Cholera toxin production inhibitors containing indole compounds and their use

Abstract

To provide a cholera toxin production inhibitor and its use for realizing cholera treatment or cholera prevention by a different approach, which is not the conventional symptomatic treatment and antibacterial treatment. 【Solution means】A cholera toxin production inhibitor containing a compound represented by the following formula (1). TIFF2026058903000007.tif47164 (In formula (1), R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 each independently represent a hydrogen atom, a hydroxyl group, or an alkoxy group having an alkyl group with 1 to 4 carbon atoms, and R 3 represents a hydrogen atom, a formyl group, or a carboxyl group. However, the case where all of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are all hydrogen atoms is excluded.)

Description

Technical Field

[0001] This specification relates to an inhibitor of cholera toxin production containing an indole compound and its use.

Background Art

[0002] Cholera is an acute bacterial infectious disease caused by Vibrio cholerae that produces cholera toxin. Cholera has caused serious damage in developing countries. According to the estimation of the World Health Organization (WHO), there are 1.3 million to 4 million patients annually, and the number of deaths is 20,000 to 140,000.

[0003] The treatment of cholera mainly focuses on symptomatic treatment with oral rehydration solutions. Treatment with antibacterial drugs is also carried out, but the increase in drug-resistant Vibrio cholerae has become a problem. Furthermore, although antibacterial drug treatment reduces the excretion period, the effect of improving diarrhea is poor. Therefore, in fact, it is not recommended in developing countries with scarce medical resources (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] This specification provides an inhibitor of cholera toxin production and its use for realizing cholera treatment or cholera prevention by a different approach, which is not the conventional symptomatic treatment and antibacterial drug treatment.

Means for Solving the Problems

[0006] The inventors of the present invention focused on Isatis tinctoria, a plant of the genus Isatis in the family Brassicaceae. The root of Isatis tinctoria is used as Banlangen for the treatment of infectious diseases and the like. The inventors of the present invention examined the growth inhibitory effect on Vibrio cholerae that produces cholera toxin for certain indole compounds derived from Isatis tinctoria, but there were cases where the growth inhibitory effect could not be found. On the other hand, these indole compounds were found to inhibit the production of cholera toxin in Vibrio cholerae that produces cholera toxin. Based on these findings, the present specification discloses the following means.

[0007] [1] A cholera toxin production inhibitor containing a compound represented by the following formula (1).

Chemical formula

[0008] [Figure 1] This diagram shows the separation schemes for ISA1 and ISA2. [Figure 2] This is a diagram showing the ISA3 isolation scheme. [Figure 3A] This figure shows the inhibitory effects of ISA1, ISA2, and ISA3 on the growth of cholera toxin-producing Vibrio cholerae O1ElTor type. [Figure 3B] This figure shows the inhibitory effects of ISA1 and ISA2 on the growth of cholera toxin-producing Vibrio cholerae O1 classical and O139 types. [Figure 4] This diagram shows the minimum inhibitory concentrations of ISA1, ISA2, and ISA3 for various types of bacteria. [Figure 5] This figure shows the inhibitory effects of ISA1 and ISA2 on cholera toxin production in cholera toxin-producing Vibrio cholerae O1 ElTor type, O1 classical type, and O139 type. [Figure 6] This figure shows the inhibitory effects of ISA1, ISA2, and ISA3 on cholera toxin production in Vibrio cholerae O1 ElTor type. [Figure 7] This figure shows the relationship between the amount of cholera toxin produced in cholera toxin-producing Vibrio cholerae O1 ElTor type and the concentrations of ISA1 and ISA2. [Figure 8]This figure shows the transcriptional repressive effects of ISA1 and ISA2 on cholera toxin-related genes in cholera toxin-producing Vibrio cholerae O1 ElTor type. [Figure 9] This figure shows the cytotoxicity of ISA1 and ISA2 against various eukaryotic cells. [Modes for carrying out the invention]

[0009] The cholera toxin production inhibitors disclosed herein (hereinafter also simply referred to as inhibitors) contain a compound represented by formula (1) above, or contain kale or a part thereof, or an extract thereof. The inhibitors suppress the production of cholera toxin by cholera-producing Vibrio cholerae. Cholera toxin is thought to be taken up by human intestinal epithelial cells, causing large amounts of electrolytes and water to leak from cells and tissues. Suppressing the production of cholera toxin in the intestinal tract during cholera outbreaks can be a means of avoiding or suppressing diarrhea and dehydration caused by diarrhea.

