WO2020111239A1 - Anti-inflammatory agent - Google Patents

Abstract

The present invention provides a means and a method that are useful for inhibiting inflammation (for example, preventing and treating inflammatory bowel disease). More specifically, provided are the following inventions. [I] An anti-inflammatory agent comprising 5-hydroxyindoleacetate. [II] A method for screening an anti-inflammatory agent, said method comprising: (1) measuring the amount of 5-hydroxyindoleacetate in a sample which is collected from a mammal administered with a test substance; and (2) selecting a test substance, which causes an increase in the amount of 5-hydroxyindoleacetate in the sample, as an anti-inflammatory agent. [III] A method for screening an anti-inflammatory agent, said method comprising: (1) culturing mammal-derived cells capable of producing 5-hydroxyindoleacetate in a medium containing a test substance; (2) measuring the amount of 5-hydroxyindoleacetate in the medium; and (3) selecting a test substance, which causes an increase in the amount of 5-hydroxyindoleacetate in the medium, as an anti-inflammatory agent.

Description

Anti-inflammatory agent

The present invention relates to anti-inflammatory agents and the like.

In recent years, prevention of lifestyle-related diseases [eg, metabolic syndrome, type 2 diabetes (T2D) and inflammatory bowel disease (IBD)] by nutritional intervention is common, and consumers are conscious of nutritional intake in order to maintain their health. is doing. Traditional Japanese foods such as natto, miso, and matcha are famous for promoting health. They have a positive effect on the intestinal environment and act as prebiotics and/or probiotics by maintaining a healthy host condition.

For example, rice bran (RB) contained in brown rice is one of the commonly consumed Japanese foods, and it has been reported that it can suppress colitis (Non-Patent Documents 1 to 4). RB is a rich source of bioactive ingredients such as dietary fiber, vitamins, free amino acids, and antioxidants (γ-oryzanol and ferulic acid) that may promote gastrointestinal health and anti-colitis effects. (Non-patent documents 2, 3, 5, 6). However, due to the complexity of RB components, the molecular mechanism of RB-mediated suppression of colitis has not yet been elucidated.

Al-Fayez M et al. , Cancer Chemother Pharmacol. 2006;58(6):816-25. Islam MS et al. , Br J Pharmacol. 2008;154(4):812-24. Komiyayama et al. , Scan J Gastroenterol. 2011;46(1):40-52. Shafie NH et al. , Int J Mol Sci. 2013;14(12):23545-58. Choi J et al. , Nutr J. 2014; 13(1):35. Islam J et al. , Nutrients. 2017; 9(7):747.

The object of the present invention is to provide means and methods useful for suppressing inflammation (eg, prevention/treatment of inflammatory bowel disease).

As a result of diligent studies, the present inventors have found that suppression of colitis by RB can be caused by the production of 5-hydroxyindoleacetic acid (5-HIAA) by intestinal bacteria, and 5-HIAA has an anti-inflammatory effect. Found to have.
The present inventors also developed an anti-inflammatory agent by screening a test substance that increases the amount of 5-HIAA in a mammal or cells derived from a mammal in view of the fact that 5-HIAA has an anti-inflammatory effect. I found that I could do it.

That is, the present invention is as follows.
[1] An anti-inflammatory agent containing 5-hydroxyindoleacetic acid.
[2] The anti-inflammatory agent according to [1], wherein the anti-inflammatory agent is an anti-inflammatory agent against intestinal inflammation.
[3] The anti-inflammatory agent according to [2], wherein the intestinal inflammation is inflammatory bowel disease.
[4] The anti-inflammatory agent according to any one of [1] to [3], which is an oral composition or a composition for rectal administration.
[5] The anti-inflammatory agent according to any one of [1] to [4], which is a medicine or food.
[6] A method for screening an anti-inflammatory agent, including the following:
(1) measuring the amount of 5-hydroxyindoleacetic acid in a sample collected from a mammal to which the test substance was administered; and (2) a test substance that increases the amount of 5-hydroxyindoleacetic acid in the sample. , To be selected as an anti-inflammatory agent.
[7] The method according to [6], wherein the sample is feces.
[8] A method for screening an anti-inflammatory agent, including the following:
(1) Culturing mammalian-derived cells in a medium containing a test substance;
(2) measuring the amount of 5-hydroxyindoleacetic acid in the medium;
(3) Select a test substance that increases the amount of 5-hydroxyindoleacetic acid in the medium as an anti-inflammatory agent.
[9] The method of [8], wherein the mammal-derived cell having the ability to produce 5-hydroxyindoleacetic acid is an enterobacteria.

According to the present invention, suppression of inflammation (eg, prevention/treatment of inflammatory bowel disease) can be realized by 5-HIAA.
Further, according to the present invention, anti-inflammatory agents can be screened by using the amount of 5-HIAA as an index.

