JP2022135978A - Agent for inhibiting phenol production by tyrosine phenol-lyase and/or indole production by tryptophan indole-lyase - Google Patents

Abstract

To provide a low-molecular-weight compound having the action of inhibiting phenol production by TPL and/or indole production by TIL, wherein the compound can be taken safely and can be prepared at relatively low cost.SOLUTION: An agent for inhibiting phenol production by TPL and/or indole production by TIL contains one or more compounds selected from a compound represented by the following formula (I) and a physiologically acceptable salt thereof (where R1 is OH or the like, R2 is H, OH, a monosaccharide, or a disaccharide).SELECTED DRAWING: None

Description

The present invention is an inhibitor of phenol production by tyrosine phenol-lyase (hereinafter sometimes referred to as "TPL"), which contains a specific lignan compound contained in sesame seeds or a derivative thereof as an active ingredient. and/or inhibitors of indole formation by tryptophan indole-lyase (hereinafter sometimes referred to as "TIL").

Diabetic nephropathy is one of the complications that develops with the progression of type 2 diabetes, which has a large number of sufferers among diabetes, and is a disease in which renal function declines. Treatment of diabetic nephropathy requires an annual cost of 1.5 trillion yen, and the biggest factor is dialysis treatment. Although many causative substances of diabetic nephropathy have been identified so far, the current situation is that no therapeutic agent has been developed yet.

Recently, phenyl sulfate has been identified as a biomarker capable of determining the onset risk and disease stage of diabetic nephropathy (Patent Document 1), and such phenyl sulfate is involved in renal damage due to diabetic nephropathy. It has also been reported that there are (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 in the intestine. Since the produced phenol is considered to be converted to phenyl sulfate through metabolism in the body, inhibition of phenol production by TPL is expected to ameliorate nephropathy due to diabetic nephropathy. In fact, it has been reported that when 2-aza-DL-tyrosine, a TPL inhibitor, is administered to renal failure model mice, the blood phenyl sulfate concentration is reduced and renal function is improved (Patent Document 2). , Non-Patent Document 1). However, it was not known that a specific lignan compound contained in sesame seeds has an inhibitory effect on phenol production by TPL. Similarly, indole-related compounds are also suspected to be involved in renal disorders. was also hitherto unknown.

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.

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

Since sesame seeds contain a large amount of antioxidant components, they are widely used as foods, beverages and pharmaceuticals. Metabolites called lignans (that is, sesame lignan compounds) are known as typical antioxidant components derived from sesame seeds, and are also called plant-specific metabolites. A sesame lignan compound has a structure in which two molecules of phenylpropanoid are bonded between carbon atoms in the center of side chains, and many of them have a phenolic basic skeleton that serves as a substrate for the enzymatic reaction of TPL. Therefore, we focused on sesame lignan compounds and continued intensive research to solve the above problems. It was found to have an inhibitory effect on indole formation by the TIL enzyme using tryptophan as a substrate. Furthermore, it was confirmed that the specific sesame lignan compound has an effect of inhibiting phenol production by TPL in various TPL-producing bacteria and an effect of inhibiting indole production by TIL in various TIL-producing bacteria. The present invention has been completed based on these findings.

That is, the present invention is as follows.
[1] Phenol production by tyrosine phenol lyase and/or tryptophan indole lyase containing one or more compounds selected from compounds represented by the following formula (I) and physiologically acceptable salts thereof inhibitor of indole formation by

（式中、Ｒ^１は、ＯＨ、又は以下の式（II）で表される基を表し、Ｒ^２は、Ｈ、ＯＨ、単糖類、又は二糖類を表す。）

(In the formula, R ¹ represents OH or a group represented by formula (II) below, and R ² represents H, OH, a monosaccharide, or a disaccharide.)

〔２〕化合物が、チロシンフェノールリアーゼによるフェノール生成を不競合阻害する、上記〔１〕に記載の阻害剤。
〔３〕化合物が、トリプトファンインドールリアーゼによるインドール生成を競合阻害又は混合阻害する、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、セサミン、セサミノール、セサモール、セサミノールモノグルコシド、セサミノール（１→２）ジグルコシド、及びセサミノール（１→６）ジ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〕～〔３〕のいずれかに記載の阻害剤。

[2] The inhibitor of [1] above, wherein the compound uncompetitively inhibits phenol production by tyrosine phenol lyase.
[3] The inhibitor of [1] or [2] above, wherein the compound competitively inhibits or mixedly inhibits indole formation by tryptophan indole lyase.
[4] The compound represented by formula (I) is one selected from sesamin, sesaminol, sesamol, sesaminol monoglucoside, sesaminol (1→2) diglucoside, and sesaminol (1→6) diglucoside or two or more compounds, the inhibitor according to any one of the above [1] to [3].

As another embodiment of the present invention,
One or two or more compounds selected from compounds represented by the following formula (I) and physiologically acceptable salts thereof (hereinafter sometimes referred to as the "present sesame lignan compound") are added to phenol by TPL. a method of inhibiting production and/or indole production by TIL, comprising administering to a subject in need of such inhibition; and
a subject sesame lignan compound for use in inhibiting phenol production by TPL and/or indole production by TIL;
Use of the subject sesame lignan compounds for the manufacture of inhibitors of phenol formation by TPL and/or indole formation by TIL;
can be mentioned.

In formula (I), ^R1 represents OH or a group represented by formula (II) below, and ^R2 represents H, OH, a monosaccharide, or a disaccharide.