[0010] Furthermore, even before the onset of cholera symptoms, administering or ingesting inhibitors in advance can suppress the production of cholera toxin when cholera-producing Vibrio cholerae multiplies upon reaching the intestinal tract. Therefore, even if infected with cholera, the production of cholera toxin can be suppressed, and diarrhea can be avoided or suppressed.

[0011] The above disclosures in this specification will be described in detail below.

[0012] (Cholera toxin production inhibitor) The inhibitor may contain a compound represented by formula (1).

[0013] [ka]

[0014] In equation (1), R 1 , R 2 , R 4 , R 5 , R 6, and R 7 Each of these independently represents a hydrogen atom, a hydroxyl group, or an alkoxy group having an alkyl group with 1 to 4 carbon atoms. In this specification, the alkyl group having 1 to 4 carbon atoms is a linear or branched alkyl group, and examples include a methyl group, an ethyl group, an n-propyl group, a 2-propyl group, an n-butyl group, a sec-butyl group, an iso-butyl group, and a tert-butyl group. The alkoxy group may be any alkoxy group having one of these alkyl groups, for example, a methoxy group or an ethoxy group.

[0015] For example, R 4 , R 5 , R 6 , and R 7 Each of these is independently a hydrogen atom or the alkoxy group, and also, for example, R 4 , R 5 , R 6 , and R 7 These are all hydrogen atoms.

[0016] For example, R 2 is a hydrogen atom or the aforementioned alkoxy group, and is also, for example, a hydrogen atom.

[0017] For example, R 1 is a hydrogen atom or the aforementioned alkoxy group, and is also, for example, a hydrogen atom or a methoxy group.

[0018] For example, R 3 This is either a formyl group or a carboxyl group.

[0019] The compound represented by formula (1) is R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 For each, possible combinations of functional groups can be made, but for example, R 1 R is a hydrogen atom or the aforementioned alkoxy group, 3 R is a formyl group or a carboxyl group. 2 , R 4 , R5 , R 6 and R 7 Examples include compounds in which each is independently a hydrogen atom or the aforementioned alkoxy group, or both are hydrogen atoms.

[0020] Compounds represented by formula (1) also include compounds represented by the following formulas (1a), (1b), and (1c). The compound represented by formula (1a) (N-methoxyindole-3-carboxyaldehyde) is also hereinafter referred to as ISA1, the compound represented by formula (1b) (3-formylindole) is also hereinafter referred to as ISA2, and the compound represented by formula (1c) (3-carboxyindole) is also hereinafter referred to as ISA3.

[0021] [ka]

[0022] The compound represented by formula (1) is a cholera toxin-producing bacterium, V. cholerae, which may not inhibit, or only not strongly inhibit, the growth of serotypes O1ElTor, O1 Classical, and O139. For example, ISA1 and ISA2 have a weak tendency to inhibit the growth of O1ElTor, O1 Classical, and O139 cholera, as well as Staphylococcus and Escherichia coli. Also, for example, ISA3 has a weak tendency to inhibit the growth of Staphylococcus and Escherichia coli, but does have a tendency to inhibit the growth of O1ElTor, O1 Classical, and O139 cholera.

[0023] On the other hand, the compound represented by formula (1) inhibits the production of cholera toxin by cholera toxin-producing Vibrio cholerae. For example, ISA1, ISA2, and ISA3 inhibit the production of cholera toxin in Vibrio cholerae O1 ElTor, Vibrio cholerae O1 Classica, and Vibrio cholerae O139. In particular, they effectively inhibit the production of cholera toxin in Vibrio cholerae O1 ElTor. In some cases, they can suppress the production of cholera toxin in Vibrio cholerae O1 ElTor.

[0024] The compound represented by formula (1) can be obtained by synthesis. Furthermore, the compound represented by formula (1) can be obtained from or a part thereof. The compound represented by formula (1) is typically found in the roots of ikat, for example. For instance, it is found in extracts of ikat roots using organic solvents such as acetone or methanol. For example, ISA1 and ISA2 may be found in acetone extracts, while ISA3 may be found in methanol extracts.

[0025] To obtain the compound represented by formula (1) from Isatis tinctoria, first, Isatis tinctoria or a portion thereof is cut to an appropriate size, and then the components are extracted by cold immersion in a suitable leaching agent (e.g., acetone, ethanol, methanol, etc.) for a certain period of time to obtain the filtrate. Next, the leached solution is separated and purified as appropriate, either as is or by removing the solvent under reduced pressure as necessary.