FIG. 1 is a diagram showing an outline of an animal experiment. FIG. 2-1 is a diagram showing the effect of a rice bran (RB) diet on weight loss. In Figures 2-1 to 2-3, significance between the control and each group is shown. ^* P<0.05, ^** P<0.01, ^*** P<0.001. FIG. 2-2 is a diagram showing the influence of the RB diet on the disease activity index (DAI). See Figure 2-1 for significance. FIG. 2-3 is a diagram showing the effect of an RB diet on colon length. See Figure 2-1 for significance. [Fig. 2-4] Fig. 2-4 is a diagram showing the influence of an RB diet on colon morphology. FIG. 2-5 is a diagram showing the influence of an RB diet on colorectal histology. Magnification is 200x and bars represent 100 μm. FIG. 2-6 is a diagram showing the influence of the RB diet on the gene expression level. FIG. 3-1 is a diagram showing the influence of the fractional component of the RB diet on weight loss. Significance between control and each group is shown in FIGS. 3-1 to 3-3. ^* P<0.05, ^** P<0.01, ^*** P<0.001. FIG. 3-2 is a diagram showing the influence of the fractional component of the RB food on DAI. FIG. 3-3 is a diagram showing the influence of the fractional component of the RB diet on the colon length. [Fig. 3-4] Fig. 3-4 is a diagram showing the influence of a fractional component of an RB diet on colon morphology. FIG. 3-5 is a figure which shows the influence of the fraction component of RB food on intestinal microflora. FIG. 4-1 is a diagram showing changes in the composition of fecal bacterial flora by RB in DSS-induced colitis mice. The relative abundance of Fecal Bacteria detected in the top 20 relative abundances above 1% of the total sequence is represented by a bar graph. FIG. 4-2 is a diagram showing changes in the diversity of Simpson. In Figures 4-2 and 4-3, the significance between control and each group is shown. *P<0.05, **P<0.01. FIG. 4-3 is a box plot of a part of the family Bacteria that significantly changed on day 0. FIG. 5-1 is a diagram showing a principal component analysis (PCA) of fecal metabolite data. FIG. 5-2 is a diagram showing the top 10 and bottom 10 factor loadings of PC2 in the principal component analysis (PCA) of fecal metabolite data. FIG. 5-3 is a diagram showing metabolites related to tryptophan metabolism in feces. FIG. 5-4 is a diagram showing the concentration of 5-HIAA in feces. In Figure 5-5, the significance between control and each group is shown. *P<0.05, **P<0.01. FIG. 6-1 is a diagram showing the relative expression level of Cyp1a1 when AhR forced expression RAW264.7 was treated with 5-HIAA. FIG. 6-2 is a diagram showing the concentration of 5-HIAA in the feces of GF mice in which each bacterial group has been established.

1. Anti-inflammatory Agent The present invention provides an anti-inflammatory agent including 5-HIAA.

5-HIAA is a metabolite of serotonin, and is biosynthesized from serotonin in predetermined cells (eg, mammalian cells capable of producing serotonin, indigenous microorganisms such as intestinal bacteria). Therefore, 5-HIAA can be prepared using serotonin-producing cells (eg, natural cells, or transformed cells incorporating the biosynthetic system as described above). 5-HIAA can also be synthesized by organic chemistry methods. Alternatively, commercially available 5-HIAA may be used.

The inflammation to which the anti-inflammatory agent of the present invention can be applied is not particularly limited as long as it is any inflammation that can occur in mammals. Examples of the site where such inflammation may occur include the intestine (eg, small intestine, large intestine), nasal cavity, bronchus, skin, and oral cavity. The site is preferably in the intestine, more preferably in the large intestine.

Preferably, the inflammation is inflammation in an inflammatory disease. Examples of such an inflammatory disease include inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease, Behcet's disease), stomatitis, nephritis, and autoimmune disease (eg, allergic disease). As inflammation, inflammatory bowel disease is preferred. Therefore, the anti-inflammatory agent of the present invention can be used as a prophylactic or therapeutic agent for such inflammatory diseases.

The amount of 5-HIAA used as an anti-inflammatory agent is not particularly limited as long as it can exert an anti-inflammatory effect, but it is as follows as a guideline. In the examples, the concentration of 5-HIAA in feces collected from a mammal (mouse) of inflammatory bowel disease model was about 100 nmol/g, and the concentration of 5-HIAA in feces collected from a RB-fed mammal (mouse) was 5- It has been confirmed that the concentration of HIAA is about 800 nmol/g. That is, it is considered desirable that the concentration of 5-HIAA at the target site in mammals is in the range of 300 nmol/g or more (eg, 300 to 1500 nmol/g). For example, in order to achieve such a range of concentrations in the intestine of a mammal, the dose or intake of 5-HIAA in the mammal is, for example, 0.01 to 100 g/kg, preferably 0.5 to 50 g/kg. kg, more preferably 1 to 10 g/kg. Therefore, the anti-inflammatory agent of the present invention may include such an amount of 5-HIAA. Alternatively, when 5-HIAA is applied to the intestine of a mammal or a site other than the intestine, it is, for example, 0.01 to 1000 mg/ml, preferably 0.1 to 100 mg/ml, more preferably 1 The anti-inflammatory agent of the present invention may include 5-HIAA in such a concentration range so that 5-HIAA is applied in the concentration range of ˜10 mg/ml.