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 whose precursors are phenols produced by TPL in TPL-producing bacteria (e.g., phenyl sulfate; the same shall apply hereinafter). a method of preventing or treating the condition or disease, comprising steps;
The present sesame lignan compound is used to treat symptoms or diseases caused by uremic substances whose precursor is indole produced by TIL in TIL-producing bacteria (e.g., indoxyl sulfate, indole-3-acetic acid, hereinafter the same). a method of preventing or treating a condition or disease, including administering to a subject in need of such prevention or treatment;
a subject sesame lignan compound for use in the prevention or treatment of a condition or disease caused by a phenol precursor uremic substance produced by TPL in TPL-producing bacteria; and
A subject sesame lignan compound for use in the prevention or treatment of a condition or disease caused by an indole-precursor uremic substance produced by TIL in TIL-producing bacteria; and
Use of the present sesame lignan compound for producing a preventive or therapeutic agent for symptoms or diseases caused by uremic substances whose precursors are phenols produced by TPL in TPL-producing bacteria; and
Use of the present sesame lignan compound for producing a preventive or therapeutic agent for symptoms or diseases caused by uremic substances whose precursor is indole produced by TIL in TIL-producing bacteria;
can be mentioned.

The present sesame lignan compound has an action of inhibiting phenol production by TPL derived from TPL-producing bacteria and an action of inhibiting indole production by TIL derived from TIL-producing bacteria. In addition, since the sesame lignan compound of the present invention is a low-molecular-weight compound related to sesame-derived components used as food, it is highly safe when ingested, and the production cost is relatively low. For this reason, when the present sesame lignan compound is ingested, it is possible to prevent or treat symptoms or diseases caused by uremic substances whose precursor is phenol produced by TPL in TPL-producing bacteria in the body such as the intestine, and to prevent or treat symptoms or diseases caused by TIL-producing bacteria. It is possible to obtain preventive and therapeutic effects for symptoms or diseases caused by uremic substances whose precursor is indole produced by TIL in .

FIG. 4 shows the result of purifying TPL from recombinant Escherichia coli JM109 strain expressing TPL using ammonium sulfate (ammonium sulfate) precipitation method. Lane 0 shows "molecular weight marker", lane 1 shows "crude enzyme solution", lane 2 shows "0-30% ammonium sulfate saturated fraction", lane 3 shows "30-70% ammonium sulfate saturated fraction" minutes" and lane 4 shows the "70-100% ammonium sulfate saturated fraction". Arrowheads indicate the 51.4 kDa TPL-derived band. TPL and its substrate L-tyrosine were combined with eight sesame lignan compounds (sesaminol [SML], sesaminol monoglucoside [SMG], sesaminol [1→2] diglucoside [1-2 SDG], sesaminol [1 → 6] Diglucoside [1-6 SDG], sesaminol triglucoside [STG], sesamin, sesamolin, and sesamol), incubation in the presence of, is a diagram showing the results of measuring the phenol production level. "*" and "**" in the figure are statistically significant (p < 0.05 and p<0.01). TIL and its substrate L-tryptophan were incubated in the presence of three instant sesame lignan compounds (sesamine [Figure 3A], SML [Figure 3B], and sesamol [Figure 3C]) and indole production levels were measured. It is a figure which shows a result. "*" and "**" in the figure indicate a statistically significant difference from the results of incubation in the absence of the above three sesame lignan compounds ("-" in the figure). (p<0.05 and p<0.01). TIL and its substrate L-tryptophan were incubated in the presence of two sesame lignan compounds (1-2 SDG [Fig. 4A] and 1-6 SDG [Fig. 4B]), and the indole production level was measured. It is a figure which shows. "**" in the figure indicates a statistically significant difference (p<0 .01). E. coli JM109 strain-derived TPL-producing bacteria and L-tyrosine, which is a substrate for TPL, were incubated in the presence of three sesame lignan compounds (sesamine [Fig. 5A], SML [Fig. 5B], and sesamol [Fig. 5C]). and shows the results of measuring the phenol production level. "*" and "**" in the figure indicate a statistically significant difference from the results of incubation in the absence of the above three sesame lignan compounds ("-" in the figure). (p<0.05 and p<0.01). TPL-producing bacteria derived from Fusobacterium periodonticum strain JCM12991 and L-tyrosine, which is a substrate of TPL, in the presence of the present sesame lignan compound (sesamol) for 30 minutes (Fig. 6A), 1 hour (Fig. 6B), and 3 hours. (FIG. 6C) It is a figure which shows the result of incubating and measuring a phenol production level. "*" and "**" in the figure indicate statistically significant differences (p<0.05 and p<0.01), respectively (same below). TPL-producing bacteria derived from Citrobacter coseri strain JCM1658 and L-tyrosine, which is a substrate of TPL, were treated in the presence of the present sesame lignan compound (sesamole) for 30 minutes (Fig. 7A), 1 hour (Fig. 7B), 3 hours (Fig. 7C), and incubation for 24 hours (Fig. 7D), and the results of measuring phenol production levels. Clostridium cochlearium JCM1396 strain-derived TPL-producing bacteria and L-tyrosine, which is a substrate of TPL, were treated in the presence of the present sesame lignan compound (sesamole) for 30 minutes (Fig. 8A), 1 hour (Fig. 8B), and 3 hours (Fig. 8C), and incubation for 24 hours (Fig. 8D), and the results of measuring phenol production levels. Escherichia coli BL21 (DE3) strain-derived TIL-producing bacteria and L-tryptophan, which is a substrate of TIL, in the presence of the present sesame lignan compound (sesamole) for 30 minutes (Fig. 9A), 1 hour (Fig. 9B), 3 hours (Fig. 9C), and incubation for 24 hours (FIG. 9D) to measure indole production levels. Citrobacter coseri strain JCM1658-derived TIL-producing bacteria and L-tryptophan, which is a substrate for TIL, were treated in the presence of the present sesame lignan compound (sesamole) for 30 minutes (Fig. 10A), 1 hour (Fig. 10B), and 3 hours (Fig. 10B). 10C), and incubation for 24 hours (FIG. 10D) to measure the level of indole production. TIL-producing bacteria derived from Bacteroides thetaiotaomicron JCM5827 strain and L-tryptophan, which is a substrate of TIL, in the presence of the present sesame lignan compound (sesamol) for 30 minutes (Fig. 11A), 1 hour (Fig. 11B), 3 hours ( FIG. 11C) and incubation for 24 hours (FIG. 11D) and measurement of indole production levels. FIG. 12 shows the results of analysis of phenol concentration in mouse feces of 3 groups (control group [FIG. 12A], m-Tyr-administered group [FIG. 12B], and present sesame lignan compound-administered group [FIG. 12C]). "**" in the figure indicates that there is a statistically significant difference (p<0.01).

