symptoms, drug sensitivity, species, etc. of the subject (mammal). The calculated concentration ranges from 0.1 μg to 200 mg/kg (body weight)/day. The inhibitor may be taken once or in multiple doses (for example, 2 to 4 times) per day.

As used herein, "additives" include conventional physiologically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonic agents, additives, Examples of ingredients include coating agents, solubilizers, lubricants, sliding agents, solubilizing agents, lubricants, flavoring agents, sweeteners, solvents, gelling agents, and nutrients. Specifically, such ingredients include water, physiological saline, animal fat and oil, vegetable oil, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropylcellulose, polyalkylene glycol, Examples include polyvinyl alcohol and glycerin.

The present inhibitor may contain components other than the present sesame lignan compound as an inhibitory component of phenol production by Had, but since the present sesame lignan compound alone exhibits an excellent inhibitory effect, Preferably, it does not contain the above-mentioned inhibitory components (eg, compounds such as proteins, amino acids, DNA, RNA, polymers, etc.; plant-derived extracts [eg, sesame lignan compounds]).

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

1. Confirmation that the sesame lignan compound inhibits phenol production by Had (1)
Lignan compounds contained in sesame seeds (ie, sesame lignan compounds) were analyzed using Had derived from Bacillus subtilis strain JCM1465 to determine whether they have the effect of inhibiting phenol production by Had.

1-1 Materials and methods [Preparation of Had crude enzyme solution]
A crude Had enzyme solution was prepared according to the following procedures [1] to [7] and used in the subsequent [Had inhibitory activity test].
[1] Had-producing E. coli strain BL21 was grown in 50 mL of a semi-solid liquid medium for GAM glycolysis (manufactured by Nissui) containing 0.1% glycerol, 1% 4-hydroxybenzoic acid, and 100 μg/mL ampicillin. The cells were cultured with shaking at 37° C. and 120 rpm until OD ₆₀₀ =0.5.
[2] After rapidly cooling on ice, the mixture was allowed to stand at 15°C for 30 minutes.
[3] 1mM IPTG was added and cultured statically (anaerobic culture) at 15°C for 24 hours in a jar containing Aneropack.
[4] The culture solution containing the cultivated Had-producing E. coli strain BL21 was collected by centrifugation (15,000 x g, 10 minutes, 4°C), and after removing the supernatant (liquid medium), the reaction buffer was (a solution containing 50mM KPB [pH 7.0] and 2mM 2-mercaptoethanol).
[5] Bacteria were collected by centrifugation (15,000 x g, 10 minutes, 4°C), and after removing the supernatant (the above reaction buffer), they were suspended in the above reaction buffer.
[6] Bacteria were collected by centrifugation (15,000 x g, 10 minutes, 4°C), and after removing the supernatant (the above reaction buffer), they were suspended in the above reaction buffer.
[7] After performing ultrasonic disruption using SONIFIER (registered trademark) SFX250 (manufactured by EMERSON), centrifugation was performed, and the supernatant was collected as a Had crude enzyme solution.

[Had inhibitory activity test]
The Had inhibitory activity test was conducted according to the following procedures [1] to [4].
[1] Reaction buffer (200 mM potassium phosphate buffer [KPB] [pH 7.0], and 95 μL of a solution containing 4 mM 2-mercaptoethanol) was incubated at 37° C. for 10 minutes. As a comparison, a reaction buffer containing 4-hydroxybenzoic acid and no sesame lignan compound was similarly incubated.
[2] 5 μL of Had's crude enzyme solution was added and incubated at 37° C. for 3 hours.
[3] After adding 100 μL of a reaction stop solution (a solution containing 40% acetonitrile and 4% trifluoroacetic acid [TFA]), the mixture was centrifuged (15,000×g, 10 minutes, 4° C.).
[4] The supernatant was collected and subjected to liquid chromatography (HPLC) analysis according to the conditions shown in Table 2 to quantify the amount of phenol. The amount of phenol was calculated as the relative value of the phenol concentration in the reaction buffer containing various sesame lignan compounds, with the phenol concentration in the reaction buffer containing no sesame lignan compound being 100 (see the vertical axis in FIG. 1).

表中、ＳＭＧは、文献「The Plant Journal, (2019), doi: 10.1111/tpj.14586」記載の方法に従って精製し、残りの７種類の化合物（セサミン、ＳＭＬ、セサモール、１－２ ＳＤＧ、１－６ ＳＤＧ、ＳＴＧ、及びセサモリン）については、長良サイエンス社製のものを使用した。

In the table, SMG was purified according to the method described in the literature "The Plant Journal, (2019), doi: 10.1111/tpj.14586", and the remaining seven compounds (sesamin, SML, sesamol, 1-2 SDG, 1 -6 SDG, STG, and sesamolin) manufactured by Nagara Science Co., Ltd. were used.