In a particular embodiment, the anti-inflammatory agent of the invention may be provided in the form of a composition.

For example, the anti-inflammatory agent of the present invention, when provided in the form of a pharmaceutical composition, may contain a pharmaceutically acceptable carrier in addition to 5-HIAA. Examples of the pharmaceutically acceptable carrier include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, and other excipients, cellulose, methyl cellulose, hydroxypropyl cellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate, aerosil, talc, lauryl Lubricants such as sodium sulfate, citric acid, menthol, glycyrrhizin/ammonium salt, glycine, fragrances such as orange powder, sodium benzoate, sodium bisulfite, preservatives such as methylparaben, propylparaben, citric acid, sodium citrate, acetic acid Stabilizers such as methylcellulose, polyvinylpyrrolidone, aluminum stearate and the like, dispersants such as surfactants, water, physiological saline, diluents such as orange juice, cacao butter, polyethylene glycol, white kerosene, etc. Examples include base waxes, but are not limited thereto. The pharmaceutical composition is a solid (eg, capsule, tablet, powder, granule), semisolid (eg, gel), liquid (eg, oral solution, or rectal solution, eye drop, Parenteral liquids such as ear drops and nasal agents).

In certain preferred embodiments, the anti-inflammatory agent of the invention in the form of a pharmaceutical composition comprises, in addition to 5-HIAA, another substance having an anti-inflammatory effect, and/or a microorganism having an anti-inflammatory effect. You may stay. Examples of other substances having an anti-inflammatory action include drugs such as steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs (NSAIDs); extracts from natural products such as plants and animals; antibody drugs. Examples of the microorganism having an anti-inflammatory action include Clostridium bacteria, Bacteroides bacteria, Lactobacillus bacteria, Bifidobacterium bacteria and the like.

When the anti-inflammatory agent of the present invention is provided in the form of a food composition, 5-HIAA may be provided in a form added to food, or may be used as a main component such as a supplement. .. Examples of foods include liquids (eg, beverages, alcohols), semisolids (eg, yogurt, jelly), and solids (eg, snacks, chocolate).

In certain preferred embodiments, the anti-inflammatory agent of the invention in the form of a food composition comprises, in addition to 5-HIAA, another substance having an anti-inflammatory effect, and/or a microorganism having an anti-inflammatory effect. You may stay. Such other substances and microorganisms are the same as those described above.

In a preferred embodiment, the anti-inflammatory agent of the present invention may be an oral composition or a composition for rectal administration. Such an anti-inflammatory agent of the present invention can exert an anti-inflammatory action in the intestine of mammals. The content of 5-HIAA in the composition for oral administration and the composition for rectal administration is the same as the above-mentioned administration or intake amount of 5-HIAA. Oral compositions can be used as solids, semi-solids, or liquids as described above in pharmaceutical and food compositions. The composition for rectal administration can be used as a semi-solid material or a liquid material.

The anti-inflammatory agent of the present invention can be applied to any mammal. Such mammals include, for example, primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, rabbits), livestock and working animals (eg, cows, pigs, horses, goats, Sheep). The mammal is preferably a primate or a rodent, more preferably a primate, and even more preferably a human from the viewpoint of clinical application.

2. Method for screening anti-inflammatory agent The present invention provides a method for screening an anti-inflammatory agent using a sample collected from a mammal or cells derived from a mammal. The mammal and the anti-inflammatory agent are the same as those described above.

In one embodiment, the screening method of the present invention comprises:
(1) measuring the amount of 5-HIAA in a sample collected from a mammal to which the test substance has been administered; and (2) a test substance that increases the amount of 5-HIAA in the sample, an anti-inflammatory agent. To choose as.

In step (1), the test substance is any substance including known substances and new substances. Such test substances include, for example, organic low molecular weight compounds, compound libraries prepared using combinatorial chemistry technology, nucleic acids (eg, nucleosides, oligonucleotides, polynucleotides), sugars (eg, monosaccharides, disaccharides). Sugars, oligosaccharides, polysaccharides), lipids (eg, saturated or unsaturated linear, branched and/or cyclic fatty acids), amino acids, proteins (eg, oligopeptides, polypeptides, antibodies or fragments thereof), solid Random peptide libraries prepared by phase synthesis or the phage display method, or natural components derived from microorganisms, plants and animals, marine organisms, etc. may be mentioned. Alternatively, microorganisms (eg, indigenous microorganisms such as intestinal bacteria) may be used as the test substance.