The inhibitors of the present invention comprise the subject sesame lignan compounds, i.e., one or more compounds selected from the following compounds of formula (I) and physiologically acceptable salts thereof, and "for inhibiting phenol production and/or for inhibiting indole production by TIL" (hereinafter sometimes referred to as "inhibitor"). The present inhibitor, the present sesame lignan compound, may be used alone as a feed for non-human mammals, food and drink, or pharmaceuticals (formulations), or may be mixed with additives to form a composition (i.e., non- You may use it as a feed composition for human mammals, a food-drinks composition, or a pharmaceutical composition). Examples of the food and drink include health foods (functional foods, nutritional supplements, health supplements, nutrient-enriched foods, nutrient-adjusting foods, supplements, etc.), health functional foods (specified health foods, nutritional functional foods, functional labeling foods, etc.).

In formula (I), ^R1 represents OH or a group represented by formula (II) below, and ^R2 represents H, OH, a monosaccharide, or a disaccharide.

In formula (I), examples of monosaccharides for R ² include glucose, fructose, galactose, and physiologically acceptable salts thereof. Examples of disaccharides for R ² include 1, 2-diglucoside, 1,4-diglucoside, 1,6-diglucoside, physiologically acceptable salts thereof and the like can be mentioned.

As the compound of formula (I), since its effect is demonstrated in the Examples described later, sesamin, sesaminol, sesamol, sesaminol monoglucoside, sesaminol (1→2) diglucoside, and sesaminol (1 →6) One or more compounds selected from diglucosides can be preferably exemplified.

As used herein, “TPL” means reaction 1 (L-tyrosine + water [H ₂ O] → phenol + pyruvic acid + ammonia [NH ₃ ]) and An enzyme that catalyzes the retrograde reaction 2 (phenol+pyruvate+ammonia [NH ₃ ]→L-tyrosine+water [H ₂ O]).

The present sesame lignan compound inhibits at least Reaction 1 in Reaction Formula 1, thereby inhibiting phenol production by TPL.

As used herein, "TIL" means reaction 1 (L-tryptophan + water [H ₂ O] → indole + pyruvic acid + ammonia [NH ₃ ]) and An enzyme that catalyzes the retrograde reaction 2 (indole+pyruvate+ammonia [NH ₃ ]→L-tryptophan+water [H ₂ O]).

The present sesame lignan compound inhibits at least Reaction 1 in Reaction Formula 2, thereby inhibiting indole formation by TIL.

The form of inhibition by the sesame lignan compound in Reaction Scheme 1 or Reaction Scheme 2 above is not particularly limited, and examples thereof include competitive inhibition, uncompetitive inhibition, mixed inhibition, and the like. can be mentioned.

As used herein, "competitive inhibition" means that the inhibitor (this sesame lignan compound) is not bound to the substrate (L-tyrosine for TPL, L-tryptophan for TIL) substrate in an enzyme (TPL or TIL) It refers to the form of inhibition that binds to the binding site. In competitive inhibition, the inhibitor does not bind to the enzyme-substrate complex.

As used herein, "uncompetitive inhibition" means that the inhibitor (this sesame lignan compound) is bound to the substrate (L-tyrosine in the case of TPL, L-tryptophan in the case of TIL) to an enzyme (TPL or TIL) that does not bind to means an inhibitory form that binds only to the enzyme-substrate complex without binding.

As used herein, "mixed inhibition" means an enzyme (TPL or TIL) in which the inhibitor (this sesame lignan compound) is not bound to the substrate (L-tyrosine in the case of TPL, L-tryptophan in the case of TIL), It refers to an inhibitory mode that binds to both the enzyme-substrate complex with different inhibition constants (Ki).

As the sesame lignan compound of the present invention, the effect is demonstrated in the Examples described later, and therefore, those that uncompetitively inhibit phenol production by TPL and those that competitively inhibit or mixedly inhibit indole production by TIL are preferably exemplified. be able to.

The above TPL is not particularly limited in its origin, but since its effect is demonstrated in the examples described later, TPL-producing bacteria (more specifically, TPL-producing bacteria in various organs or tissues in mammalian organisms) TPL produced by TPL (in other words, TPL derived from TPL-producing bacteria) can be preferably exemplified. Therefore, the present inhibitor can be advantageously applied as a prophylactic or therapeutic agent for symptoms or diseases caused by uremic substances whose precursors are phenols produced by TPL in TPL-producing bacteria in vivo in mammals. .