[0026] The following describes an example of obtaining the compound represented by formula (1) from Isatis tinctoria. The compound represented by formula (1) can be obtained, for example, from an acetone extract obtained by immersing the dried roots of Isatis tinctoria in acetone at room temperature for a predetermined time, and then removing the acetone under reduced pressure. The compound represented by formula (1) can be obtained in at least one of several fractions obtained by separating the acetone extract using silica gel column chromatography, for example, in a hexane:acetone ratio of 3:1 to 1:1.

[0027] Furthermore, for example, the above acetone extract can be separated into at least one of several fractions in a hexane:acetone = 3:1 ratio, and this at least one fraction can be separated into at least one of several compounds in a benzene:hexane:acetone 20:5:3 ratio to obtain a compound represented by formula (1) (e.g., ISA1). Alternatively, for example, the above acetone extract can be separated into at least one of several fractions in a hexane:acetone = 2:1 ratio, and this at least one fraction can be further separated into at least one of several compounds in a benzene:hexane:acetone 20:5:1 ratio to obtain a compound represented by formula (1) (e.g., ISA1).

[0028] Furthermore, for example, at least one of several fractions obtained with hexane:acetone = 2:1, and at least one of several compounds separated with benzene:acetone = 12:1, can be used to obtain a compound represented by formula (1) (e.g., ISA2).

[0029] Furthermore, for example, a compound represented by formula (1) can be obtained from a methanol extract obtained by heating the residue after acetone extraction of dried powder of the roots of Isatis tinctoria twice under reflux with methanol and removing the solvent under reduced pressure. For example, a compound represented by formula (1) (e.g., ISA3) can be obtained by separating the methanol extract with hexane:acetone = 2:1 by silica gel column chromatography, further separating this fraction with benzene:chloroform:ethyl acetate = 30:1:1 by silica gel column chromatography, and further separating this fraction with isopropyl ether:acetone = 10:1 to obtain at least one of the compounds.

[0030] The inhibitor only needs to contain a compound represented by formula (1), and in that respect, it includes embodiments that contain kale or a part thereof, or an extract thereof. For example, when kale or a part thereof is used as an inhibitor, the kale or a part thereof may be fresh or dried by known drying methods such as freeze-drying. Furthermore, the kale or a part thereof may be a part thereof, such as the leaves or roots. In addition, the kale or a part thereof may be in the form of suitable shredded material, paste, powder, etc.

[0031] The inhibitory agent, Isatis tinctoria or a part thereof, is an extract (leachate) prepared with an organic solvent such as acetone or methanol, although this is not particularly limited. The extract can take the form of a solution, extract, paste, powder, etc., although this is not particularly limited.

[0032] An extract of Isatis tinctoria or a part thereof can be obtained, for example, by cutting Isatis tinctoria or a part thereof (e.g., the roots) to an appropriate size, extracting the components using a suitable extracting agent (e.g., acetone, ethanol, methanol, etc.), separating the solid and liquid to obtain an extract, and then, if necessary, fractionating or purifying this extract, followed by concentration or drying. For example, the extract may be obtained by simply concentrating or drying the extract, or it may be a crude purified product of the extract.

[0033] A person skilled in the art can determine the inhibitory activity of a compound represented by formula (1) as an inhibitor by evaluating whether it inhibits toxin production in cholera toxin-producing Vibrio cholerae, and then use a specific compound. Similarly, with regard to extracts of Isatis tinctoria, a fraction or compound that can be used as an inhibitor can be identified by evaluating whether the obtained fraction or isolated compound inhibits toxin production in cholera toxin-producing Vibrio cholerae.

[0034] (Composition for the treatment or prevention of cholera) The compositions for the treatment or prevention of cholera disclosed herein (hereinafter simply referred to as "the Composition") may contain the above-mentioned inhibitor. By containing the above-mentioned inhibitor, administering or ingesting the Composition at the time of cholera onset can prevent or suppress diarrhea and dehydration caused by diarrhea. Furthermore, administering or ingesting the Composition before the onset of cholera can suppress the production of cholera toxin during the proliferation of cholera toxin-producing Vibrio cholerae in the intestinal tract, thereby preventing or suppressing diarrhea.

[0035] The form in which this composition is administered or ingested is not particularly limited. This composition can take the form of a pharmaceutical, supplement, food, or the like.

[0036] If this composition is a pharmaceutical product, the formulation form can be appropriately selected from various known formulation forms. Since this composition is preferably intended to act in the intestinal tract, it can be in the form of, for example, capsules, oral liquid preparations such as elixirs, syrups, granules, powders, tablets, pills, oral jellies, or oral application preparations.