The sample is a sample that can be collected non-invasively from a mammal or a sample that can be collected invasively from a mammal. Examples of samples that can be collected non-invasively from mammals include excrement samples (eg, feces, urine), liquid samples (eg, saliva, tears, sweat), mucosal samples (eg, intranasal mucosa, Oral mucosa). Examples of samples (eg, biopsy samples) that can be collected invasively from mammals include samples that can be collected from skin, bronchus, blood, small intestine, large intestine, cecum, and kidney. As the sample, a sample that can be collected non-invasively from a mammal is preferable, and feces is more preferable. The sample may be subjected to pretreatment. Examples of such pretreatment include dissolution in liquid, centrifugation, extraction, heating, freezing, and refrigeration.

The amount of 5-HIAA can be measured by any method. Examples of such a method include high performance liquid chromatography (HPLC), mass spectrometry, immunoassay, and colorimetric method. The amount of 5-HIAA may be measured as the 5-HIAA concentration per unit amount of sample.

The present embodiment can further include administering a test substance to a mammal before the step (1). The amount of test substance to be administered can be adjusted appropriately. For example, if it is particularly desirable to screen for potent anti-inflammatory agents, the amount of test substance to be administered can be set to a low amount (eg, 0.05-10 g/kg). Alternatively, the amount of the test substance to be administered may be set to a higher amount (eg, 10 to 100 g/kg) when it is not particularly desired to screen for potent anti-inflammatory agents.

In another embodiment, the screening method of the present invention comprises:
(1) Culturing a mammal-derived cell having the ability to produce 5-HIAA in a medium containing a test substance;
(2) measuring the amount of 5-HIAA in the medium;
(3) Selecting a test substance that increases the amount of 5-HIAA in the medium as an anti-inflammatory agent.

In step (1), mammalian-derived cells are cultured in a medium containing the test substance. The test substance is the same as that described above. The concentration of the test substance in the medium can be adjusted appropriately. For example, when the screening of a highly potent anti-inflammatory drug is particularly desired, the concentration of the test substance in the medium can be set to a low concentration (eg, 1 nM to 10 μM). Alternatively, the concentration of the test substance in the medium may be set to a higher concentration (eg, 100 nM to 10 mM) when the screening of a potent anti-inflammatory agent is not particularly desired.

The mammal-derived cell used in the step (1) is not particularly limited as long as it has the ability to produce 5-HIAA. Such a mammal-derived cell is a mammalian cell or a microorganism existing in a mammal, preferably a microorganism existing in a mammal. The microorganism present in a mammal is a resident microorganism present in a site where inflammation can occur in a mammal (eg, the above-mentioned site), and preferably an intestinal bacterium.

Residual microorganisms such as enterobacteria can be cultured in the form of a mixture with other microorganisms or in the form of a non-mixture with other microorganisms. For example, if certain intestinal bacteria are cultivated in the form of a mixture with other intestinal bacteria, such a mixture can be obtained by recovering from the faeces of mammals as described above.

Cultivation of indigenous microorganisms and other resident microorganisms can be carried out under the general culturing conditions of resident microorganisms using the same medium as that used for culturing resident microorganisms. For example, general media and culture conditions for enterobacteria are well known (eg, (1) Fukuda S. et al., J Vet Med Sci., 2002 Nov; 64(11): 987-92, ( 2) Fukuda S. et al., J Gen Appl Microbiol., 2005 Apr; 51(2): 105-13, (iii) Fukuda S. et al., Sci Rep., 2018 Jan 11; 8(1): 435).

For example, the medium can contain components such as a carbon source, a nitrogen source, an organic micronutrient source, vitamins and inorganic ions. Examples of carbon sources include carbohydrates such as monosaccharides (eg, glucose), disaccharides, oligosaccharides, and polysaccharides; invert sugar obtained by hydrolyzing sucrose; glycerol; methanol, formaldehyde, formate, carbon monoxide, and dioxide. Compounds having 1 carbon atom such as carbon; oils such as corn oil, palm oil and soybean oil; short-chain fatty acids such as acetic acid, propionic acid, butyric acid; organic acids such as succinic acid and lactic acid; animal fats and oils; animal oil; saturated Fatty acids such as fatty acids and unsaturated fatty acids; lipids; phospholipids; glycerolipids; glycerin fatty acid esters such as monoglycerides, diglycerides, triglycerides; polypeptides such as microbial proteins and plant proteins; regeneration of hydrolyzed biomass carbon sources, etc. Possible carbon sources; yeast extract; horse serum; fecal extract; meat extract; vegetable extract; stomach content extract; or a combination thereof. Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas and aqueous ammonia. As the organic micronutrient source, for example, it is desirable to add an appropriate amount of a required substance such as L-homoserine or yeast extract. Examples of vitamins include vitamins B1, B2, B3, B6, B12, C and K1. Examples of inorganic ions include potassium phosphate, magnesium sulfate, iron ions, and manganese ions.