In addition, although the origin of the TIL is not particularly limited, since its effect is demonstrated in the Examples described later, TIL-producing bacteria (more specifically, in various organs or tissues in mammalian organisms) TIL produced by TIL-producing bacteria) can be mentioned. Therefore, the present inhibitor can be advantageously applied as a prophylactic or therapeutic agent for symptoms or diseases caused by uremic substances whose precursor is indole produced by TIL in TIL-producing bacteria in vivo in mammals. .

As used herein, "TPL-producing bacteria" are preferably TPL-producing enterobacteria, etc. Specifically, for example, Pantoea bacteria (e.g., Pantoea agglomerans [P. agglomerans], Pantoea ananatis [P. ananatis]), Citrobacter bacteria (e.g., Citrobacter blackie [C. braakii], Citrobacter freundii [C. freundii]), Morganella bacteria (e.g., Morganella morganii), Clostridium bacteria (eg, Clostridium cochlearium [C. cochlearium], Clostridium tetani [C. tetani]), and the like.

As used herein, the "TIL-producing bacterium" is preferably a TIL-producing intestinal bacterium. E. albertii]), Morganella bacteria (e.g., Morganella morganii [M. morganii]), Bacteroides bacteria (e.g., Bacteroides thetaiotaomicron [B.thetaiotaomicron], Bacteroides obatas [B ovatus]), bacteria belonging to the genus Citrobacter (for example, Citrobacter koseri [C. koseri]), and the like.

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 ducts and the like.

As used herein, mammals include humans and non-human mammals (e.g., monkeys, mice, rats, dogs, cats, domestic animals [e.g., rabbits, pigs, horses, cows, sheep, goats, deer]), etc. can be mentioned.

In the present specification, "symptoms or diseases caused by uremic substances whose precursor is phenol produced by TPL in TPL-producing bacteria" and "uremia whose precursor is indole produced by TIL in TIL-producing bacteria "Symptoms or diseases caused by toxic substances" include, for example, digestive system abnormalities such as anorexia, nausea, vomiting, bad breath, stomatitis, and enteritis; Nervous system abnormalities such as neuritis, (developmental) coordination disorder, anorexia; , circulatory system abnormalities such as cardiovascular disorders; Renal anemia, chronic renal failure, tubulointerstitial disorder, acute nephritis, chronic nephritis, nephrosis, diabetic nephropathy, arteriosclerotic nephropathy, renal bone disorders (e.g., renal osteodystrophy), effusion stroke; myocardial infarction; cancer; autism; immunodeficiency; In addition, LPS (Lipopolysaccharide) such as IgA nephropathy, cystic kidney disease, liver dysfunction (e.g., fulminant hepatitis, fatty liver, NASH, NAFLD), cancer (e.g., bile carcinogenesis) is involved in the above symptoms or diseases. 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; Alzheimer's disease; also includes sarcopenia. The above symptoms or diseases are caused by uremic substances whose precursors are phenols produced by TPL in TPL-producing bacteria and uremic substances whose precursors are indole produced by TIL in TIL-producing bacteria. However, symptoms or diseases caused by factors other than these uremic substances are not included.

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

As used herein, the term "physiologically acceptable salt" means a salt that is too toxic, within the scope of sound medical, pharmaceutical, or biological judgment, to be used in contact with mammalian tissue. , salts that are suitable commensurate with a reasonable benefit/risk ratio, without irritation, allergic response, and other problems or complications. Physiologically acceptable salts include, for example, ammonium salts; alkali metal salts such as sodium salts, lithium salts and potassium salts; alkaline earth metal salts such as aluminum salts, calcium salts and magnesium salts; salts with organic bases such as -methyl-D-glucamine; salts with amino acids such as arginine, lysine and ornithine; salts generated by basic nitrogen-containing groups;

The present sesame lignan compounds also include various isomers (e.g., optical isomers, positional isomers, tautomers, etc.), solvates such as hydrates, crystal polymorphs, and prodrugs such as esters. be done. Also included in the subject sesame lignan compounds are compounds in which one or more atoms constituting the compound of formula (I) are isotopes. Examples of isotopes included in the subject sesame lignan compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, bromine, and chlorine, 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.

Subjects for ingestion (administration) of the present inhibitor are not particularly limited, and are usually mammals requiring inhibition of phenol production by TPL and/or indole production by TIL, and phenol produced by TPL in TPL-producing bacteria. Mammals in need of prevention or treatment of symptoms or diseases caused by uremic substances whose precursors are is a mammal in need of prophylaxis or treatment of

Methods for ingesting (administering) the present inhibitor to a subject include, for example, oral ingestion in the form of powders, granules, tablets, capsules, syrups, suspensions, etc. (oral ingestion ); method of injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, topical administration) in the form (dosage form) of solution, emulsion, suspension, etc. (parenteral administration); intranasal administration in the form of a spray administration method (parenteral administration);

The instant inhibitors for oral intake can be sustained-release formulations or enteric-coated formulations. Such an enteric agent comprises, for example, granules containing the sesame lignan compound as an active ingredient, an enteric coating (that is, a base [enteric component] that is resistant to gastric juice and dissolves in the small intestine). or by adding a lubricant to granules containing the present sesame lignan compound, which is an active ingredient, and compressing the tablet, and then coating the tablet with an enteric coating. can.

As a method of obtaining the present inhibitor in the form of a tablet, for example, the present sesame lignan compound as an active ingredient and optional ingredients (additives) that may be blended as necessary are mixed, and then the mixture is compressed. Examples include a method of molding to obtain a tablet, and a method of coating a tablet obtained after compression molding with an enteric component (method of forming an enteric coating), and the latter method is preferred.