1-2 Results When recombinant E. coli-derived Had and its substrate, 4-hydroxybenzoic acid, were incubated in the presence of the eight sesame lignan compounds shown in Table 1, the amount of phenol produced was reduced when seven of the sesame lignan compounds (sesamol, sesaminol [SML], sesamolin, sesaminol monoglucoside [SMG], sesamin, diglucoside [1-2 SDG], and sesaminol monoglucoside [SMG]) were used compared to incubation in the absence of these sesame lignan compounds. In particular, the amount of phenol produced was significantly reduced for four of the sesame lignan compounds (sesamol, SML, sesamolin, and SMG) (see Figure 2).
This result indicates that the above seven types of sesame lignan compounds included in the present sesame lignan compounds have an inhibitory effect on phenol production by Had.

2. Inhibition type and inhibition constant of Had by the sesame lignan compound Next, we determined the type of inhibition of Had by the sesame lignan compound, and the inhibition constant of Had by the sesame lignan compound. Specifically, 50% inhibition of phenol production by Had was observed. The concentration of the sesame lignan compound was calculated.

2-1 Method The Had inhibitory activity test of Example 1 was conducted using 100 μM of two types of sesame lignan compounds (sesamol and SML), and the phenol production rate (μM/min) (hereinafter referred to as "v") was calculated. . Next, v is plotted on the vertical axis and 4-hydroxybenzoic acid concentration (mM) (hereinafter referred to as [S]) is plotted on the horizontal axis, and an approximate curve is fitted using SigmaPlot (manufactured by Hulinx) to determine the speed. The theoretical parameters were calculated. Then, plot 1/v on the vertical axis and 1/[S] on the horizontal axis (Lineweaver Burk plot), and from the shape of the approximate straight line, determine the form of Had inhibition by various sesame lignan compounds and calculate the Had inhibition constant. I did it.

2-2 Results As a result, it was revealed that the inhibition type and inhibition constant (Ki) of Had by two types of sesame lignan compounds (sesamol and SML) are as shown in Table 3.

3. Confirmation that the sesame lignan compound inhibits phenol production by Had (2)
Regarding sesame lignan compounds, whether or not they have the effect of inhibiting phenol production by Had was determined using the E. coli BL21 strain (herein sometimes referred to as "Had-producing E. coli BL21 strain") into which the Had gene derived from Bacillus subtilis strain JCM1465 was introduced. Analyzed using Had.

3-1 Materials and Methods [Preparation of crude enzyme solution of Had]
A crude enzyme solution of Had was prepared according to the following steps (1) to (7) and used in the subsequent [Had inhibitory activity test].
[1] Had-producing Escherichia coli BL21 strain was cultured with shaking at 37°C and 120 rpm in 50 mL of semi-solid liquid medium for GAM glycolysis (manufactured by Nissui Co., Ltd.) containing 0.1% glycerol, 1% 4-hydroxybenzoic acid, and 100 μg/mL ampicillin until OD ₆₀₀ reached 0.5.
[2] After rapid cooling on ice, the mixture was allowed to stand at 15°C for 30 minutes.
[3] 1 mM IPTG was added, and the mixture was subjected to static culture (anaerobic culture) in an Anaeropack-containing jar at 15° C. for 24 hours.
[4] The culture solution containing the cultured Had-producing E. coli BL21 strain was centrifuged (15,000 × g, 10 minutes, 4°C) to harvest the bacteria. After removing the supernatant (liquid medium), the bacteria were suspended in a reaction buffer (a solution containing 50 mM KPB [pH 7.0] and 2 mM 2-mercaptoethanol).
[5] The cells were harvested by centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant (the above-mentioned reaction buffer) was removed, and the cells were suspended in the above-mentioned reaction buffer.
[6] The cells were harvested by centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant (the above-mentioned reaction buffer) was removed, and the cells were suspended in the above-mentioned reaction buffer.
[7] After ultrasonic disruption using SONIFIER (registered trademark) SFX250 (manufactured by EMERSON), the cells were centrifuged and the supernatant was collected as a crude enzyme solution of Had.

[Had inhibitory activity test]
The Had inhibitory activity test was conducted according to the following procedures [1] to [2].
[1] 4-hydroxybenzoic acid and sesamol were added to Had's crude enzyme solution to a final concentration of 5 mM, and incubated at 37°C for various times (30 minutes, 1 hour, 3 hours, or 24 hours). It was incubated in a jar containing an anero pack. As a comparison, Had's crude enzyme solution to which either 4-hydroxybenzoic acid or sesamol was added and Had's crude enzyme solution to which neither 4-hydroxybenzoic acid nor sesamol were added were incubated in the same way. did.
[2] After centrifugation (15,000 x g, 10 minutes, 4°C) and passing the supernatant through a 0.45 μm hydrophilic filter, HPLC analysis was performed according to the conditions shown in Table 2 to quantify the amount of phenol. (See Figure 2).