Explaining the culture conditions, for example, the intestinal bacterial density in the medium is, for example, 1×10 ⁶ to 1×10 ¹¹ cells/mL, preferably 1×10 ⁷ to 1×10 ¹⁰ cells/mL, and It is preferably 1×10 ⁸ to 1×10 ⁹ cells/mL. The culture temperature is, for example, 30 to 40°C. The culture period is, for example, 1 to 7 days. Although either anaerobic culture conditions or aerobic culture conditions can be utilized, anaerobic culture conditions are preferred. The oxygen concentration under anaerobic culture conditions is, for example, 0 to 5%, preferably 0 to 3%, more preferably 0 to 2%, and even more preferably 0 to 1%.

INDUSTRIAL APPLICABILITY The screening method of the present invention is useful, for example, for the development of a new drug or food having an anti-inflammatory action, and for the development of a drug or food capable of exhibiting a combined action having an anti-inflammatory action in addition to the existing action Is.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

(Method)
(1) Mice and Feed Five-week-old female C57BL/6J mice were treated with Clea Japan, Inc. Purchased from. Mice were housed under controlled light conditions (12 h light/12 h dark cycle). Tap water or 2.0% DSS (molecular weight 36,000-50,000; MP Bio Japan) was given, and AIN-93G (Oriental yeast) or 5% RB, 10% RB, 20% RB, γ-oryzanol, RB Oil and defatted RB (AIN-93G) were given. RB samples are from Ritechech Inc. Γ-oryzanol, RB oil and degreased RB, provided by Oryza Oil & Fat Chemical Inc. Offered by

(2) Animal experiments In this study, two animal experiments were conducted. In the first experiment, the effect of rice bran (RB) was verified, and the RB concentration dependence was examined. In the second experiment, the components of RB were fractionated and it was investigated which component contributed to the colitis suppressing effect of RB. In both experiments, after a one week acclimation period, mice were randomly assigned to groups of 4 or 6 (n=5-7 in each group) as follows: Control: AIN-93G diet and 2. Colitis was induced by administering 0% (w/v) DSS water for 10 weeks. 5% RB, 10% RB, 20% RB, RB, RBG (gamma oryzanol), RBO (RB oil) and RBD (defatted RB): 5% RB, 10% RB, 20% RB, 20% RB, γ- AIN-93G diet and tap water supplemented with oryzanol, RB oil and defatted RB were administered for 1 week, and then 2.0% DSS water was administered for 10 days each. During the experimental period, fecal samples from all mice were collected daily for 16S rRNA gene analysis. In addition, the severity of colitis during the DSS administration period was assessed as described in the DAI assessment below. All mice were dissected 10 days after the DSS administration. The large intestine and the large intestine contents were collected, and the large intestine length was measured. All samples were stored at -80°C.

(3) Fecal Collection Fecal samples were collected for capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CETOMS) and 16S rRNA gene analysis. All mice were placed in separate cages. Five fecal pellets per mouse daily were collected in tubes with autoclaved forceps and placed on ice. These samples were stored at -80°C until further analysis.

(4) DAI Evaluation The disease activity index (DAI) was measured daily to evaluate the severity of colitis. DAI was weight change (0:<1%;1:1-5%;2:5-10%;3:10-15%;4:>15%), feces (0:negative, 1:+, 2:++, 3:++++, 4:++++), and fecal hardness (0: normal, 2: soft, 4: diarrhea).

(5) Histopathology For histopathological analysis, a representative sample from the center of the large intestine was fixed in 10% formalin, embedded in paraffin, sectioned and treated with hematoxylin and eosin (H&E). Stained and examined at ×200 magnification.

(6) DNA extraction Fecal DNA extraction was performed according to the method described previously with some modifications (Furusawa Y et al., Nature. 2013; 504(7480): 446-50.). Fecal samples were lyophilized for 24 hours using a VD-800R lyophilizer (TAITEC). The lyophilized feces were disrupted by shaking vigorously (1,500 rpm for 10 minutes) with 3.0 mm zirconia beads (Biomedical Science) using Shake Master (Biomedical Science). A stool sample (10 mg) was diluted with 400 μL of a 1.0% (w/v) SDS/TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) solution and 400 μL of phenol/chloroform/isoamyl alcohol (25:24:1). Suspended. 0.1 mm zirconia/silica beads were added to the mixed solution, and shaken vigorously (1,500 rpm for 5 minutes) using a shake master to further break the mixture. After centrifugation at 17,800 xg for 5 minutes at room temperature, bacterial genomic DNA was purified from fecal extracts by the standard phenol/chloroform/isoamyl alcohol method. RNA was removed from the sample by RNase A treatment, then the DNA sample was purified once again by the standard phenol/chloroform/isoamyl alcohol method.