Examples of the enteric component include shellac, zein, hydroxymethylcellulose phthalate, carboxymethylcellulose, carboxymethylethylcellulose, cellulose acetate phthalate, methacrylic acid copolymer, water-insoluble ethylcellulose, aminoalkyl methacrylate copolymer, brewer's yeast cell wall (e.g. , trade name Eastrap, etc.), tapioca starch, gelatin, pectin, etc., and shellac is particularly preferred. Whether or not it is an enteric agent can be confirmed by the Japanese Pharmacopoeia 14th Edition disintegration test method.

The intake amount of the sesame lignan compound contained in the inhibitor is appropriately determined according to the age, body weight, sex, symptoms, drug sensitivity, biological species, etc. of the intake target (mammal). The converted concentration ranges from 0.1 μg to 200 mg/kg (body weight)/day. The present inhibitor may be taken in a single dose or in multiple doses (for example, 2 to 4 doses) per day.

As used herein, the term "additives" includes physiologically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonic agents, additives, Examples of compounding ingredients include coating agents, solubilizers, lubricants, glidants, solubilizers, lubricants, flavoring agents, sweeteners, solvents, gelling agents, and nutrients. Specific examples of 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 a component other than the present sesame lignan compound as a component that inhibits phenol formation by TPL and/or indole formation by TIL, but the present sesame lignan compound alone exhibits an excellent inhibitory effect. Therefore, it is preferable that the sesame lignan compound does not contain the above inhibitory components (eg, compounds such as proteins, amino acids, DNA, RNA, and polymers; plant-derived extracts [eg, sesame lignan compound]).

EXAMPLES The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

1. Confirmation that the present sesame lignan compound inhibits phenol production by TPL The lignan compound contained in sesame seeds (ie, sesame lignan compound) was analyzed to see if it has an inhibitory effect on phenol production by TPL.

1-1 Materials and methods [Preparation of TPL-containing liquid]
A TPL-containing solution was prepared according to the following procedures [1] to [4] and used in the subsequent [TPL inhibitory activity test].
[1] Escherichia coli strain JM109 (manufactured by Takara Bio Inc.) into which the tpl gene derived from Pantoea agglomerans was introduced (in this specification, it may be referred to as "recombinant Escherichia coli JM109 strain expressing TPL"). Shaking culture was carried out at 25° C. and 120 rpm for 24 hours in 50 mL of LB liquid medium containing 1 wt % tyrosine and 100 μg/mL ampicillin.
[2] The cultured E. coli strain JM109-derived TPL-producing bacteria were subjected to ultrasonication using SONIFIER (registered trademark) SFX250 (manufactured by EMERSON), followed by centrifugation, and the supernatant was collected as a crude enzyme solution. did.
[3] For the crude enzyme solution, ammonium sulfate (ammonium sulfate) precipitation is performed according to a standard method, and three fractions (0-30% ammonium sulfate saturated fraction, 30-70% ammonium sulfate saturated fraction, and 70-100% of the ammonium sulfate saturated fraction) was obtained.
[4] The above three fractions were subjected to polyacrylamide gel electrophoresis to confirm a TPL-derived band (see FIG. 1). Of the above three fractions, the "30 to 70% ammonium sulfate saturated fraction" having the highest TPL-derived band intensity was desalted with a gel filtration spin column to obtain a TPL-containing liquid.

[TPL inhibitory activity test]
The TPL inhibitory activity test was performed according to the following procedures [1] to [4].
[1] A reaction buffer (100 mM potassium phosphate buffer [KPB ] [pH 8.0], 100 μM pyridoxal-5′-phosphate [PLP], and 2 mM 2-mercaptoethanol) was incubated at 37° C. for 10 minutes. As a control, a reaction buffer solution containing L-tyrosine and not containing a sesame lignan compound was also incubated in the same manner.
[2] 2 µL of TPL-containing solution diluted 10-fold with 1M KPB (pH 8.0) was added and incubated at 37°C for 30 minutes.
[3] After addition of 100 μL of reaction stop solution (liquid 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 mass spectrometry using a liquid chromatograph mass spectrometer (LC-MS) (LCMS-8030, manufactured by Shimadzu Corporation) according to the conditions shown in Table 2 to quantify the amount of phenol. . The amount of phenol was calculated as a 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. 2).

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。以下の実施例２～６においても同様である。

In the table, SMG was purified according to the method described in the document "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), those manufactured by Nagara Science Co., Ltd. were used. The same applies to Examples 2 to 6 below.

1-2 Results When TPL and its substrate tyrosine were incubated in the presence of the eight sesame lignan compounds shown in Table 1, six sesame lignan compounds (sesamin, sesaminol [SML], sesamol, sesaminol mono glucoside [SMG], sesaminol [1→2] diglucoside [1-2 SDG], and sesaminol [1→6] diglucoside [1-6 SDG]) were incubated in the absence of these sesame lignan compounds. It was shown that the amount of phenol produced was significantly reduced compared to the case (see FIG. 2).
This result indicates that the six types of sesame lignan compounds included in the present sesame lignan compound have an effect of inhibiting phenol production by TPL.

2. Confirmation that the present sesame lignan compound inhibits indole formation by TIL Indoxyl sulfate, like phenyl sulfate, has high cytotoxicity and is known as a uremic substance that contributes to the progression of diabetic nephropathy. . In vivo, L-tryptophan (hereinafter simply referred to as "tryptophan") ingested through diet is metabolized into indole by TIL derived from intestinal bacteria, and further metabolized into indoxyl sulfate in the liver. there is Furthermore, since TIL, like TPL, is an enzyme derived from intestinal bacteria, the present sesame lignan compound, which was confirmed to have an inhibitory effect on phenol production by TPL, was also analyzed to inhibit indole production by TIL. did.