[0037] If this composition is a supplement, it can take the pharmaceutical formulation forms described above, particularly in the form of tablets or capsules. If this composition is a food, it can take the form of a food, such as a functional food or health supplement. Examples include powdered foods that can be taken orally, which may also contain powdered milk or protein, jelly-like foods, solid foods such as biscuits and cereals, and liquid foods such as beverages.

[0038] The effective use and dosage of this composition will be appropriately determined based on the age, weight, symptoms, etc., of the patient who has developed or is about to develop cholera. This composition can be administered or ingested, for example, as the compound represented by formula (1), at doses of 0.125 to 0.5 g, 3 to 4 times a day. [Examples]

[0039] The following examples illustrate the disclosures of this specification in more detail. The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosures of this specification. [Examples]

[0040] In this example, various fractions were separated from the roots of Isatis tinctoria, and indole compounds (ISA1, ISA2, ISA3) were obtained by separation and purification from specific fractions. The separation scheme is shown in Figures 1 and 2.

[0041] 1 kg of commercially available dried roots of Isatis tinctoria were immersed in 4 L of acetone for 12 hours, followed by solid-liquid separation. The solvent was then removed from the leachate under reduced pressure to obtain 3.46 g of acetone extract. As shown in Figure 1, the 3.46 g of acetone extract was divided into 12 major fractions using silica gel column chromatography (hexane:acetone (9:1, 4:1, 3:1, 2:1, 1:1) mixture, acetone, methanol). Furthermore, each fraction was separated and purified by silica gel column chromatography and thin-layer chromatography.

[0042] (Obtaining ISA1) Frac6 (139.2 mg, fraction prepared using a hexane:acetone (3:1) mixture) shown in Figure 1 was separated into six fractions using thin-layer chromatography with a benzene:hexane:acetone (20:5:3) mixture. From the separated fractions, one compound was identified from Frac6-2 as 1-methoxyindole-3-carbaldehyde (ISA1)(C 10 H9NO2 (MW: 175) was obtained (4.0 mg). ISA1 was a colorless oil.

[0043] (NMR identification data for ISA1) 1 H-NMR (acetone-d6) δ H :4.25 (3H, s, 1-OCH3), 8.39 (1H, s, H-2), 9.94 (1H, s, 3-CHO), 8.22 (1H, d, J = 8.4 Hz, H-4), 7.30 (1H, t, J = 7.1 Hz, H-5), 7.38 (1H, t, J = 7.1 Hz H-6), 7.57 (1H, d, J = 8.4 Hz, H-7) 13 C-NMR (acetone-d6) δ C:66.9 (1-OCH3), 133.6 (C-2), 114.4 (C-3), 184.1 (3-CHO), 121.8 (C-3a), 122.1 (C-4), 123.4 (C-5), 124.7 (C-6), 109.2 (C-7), 133.6 (C-7a)

[0044] Furthermore, Frac7 (141.0 mg, a fraction prepared using a hexane:acetone (2:1) mixture) shown in Figure 1 was separated into seven fractions using a benzene:hexane:acetone (20:5:1) mixture. Of the separated fractions, ISA1 was obtained as one compound from Frac7-1 (0.7 mg).

[0045] (Obtaining ISA2) Furthermore, Frac8 (129.5 mg, a fraction prepared using a hexane:acetone (2:1) mixture) shown in Figure 1 was separated into six fractions using a benzene:acetone (12:1) mixture via thin-layer chromatography. Of the separated fractions, indole-3-carbaldehyde (ISA2) (C9H7NO(MW:145)) was obtained as one compound from Frac8-4 (3.7 mg). ISA2 was a colorless oil.

[0046] (NMR identification data for ISA2) 1 H-NMR (acetone-d6) δ H :8.18 (1H, s, H-2), 10.02 (1H, s, 3-CHO), 8.21 (1H, dd, J = 1.5, 6.7 Hz H-4), 7.24 (2H, m, H-5,6), 7.52 (1H, t, J = 7.0 Hz H-7)

[0047] Furthermore, Frac9 (64.9 mg, a fraction prepared using a hexane:acetone (1:1) mixture) shown in Figure 1 was separated into four fractions using a benzene:acetone (10:1) mixture. Of the separated fractions, ISA2 was obtained as one compound from Frac9-2 (1.5 mg).