(7) 16S rRNA Gene Sequencing The 16S rRNA gene in fecal DNA samples was analyzed using the MiSeq sequencer (Illumina) according to the method previously described with some modifications (Murakami S et al., Altern. Med. 2015; 2015: 824395.). In summary, the V1-V2 region of the 16S rRNA gene is represented by the bacterial universal primer set 27Fmod (5'-AGRGTTTGATYMTGGCTCAG-3' (SEQ ID NO: 1)) (Kim SW et al., DNA Res. 2013; 20(3):241. -53.) and 338R (5'-TGCTGCCTCCCGTAGGAGT-3' (SEQ ID NO: 2)) were used to amplify from DNA isolated from feces. PCR was performed using Tks Gflex DNA Polymerase (manufactured by Takara Bio), followed by amplification for 30 cycles of 98° C. for 1 minute, 98° C. for 10 seconds, 55° C. for 15 seconds, and 68° C. for 30 seconds. Finally stretched at 68°C for 3 minutes. Amplification products were verified by agarose gel electrophoresis and purified using an Agencourt AMPure XP (Beckman Coulter), then a forward primer containing the P5 sequence, an 8 bp barcode sequence (indicated by N) unique to each sample. (5'-NNNNNNNNNTATGGTAAATTGTAGRGTTTTGATYMTGGCTCAGT-3' (SEQ ID NO: 3)), Rd1 SP sequence and 27Fmod primer and a reverse primer containing a P8 sequence including the unique 8bp barcode sequence (denoted by N) (5'-CAAGCAGAAA). -GACGGCTATACGAGATNNNNNNNNN-AGTCAGTCAGCCTGCTGCCCTCCCGAGGAGT-3' (SEQ ID NO: 4)), Rd2 SP sequence, and 338R primer. Amplified products purified using the Agencourt AMPure XP were quantified using the Quant-iTPicoGreen DNA assay (Invitrogen), and approximately equal amounts of PCR amplicons from each sample controlled by the Agilent 2100 Bioanalyzer (Agilent Technologies). A mixed sample was prepared by pooling. Finally, MiSeq sequencing was performed as per protocol. In this study, 2×300 bp paired-end sequencing was used.

(8) Metabolome Analysis Using Capillary Electrophoresis Electrospray Ionization Time-of-Flight Mass Spectrometer Metabolome analysis was performed according to the method previously described [(1) Murakami S et al. , Altern Med. 2015; 2015:824395. , And (2) Mishima E et al. , J Am Soc Nephrol. 2015;26(8):1787-94. ]. Briefly, fecal samples were lyophilized and disrupted with 3.0 mm zirconia beads as described for DNA extraction above. To extract metabolites from fecal samples, 500 μL of methanol containing an internal standard (20 μM each of methionine sulfone and D-camphor-10-sulfonic acid (CSA)) was added to 10 mg of fecal samples. The mixture was further broken with 100 mg of 0.1 mm zirconia/silica beads with vigorous shaking (1,500 rpm for 5 minutes) using a Shake Master. Next, this mixture was mixed with 200 μL of ultrapure water and 500 μL of chloroform and then shaken vigorously (1500 rpm for 5 minutes). After centrifugation at 4,600 xg for 15 minutes at 4°C, the supernatant was transferred to a centrifuge filter tube (UltrafreeMC-PLHCC 250/pk for metabolome extraction of Human Metabolome Technologies) to remove protein and lipid molecules. The filtrate was centrifuged and dissolved in 100 μL of ultrapure water containing the reference compound (3-aminopyrrolidine and trimesic acid 200 μM respectively) immediately before CE-TOFMS analysis.

Ionic metabolites were analyzed using CE-TOFMS in both positive and negative modes. All CE-TOFMS experiments were performed using the Agilent CE Capillary Electrophoresis System (Agilent Technologies). Annotation tables were created from measurements of standard compounds and aligned with the dataset according to similar m/z values and standardized migration times. The peak areas were then normalized to the peak areas of the internal standard methionine sulfone or CSA for the cationic and anionic metabolites, respectively. The concentration of each metabolite was calculated based on the relative peak area and concentration of the standard compound. The peak annotation and quantification were confirmed by in-house software (MasterHands) (Sugimoto M et al., Metabolomics. 2010; 6(1): 78-95.). Metabolite set enrichment analysis (MSEA) was performed using metabolites from control and 10% RB on day 0 of DSS administration [(1) Xia J et al. , Nucleic Acids Res. 2010; 38 (SUPPL. 2). , And (2) Xia J et al. , In: Current protocols in bioinformatics. 2016. p. 14.10.1-14.10.91. ].

(9) Statistical analysis Differences between two or more groups were evaluated by Student's t-test or Dunnett's test, respectively. Weight change, DAI, was analyzed by a two-way repeated measures ANOVA, followed by the Holm-Bonferroni procedure, which uses anovakun version 4.8.0. Principal component analysis (PCA) was performed by SIMCAP 13.0.3 (Umetrics). Hierarchical clustering and heat maps were drawn using MeV TM4 software 4.7.4.