2-1 Materials and methods [TIL-containing liquid]
As the TIL-containing solution, a solution containing E. coli-derived TIL (10,000 U/mL) (manufactured by Sigma-Aldrich) was used in the following [TIL inhibitory activity test].

[TIL inhibitory activity test]
The TIL inhibitory activity test was performed according to the following procedures [1] to [4].
[1] Reaction buffer solution (100 mM 98 μL of a solution containing KPB [pH 8.0], 100 μM PLP, and 2 mM 2-mercaptoethanol) was incubated at 37° C. for 10 minutes. As a control, a reaction buffer containing tryptophan but not containing a sesame lignan compound was also incubated in the same manner.
[2] 2 μL of TIL-containing solution was added to a final concentration of 100 U/mL, and incubated at 37° C. for 30 minutes.
[3] After adding 100 μL of a reaction stop solution (liquid containing 40% acetonitrile and 4% trifluoroacetic acid), the mixture was centrifuged (15,000×g, 10 minutes, 4° C.).
[4] The supernatant was collected and subjected to mass spectrometry using LC-MS under the conditions shown in Table 2 to quantify the indole produced by TIL. Taking the indole concentration in the reaction buffer containing no sesame lignan compound as 100, the relative values of the indole concentration in the reaction buffer containing various sesame lignan compounds were calculated (see vertical axes in FIGS. 3 and 4).

2-2 Results When TIL and its substrate, tryptophan, were incubated in the presence of the five sesame lignan compounds (sesamine, SML, sesamol, 1-2 SDG, and 1-6 SDG), It was shown that the amount of indole produced was significantly reduced in a concentration-dependent manner of the present sesame lignan compound compared to the case of incubation at . (see FIGS. 3 and 4).
This result indicates that the five types of sesame lignan compounds included in the present sesame lignan compounds have an inhibitory effect on the formation of indole by TIL.

3. Inhibition Mode and Inhibition Constant of TPL and TIL by the Subject Sesame Lignan Compound Next, the determination of the mode of inhibition of TPL and TIL by the subject sesame lignan compound and the inhibition constant of TPL and TIL by the subject sesame lignan compound, specifically, phenol production by TPL The present sesame lignan compound concentration at which 50% inhibition of indole formation by TIL is observed and the present sesame lignan compound concentration at which 50% inhibition of indole formation by TIL is observed were calculated.

3-1 Method TPL inhibitory activity test of Example 1 and TIL inhibitory activity of Example 2 using 15 μM of five sesame lignan compounds (sesamine, SML, sesamol, 1-2 SDG, and 1-6 SDG) A test was conducted and the phenol production rate (μM/min) and the indole production rate (μM/min) (hereinafter collectively referred to as "v") were calculated. Next, plot v on the vertical axis and tyrosine concentration (μM) or tryptophan concentration (μM) on the horizontal axis (hereinafter collectively referred to as [S]), and approximate using SigmaPlot (manufactured by Hulinks). Curves were fitted and kinetic parameters were calculated. After that, 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 type of inhibition of TPL and TIL by various sesame lignan compounds, and TPL and TIL. Inhibition constants were calculated.

3-2 Results As a result, the inhibition modes and inhibition constants (Ki) of TPL and TIL by five sesame lignan compounds (sesamine, SML, sesamol, 1-2 SDG, and 1-6 SDG) are shown in Table 3. It became clear that

4. Confirmation that the present sesame lignan compound inhibits phenol production by TPL-producing bacteria (1)
It was analyzed whether the subject sesame lignan compound, which has an inhibitory effect on phenol production by TPL, also exerts an inhibitory effect on TPL-producing cells.

4-1 Method Specifically, analysis was performed according to the following procedures [1] to [7].
[1] A recombinant Escherichia coli JM109 strain expressing TPL was precultured in 3 mL of LB liquid medium containing 100 μg/mL ampicillin at 37° C. and 120 rpm for 16 hours.
[2] 300 μL of the pre-cultured liquid medium was added to 100 mL of LB liquid medium containing 100 μg/mL ampicillin and 0.2 wt % tyrosine, and cultured at 28° C. and 120 rpm for 24 hours.
[3] The cells were collected by centrifugation (15,000×g, 5 minutes, 4° C.), the supernatant (liquid medium) was removed, and the cells were suspended in 5 mL of 0.85% sodium chloride solution.
[4] Collect bacteria by centrifugation (15,000 x g, 5 minutes, 4 ° C.), remove the supernatant (0.85% sodium chloride solution), and suspend in 10 mL of sterile distilled water (SDW) did.
[5] Aliquot 500 μL each, collect bacteria by centrifugation (15,000 × g, 5 minutes, 4 ° C.), remove the supernatant (SDW), add 500 μM tyrosine and 3 at the concentration shown in FIG. Suspended in 100 μL of reaction buffer (solution containing 100 mM KPB [pH 8.0], 100 μM PLP, and 2 mM 2-mercaptoethanol) containing the sesame lignan compounds of the present invention (sesamine, SML, and sesamol) . As a control, the cells were similarly suspended in a reaction buffer solution containing tyrosine and not containing the present sesame lignan compound.
[6] After incubating at 37°C for 3 hours, 100 µL of reaction stop solution (solution containing 40% acetonitrile and 4% TFA) was added, followed by centrifugation (15,000 xg, 10 minutes, 4°C). .
[7] 50 μL of the supernatant was recovered and subjected to mass spectrometry using LC-MS according to the conditions shown in Table 2 to quantify the produced phenol. Taking the concentration of phenol in the reaction buffer containing no sesame lignan compound as 100, the relative value of the phenol concentration in the reaction buffer containing the three types of sesame lignan compounds was calculated (see the vertical axis in FIG. 5).