3-2 Results When Had-producing E. coli strain BL21-derived Had was incubated with its substrate 4-hydroxybenzoic acid in the presence of the present sesame lignan compound (sesamol) for at least 30 minutes, when it was incubated in the absence of the present sesame lignan compound It was shown that the amount of phenol produced was significantly reduced compared to the above (see Figure 2).
This result shows that the present sesame lignan compound inhibits phenol production by Had in a short period of time.

4. Confirmation that the present sesame lignan compound inhibits phenol production by Had-producing bacteria It was analyzed whether the present sesame lignan compound, which has an inhibitory effect on phenol production by Had, also exerts an inhibitory effect on Had-producing bacteria.

4-1 Materials and methods [Had-producing bacteria]
As Had-producing bacteria, there are four types of intestinal bacteria that produce Had, specifically, Had-producing bacteria derived from Citrobacter koseri JCM1658 strain, and Had-producing bacteria derived from Kluyvera intermedia JCM1238 strain. bacteria (all obtained from the RIKEN BioResource Research Center), Had-producing bacteria derived from Enterobacter sp. ALC0001 strain, and Had-producing bacteria derived from Shigella sp. ALC0002 strain (both were research strain) was used in the following [Had inhibitory activity test].

[Had inhibitory activity test]
The Had inhibitory activity test was carried out according to the following procedures [1] to [5].
[1] Had-producing bacteria were statically cultured (anaerobic culture) in a semi-solid liquid medium for GAM sugar decomposition (manufactured by Nissui Co., Ltd.) containing 0.1% glycerol and 1% 4-hydroxybenzoic acid at 37°C for 24 hours in a jar containing an Anaeropack.
[2] The culture solution containing the cultured cells was centrifuged (15,000 × g, 10 minutes, 4 ° C.) to collect the cells, and after removing the supernatant (liquid medium), the cells were suspended in a reaction buffer (a solution containing 50 mM KPB [pH 7.0] and 2 mM 2-mercaptoethanol).
[3] The cells were harvested by centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant (the above-mentioned reaction buffer) was removed, and the cells were suspended in the above-mentioned reaction buffer.
[4] The bacteria were harvested by centrifugation (15,000×g, 10 min, 4° C.), and the supernatant (reaction buffer) was removed. The Had-producing bacteria were then suspended in the reaction buffer containing 5 mM 4-hydroxybenzoic acid and 5 mM sesamol to give an OD ₆₀₀ of 2.0, and incubated in a jar containing an Anaeropack for various times (30 min, 1 h, 3 h, or 24 h) at 37° C. As comparative controls, Had-producing bacteria were suspended in the reaction buffer containing either 4-hydroxybenzoic acid or sesamol, and in the reaction buffer containing neither 4-hydroxybenzoic acid nor sesamol, and incubated in the same manner.
[5] After centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant was passed through a 0.45 μm hydrophilic filter, and then HPLC analysis was carried out according to the conditions shown in Table 2 to quantify the amount of phenol (see FIGS. 3 to 6).

4-2 Results When four types of Had-producing bacteria (Citrobacter koseri JCM1658 strain-derived Had-producing bacteria, Kluyvera intermedia JCM1238 strain-derived Had-producing bacteria, Enterobacter sp. ALC0001 strain-derived Had-producing bacteria, and Shigella sp. ALC0002 strain-derived Had-producing bacteria) were incubated for at least 30 minutes in the presence of their substrate, 4-hydroxybenzoic acid, and the present sesame lignan compound (sesamol), the amount of phenol produced was significantly reduced compared to when incubated in the absence of the present sesame lignan compound (see Figures 3 to 6).
This result indicates that the present sesame lignan compounds such as sesamol are taken up into the cells of enterobacteria that produce Had, and inhibit the production of phenol by Had produced by the Had-producing bacteria.

The present invention contributes to the prevention and treatment of symptoms and diseases caused by uremic substances (eg, phenyl sulfate) whose precursor is phenol produced by Had in Had-producing bacteria in vivo.

Claims (4)

Inhibition of phenol production by 4-hydroxybenzoic acid decarboxylase, comprising one or more compounds selected from the group consisting of compounds represented by the following formula (I) and physiologically acceptable salts thereof: agent.

(In the formula, R ¹ represents OH, a group represented by the following formula (II) or (III), and R ² represents H, OH, a monosaccharide, a (1→2) disaccharide, or a trisaccharide. represent.)

2. The inhibitor according to claim 1, wherein ^R2 is H, OH, or a monosaccharide. The inhibitor according to claim 1 or 2, wherein the compound represented by formula (I) is one or more compounds selected from sesamol, sesaminol, sesamolin, and sesaminol monoglucoside. The inhibitor according to claim 1 or 2, wherein the compound represented by formula (I) mixedly inhibits phenol production by 4-hydroxybenzoic acid decarboxylase.