(10) AhR in vitro test Mouse-derived RAW264.7 cells (RAW/Neo) or RAW cells forcibly expressing AhR (RAW/AhR) were treated with 5-HIAA only as a solvent (DMSO), 2 μM, 20 μM, and 200 μM medium. The relative expression level of Cyp1a1 when added and cultured for 24 hours was measured.

(11) Gnotobiot test A solvent alone or a group of Enterococcus bacteria, a group of Lactobacillus bacteria, a group of Bacteroides bacteria, or a group of Clostridium bacteria was orally administered to sterile mice. Feces were collected one month after administration and the concentration of 5-HIAA was measured using CE-TOFMS.

(12) FISH analysis A large intestine was collected and treated by the Methacarn fixing method to prepare a paraffin section. The prepared paraffin section was deparaffinized with xylene, and then the 16S rRNA target was stained with Clostridium, which specifically increases in rice bran-fed mice, using the Fluorescence in situ hybridization (FISH) method. For FISH staining, Erec482 (5'-GCTTCTTAGTCARGTTACCG-3') fluorescently labeled FISH probe that specifically stains Clostridium cluster XIV was used. After Clostridium is stained with FISH, DNA and mucin are stained with DAPI and fluorescently labeled Wheat Germ Agglutinin (WGA) lectin, respectively.

(Results and discussion)
(1) Ingestion of RB suppresses colitis (Fig. 2)
In order to evaluate the effects of RB diet on weight loss, DAI, shortening of colon, and colitis in RB-fed mice, animal experiments were conducted using a DSS-induced colitis mouse model. Although the body weight was reduced in the control, the body weight was suppressed in the RB-administered group (FIG. 2-1). Similarly, there was a significant difference in DAI between the control group and the RB intake group (Fig. 2-2). A significant difference was observed in the large intestine length between the control group and the RBG intake group (FIGS. 2-3 and 2-4). Furthermore, there was a difference in the amount of blood in the large intestine (Fig. 2-3). Tissue sections of the large intestine showed distortion of the supracryptic fossa and reduced leukocyte and neutrophil mucosa and infiltration in the RB-fed group of mice (FIGS. 2-5). Taken together, these results suggest that the RB diet can suppress DSS-induced colitis.

(2) RB degreasing component suppresses colitis (Fig. 3)
Three components, RBG, RBD and RBO, were used to identify the active components that contribute to the colitis suppressing effect of RB. RBD, which consists of fat-free RB, has a significant colitis-suppressing effect on body weight, DAI, and colon length and morphology (FIGS. 3-1 to 3-4). In particular, the observed colitis suppressing effect was similar to that of whole RB (RB). In contrast, RBG and RBO, which were previously thought to have a colitis suppressing effect, did not show a significant colitis suppressing effect. These results suggest that the suppressive effect of RB on colitis may be mainly due to its defatted components.

(3) Changes in fecal bacterial flora due to RB intake (Fig. 4)
To assess the kinetics of fecal microbial composition with RB diet intervention, 16S rRNA gene analysis was performed using stool samples taken daily during the experiment. 59,662 OTAs (operational taxonic units) were constructed from 308 samples (some stool samples were lost during stool collection). Taxonomic profiling of faecal flora at the family level is shown (Fig. 4-1). It was observed that the composition of the fecal flora of DSS-induced colitis mice changed dramatically. Alpha diversity in the RB-fed group of mice was significantly higher or delayed compared to controls (Figure 4-2). To investigate changes in fecal flora, the relative abundance of each bacterial family on day 0 was compared between control and other groups. Compared to the control group, 20 families were significantly increased in the RB-administered group, particularly Bifidobacterium lacae, Lactobacilraceae and Turicibacterium aceae were significantly increased, and Bacteroideaceae, Erysiperitrichaceae and Enterobacteriaceae (Figure 3) were significantly decreased.