4-2 Results When the recombinant E. coli JM109 strain expressing TPL and tyrosine, which is a substrate of TPL, are incubated in the presence of the three sesame lignan compounds (sesamin, SML, and sesamol), It was shown that the amount of phenol produced was significantly reduced in a concentration-dependent manner of the present sesame lignan compound as compared with the case of incubation at (see FIG. 5).

This result indicates that the present sesame lignan compound was incorporated into the cells of the recombinant E. coli JM109 strain expressing TPL and inhibited phenol production by TPL produced by the strain.

5. Confirmation that the present sesame lignan compound inhibits phenol production by TPL-producing bacteria (2)
Next, it was analyzed whether the present sesame lignan compound inhibits phenol production also in TPL-producing bacteria other than Escherichia coli, and whether it inhibits indole production by TIL in TIL-producing bacteria.

5-1 Materials and methods [TPL-producing bacteria, TIL-producing bacteria]
As TPL-producing bacteria, three representative types of intestinal bacteria that produce TPL, specifically, Fusobacterium periodonticum JCM12991 strain-derived TPL-producing bacteria, Citrobacter koseri JCM1658 strain The derived TPL-producing bacteria and Clostridium cochlearium JCM1396 strain-derived TPL-producing bacteria (all manufactured by RIKEN BioResource Research Center) were used in the following [TPL/TIL inhibitory activity test].
In addition, as TIL-producing bacteria, three typical types of intestinal bacteria that produce TIL, specifically, E. coli BL21 (DE3) strain-derived TIL-producing bacteria (manufactured by Takara Bio Inc.), Citrobacter koseri ) JCM1658 strain-derived TIL-producing bacterium (manufactured by RIKEN BioResource Research Center) and Bacteroides thetaiotaomicron JCM5827 strain-derived TIL-producing bacterium (manufactured by RIKEN BioResource Research Center) Inhibitory activity test].

[TPL/TIL inhibitory activity test]
The TPL/TIL inhibitory activity test was performed according to the following procedures [1] to [6].
[1] TPL-producing bacteria or TIL-producing bacteria are grown in a liquid medium containing 1% glycerol and 0.1% tyrosine for GAM glycolysis (manufactured by Nissui) from which agar has been removed, in a liquid medium at 37°C for 24 hours. , was statically cultured (anaerobic culture) in a jar containing an aneropack.
[2] The culture solution containing the cultured cells was collected by centrifugation (15,000 x g, 10 minutes, 4 ° C.), and after removing the supernatant (liquid medium), the reaction buffer (100 mM of KPB [pH 8.0], 200 μM PLP, and 2 mM 2-mercaptoethanol).
[3] Bacteria were collected by centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant (the above reaction buffer) was removed, and the cells were suspended in the above reaction buffer.
[4] Collect bacteria by centrifugation (15,000 x g, 10 minutes, 4 ° C.), remove the supernatant (reaction buffer), and remove TPL-producing bacteria so that OD ₆₀₀ = 2.0 was suspended in the reaction buffer containing 5.5 mM tyrosine and 5 mM of the present sesame lignan compound (sesamole) ([substrate +, sesamol +] in FIGS. 6 to 8), and TIL-producing bacteria were added to 5.5 mM of tryptophan and 5 mM of the present sesame lignan compound ([substrate+, sesamol+] in FIGS. 9-11). As a control, TPL-producing bacteria were treated with three types of reaction buffers, specifically, the reaction buffers containing neither tyrosine nor the present sesame lignan compound (sesamol) ([Substrate -, sesamol-]), the reaction buffer containing tyrosine and not containing the subject sesame lignan compound (sesamole) ([substrate +, sesamol-] in FIGS. 6 to 8), and not containing tyrosine and not containing the subject Similarly, the TIL-producing bacteria were also suspended in the reaction buffer containing the sesame lignan compound (sesamol) ([substrate-, sesamol+] in FIGS. Specifically, the above reaction buffer solution ([substrate-, sesamol-] in FIGS. 9 to 11) containing neither tryptophan nor the subject sesame lignan compound (sesamol), a tryptophan and the subject sesame lignan compound (sesamol) The reaction buffer without tryptophan ([substrate +, sesamol-] in FIGS. 9 to 11), and the reaction buffer without tryptophan and containing the present sesame lignan compound (sesamole) ([substrate in FIGS. 9 to 11 -, sesamol +]) were similarly suspended.
[5] After stationary culture (anaerobic culture) in an aneropack-containing jar at 37°C for each time (30 minutes, 1 hour, 3 hours, or 24 hours), centrifugation (15,000 x g, 10 minutes) , 4°C).
[6] After passing the supernatant through a 0.45 μm hydrophilic filter, mass spectrometry using LC-MS was performed according to the conditions shown in Table 2. For experiments using TPL-producing bacteria, the amount of phenol was quantified, For experiments using TIL-producing bacteria, the amount of indole was quantified.

5-2 Results Three types of TPL-producing bacteria (TPL-producing bacteria derived from Fusobacterium periodonticum JCM12991 strain, TPL-producing bacteria derived from Citrobacter coseri strain JCM1658, and TPL-producing bacteria derived from Clostridium cochlearium JCM1396 strain) and TPL Incubation of the substrate tyrosine in the presence of the instant sesame lignan compound (sesamole) for at least 30 minutes was shown to significantly reduce the amount of phenol produced compared to incubation in the absence of the instant sesame lignan compound. (See Figures 6-8).

In addition, three types of TIL-producing bacteria (TIL-producing bacteria derived from E. coli BL21 (DE3) strain, TIL-producing bacteria derived from Citrobacter coseri JCM1658 strain, and TIL-producing bacteria derived from Bacteroides thetaiotaomicron JCM5827 strain) and TIL substrates Incubation of a tryptophan in the presence of the subject sesame lignan compound (sesamole) for at least 30 minutes was shown to significantly reduce the amount of indole produced compared to incubation in the absence of the subject sesame lignan compound (Fig. 9-11).

This result shows that the present sesame lignan compound such as sesamol is taken up into the cells of typical intestinal bacteria that produce TPL and TIL, and phenol production by TPL produced by TPL-producing bacteria and TIL produced by TIL-producing bacteria It shows that the indole formation by

6. Confirmation that the sesame lignan compound inhibits phenol production in vivo The sesame lignan compound, which has an inhibitory effect on phenol production by TPL in vitro, also exerts an inhibitory effect on phenol production in vivo. analyzed whether to

6-1 Materials and Methods Twenty-one ICR mice (obtained from CLEA Japan, Inc.) were acclimatized for 10 days. ) to AIN76 (tyrosine-free feed) (manufactured by CLEA Japan, Inc.). In addition, 7 days out of 10 days of acclimatization were co-housing, and 3 days before administration of the subject sesame lignan compound, animals were randomly divided into 3 groups of 7 each (control group, m-Tyr administration group, and subject sesame lignan compound administration group). rice field. Mice in the control group were allowed to drink water ad libitum, and mice in the m-Tyr administration group had the effect of lowering the blood phenylsulfate concentration, and L-m-tyrosine (Astatech), which is considered to be a TPL inhibitor. Co., Ltd.) (Non-Patent Document 1, Patent 6801894) (water containing 36 μg/mL Lm-tyrosine) was allowed to drink freely. The dosing test was started by allowing the animals to drink water containing sesamol ad libitum, and then ad libitum drinking of these waters was continued. On days 0, 4, and 7 after administration, mice in each group were weighed and feces were collected. The bedding was changed on the day before collection of feces. 50 mg of collected stool was weighed into a 2.0 mL tube (manufactured by Biomedical Science) containing stainless steel beads (5 mm in diameter) and zirconia beads (manufactured by Biomedical Science) in advance, and 10 μL/stool of 50% ethanol was added thereto. mg, ice-cooled for 1 hour, and pulverized at 1,500 rpm for 10 minutes using Shake Master Neo (manufactured by Biomedical Science). After grinding, it was centrifuged at 8,000 rpm for 10 minutes and the supernatant was transferred to a 1.5 mL tube. The same amount of 50% ethanol as before was added to the tube again, and the mixture was pulverized, centrifuged, and the supernatant was collected in the same 1.5 mL tube. After centrifugation at 15,000 rpm for 10 minutes, the supernatant was filtered through a 0.45 μm PVDF filter (manufactured by Millex) and then diluted appropriately with ultrapure water (MilliQ). The amount of phenol contained in feces was measured according to the conditions shown in .

6-2 Results First, it was confirmed that there was no significant change in the body weight of the mice in the three groups on days 0, 4 and 7 after administration.
On the other hand, as a result of analyzing the phenol concentration in the mouse feces of the above three groups on the 0th day, the 4th day, and the 7th day after administration, the phenol concentration in the mouse feces of the control group was Although there was almost no change on day 7 and day 7, the phenol concentration in mouse feces of the m-Tyr administration group and the phenol concentration in mouse feces of the present sesame lignan compound administration group both changed over time. A decrease was observed (see Figure 12). In particular, in the m-Tyr administration group, no significant decrease in fecal phenol concentration was observed, whereas in the present sesame lignan compound administration group, the fecal phenol concentration was significantly reduced 7 days after administration. was shown to decrease to In addition, on day 4 after administration, the median value of phenol concentration in feces was approximately half of the median value of phenol concentration in feces on day 0 after administration (see FIG. 12).

These results indicate that the present sesame lignan compound, which has an inhibitory effect on phenol production by TPL, also exerts an inhibitory effect on phenol production in vivo. superior to tyrosine. Furthermore, it is suggested that the present sesame lignan compound has an effect of suppressing phenol production by intestinal bacteria existing in vivo.

The present invention relates to the prevention and treatment of symptoms or diseases caused by uremic substances (e.g., phenyl sulfate) whose precursor is phenol produced by TPL in TPL-producing bacteria in vivo, and in TIL-producing bacteria in vivo. It contributes to the prevention and treatment of symptoms or diseases caused by uremic substances (eg, indoxyl sulfate, indole-3-acetic acid) whose precursor is indole produced by TIL.

Claims (4)

Phenol production by tyrosine phenol lyase and/or indole production by tryptophan indole lyase, containing one or more compounds selected from compounds represented by the following formula (I) and physiologically acceptable salts thereof inhibitor of

(In the formula, R ¹ represents OH or a group represented by formula (II) below, and R ² represents H, OH, a monosaccharide, or a disaccharide.)

2. The inhibitor of claim 1, wherein the compound uncompetitively inhibits phenol production by tyrosine phenol lyase. 3. The inhibitor of claim 1 or 2, wherein the compound competitively or mixedly inhibits indole formation by tryptophan indole lyase. The compound represented by formula (I) is one or two selected from sesamin, sesaminol, sesamol, sesaminol monoglucoside, sesaminol (1→2) diglucoside, and sesaminol (1→6) diglucoside The inhibitor according to any one of claims 1 to 3, which is the above compound.

Images