(4) Changes in fecal metabolites due to RB intake (Fig. 5)
Metabolome analysis was performed using fecal samples collected on days 0, 5, and 10 to assess the effects of RB intake on the molecular level. Metabolome analysis by CE-TOFMS and Gas Chromatography Mass Spectrometry (GC-MS) identified 360 metabolites using feces from at least one mouse per group per time point. The concentrations of these metabolites were compared in controls and other groups (data not shown). The temporal changes in the fecal metabolite profiles of control and RB mice in PCA were in different directions (Fig. 5-1). This indicates that fecal metabolomic profile was affected by DSS, but RB uptake suppressed that effect. 41 metabolites including short-chain fatty acids such as acetate and butyrate were significantly changed in the RB-fed mice compared with the control (FIG. 5-2). SCFA produced by fermentation of dietary fiber by intestinal microbiota induces apoptosis in neutrophils (Maslowski KM et al., GPR43. Nature. 2009; 461(7268):1282-6.) and colon regulatory. It suppresses colitis in mice through T cell (Treg) differentiation (Furusawa Y et al., Nature. 2013; 504(7480): 446-50.). Moreover, increasing the concentration of SCFAs results in a decrease in pH. In the presence of lower pH, the number of pathogenic microorganisms and the solubility of bile acids are reduced, resulting in a decrease in the tumor promoter activity of secondary bile acids [(1) Cook SI et al. , Aliment Pharmacol Ther. 1998; 12(6):499-507. , (2) Grubben MJAL et al. , Dig Dis Sci. 2001;46(4):750-6. , And (3) Wong JM et al. , J Clin Gastroenterol. 2006;40(3):235-43. ]. Therefore, increased intake of fermentable dietary fiber such as RB may be beneficial in suppressing colitis [(1) Daou C et al. , J Food Sci Technology. 2014;51(12):3878-85. , And (2) Park HY et al. , Planta Med. 2016; 82(07):606-11. ]. In our study, the concentration of SCFAs increased with RB consumption. Acetate, the most prominent SCFA, is important for lowering colonic pH [(1) Duncan SH et al. , Br J Nutr. 2004;91(06):915-23. , And (2) Ye H et al. , Sci Rep. 2016; 6:20329. ], whereby the number of pathogenic microorganisms and the solubility of bile acids have anti-inflammatory properties and promote the integrity of intestinal epithelium in mice [(1) Fukuda S et al. , Nature. 2011;469(7331):543-7. , And (2) Macia L et al. , Nat Commun. 2015; 6 (August): 6734. ]. Also, on day 0, MSEA was performed using control and 10% RB metabolome data (Table 1). Vitamin B6 metabolism, tryptophan metabolism and ubiquinone biosynthesis were significantly changed between control and 10% RB. Among tryptophan metabolism-related metabolites, 5-hydroxyindoleacetic acid (5-HIAA) was increased by RB intake (FIGS. 5-3 and 5-4).

(5) Suppression of colitis by 5-HIAA (Fig. 6)
To investigate whether 5-HIAA is an AhR ligand, AhR forced expression RAW264.7 cells were treated with 5-HIAA (concentrations 2, 20, and 200 μM in the medium), and the expression amount of Cyp1a1 gene was increased as compared with the control. (Fig. 6-1). Further, colonization of the bacterium belonging to the genus Clostridium in GF mice produced more 5-HIAA than when colonized with germ-free mice or other bacteria (Fig. 6-2). As a result of FISH analysis of the cross section of the large intestine, the group of bacteria belonging to the genus Clostridium was localized at the fibrous site of RB. Previous studies have shown that tryptophan metabolism by the gut microbiota plays a role in the AhR-mediated mucosal immune response by regulating the production of IL-22, a cytokine with a well-known effect on intestinal homeostasis. It has been suggested [(1) Lamas B et al. , Nat Med. 2016;22(6):598-605. , (2) Rutz S et al. , Immunol Rev. 2013;252(1):116-32. , And (3) Zelante T et al. , Immunity. 2013; 39(2):372-85. ]. Furthermore, in IBD patients, impaired microbial production of AhR ligands is observed (Lamas B et al., Nat Med. 2016;22(6):598-605.). In our study, tryptophan metabolism was significantly upregulated by RB consumption. Therefore, tryptophan metabolism from the intestinal flora may be a target for the development of new therapeutic agents for patients with colitis.

The anti-inflammatory agent of the present invention is useful for suppressing inflammation (eg, prevention/treatment of inflammatory bowel disease).
The screening method of the present invention is useful for developing anti-inflammatory agents.

Claims (9)

An anti-inflammatory agent containing 5-hydroxyindoleacetic acid. The anti-inflammatory agent according to claim 1, which is an anti-inflammatory agent against intestinal inflammation. The anti-inflammatory agent according to claim 2, wherein the intestinal inflammation is inflammatory bowel disease. The anti-inflammatory agent according to any one of claims 1 to 3, which is an oral composition or a composition for rectal administration. The anti-inflammatory agent according to any one of claims 1 to 4, which is a medicine or food. Methods for screening anti-inflammatory agents, including:
(1) measuring the amount of 5-hydroxyindoleacetic acid in a sample taken from a mammal to which the test substance was administered; and (2) a test substance that increases the amount of 5-hydroxyindoleacetic acid in the sample. , To be selected as an anti-inflammatory agent. The method according to claim 6, wherein the sample is feces. Methods for screening anti-inflammatory agents, including:
(1) culturing mammalian-derived cells capable of producing 5-hydroxyindoleacetic acid in a medium containing a test substance;
(2) measuring the amount of 5-hydroxyindoleacetic acid in the medium;
(3) Select a test substance that increases the amount of 5-hydroxyindoleacetic acid in the medium as an anti-inflammatory agent. The method according to claim 8, wherein the mammal-derived cell having the ability to produce 5-hydroxyindoleacetic acid is an intestinal bacterium.

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