[0048] (Obtaining ISA3) After acetone extraction from the roots of commercially available Isatis tinctoria, 2 kg of the roots were further heated twice under reflux with methanol, and the solvent was removed by vacuum distillation to obtain a methanol extract (19.12 g). Next, as shown in Figure 2, this extract was divided into 12 major fractions by silica gel column chromatography (hexane:acetone (9:1, 4:1, 3:1, 2:1, 1:1) mixture, acetone, methanol). Furthermore, in Figure 2, a portion of Frac.M8 (hexane:acetone (2:1) mixture separation fraction, 770.5 mg) was separated into 9 fractions by silica gel chromatography (benzene:chloroform:ethyl acetate (30:1:1) mixture), and of these, FracM8-7 was further separated and purified by thin-layer chromatography (isopropyl ether:acetone (10:1) mixture) to obtain indole 3-carboxylic acid (ISA3, C9H7NO2 (MW:161)) (1.3 mg). ISA3 was a colorless, oily substance.

[0049] (NMR identification data for ISA2) 1 H-NMR (acetone-d6) δ H :8.02 (1H, s, H-2), 8.14 (1H, m, H-4), 7.19 (2H, m, H-5,6), 7.49 (1H, m, H-7) [Examples]

[0050] The following tests were performed on the indole compounds ISA1, ISA2, and ISA3 obtained in Example 1.

[0051] (Test items and methods) (1) Inhibitory effect on the growth of cholera toxin-producing Vibrio cholerae Muller Hinton Broth (MHB, Becton Dickinson and Company) 1×10 in 1mL 5Test bacteria were added to achieve a CFU (colony forming unit) / ml ratio, and compounds of each concentration were added, followed by static incubation at 37°C. The bacterial suspensions before incubation and at 2, 4, 6, and 24 hours after incubation were diluted with phosphate-buffered saline (PBS), spotted onto ordinary agar plates, and incubated overnight at 37°C. The number of colonies was measured the following day, and the number of bacteria (CFU) in the medium at each time point was calculated. The test strains used were Vibrio cholerae strain N16961 (O1, ElTor type), Vibrio cholerae strain 569B (O1, Classical), and Vibrio cholerae strain SG24 (O139). The results are shown in Figure 3.

[0052] As shown in Figures 3A and 3B, ISA1 did not inhibit the growth of O1 ElTor and O139 strains, but it tended to slightly inhibit the growth of O1 classical strains. ISA2 did not inhibit the growth of any of the O1 ElTor, O1 classical, or O139 strains. ISA3 tended to inhibit the growth of O1 ElTor strains. However, regrowth was observed after prolonged culture.

[0053] (2) Measurement of minimum inhibitory concentration (MIC) Minimum inhibitory concentration (MIC) was measured using the microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Muller-Hinton Broth (MHB, Becton Dickinson and Company) was used as the culture medium. The test strains were Staphylococcus aureus N315 (methicillin-resistant Staphylococcus aureus), Staphylococcus aureus 209P (methicillin-sensitive Staphylococcus aureus), Escherichia coli JCM1649, Vibrio cholerae N16961 (O1, ElTor type), Vibrio cholerae 569B (O1, Classical), and Vibrio cholerae SG24 (O139). The results are shown in Figure 4.

[0054] As shown in Figure 4, ISA1 and ISA2 showed similar MICs against two species of Staphylococcus, one species of Escherichia coli, and cholera toxin-producing Vibrio cholerae. In contrast, ISA3 showed a similar MIC to ISA1 and ISA2 against Escherichia coli, but a lower MIC against each species of cholera toxin-producing Vibrio cholerae compared to Staphylococcus and Escherichia coli. In particular, against the O1ElTor type and O1 classical type, the MIC was 12.5–50 μM, clearly indicating growth inhibitory activity. These results were consistent with the results of the effects on Vibrio cholerae growth shown in Figure 3.

[0055] (3) Effects on the production of cholera toxin The effect of latex agglutination on cholera toxin production in cholera toxin-producing Vibrio cholerae was investigated using the Denka Seiken VET-RPLA bacterial toxin detection kit. Bacteria grown on agar were suspended in 3 mL of YEP broth and incubated overnight at 30°C. The following day, 180 μL of YEP broth, 20 μL of cultured bacteria, and 2 μL of dimethyl sulfoxide (DMSO, FUJIFILM Wako Pure Chemical) or 2 μL of each compound (6.25-100 μM) were mixed and incubated overnight at 30°C. The toxin in the culture supernatant was quantified using the aforementioned kit. The results are shown in Figures 5-7.

[0056] YEP broth was prepared by mixing 0.1% yeast extract (Becton Dickinson and Company), 0.1% tryptone (Becton Dickinson and Company), and 0.05% NaCl (Nakalai-Tesque).

[0057] As shown in Figures 5 and 6, ISA1, ISA2, and ISA3 all suppressed the production of cholera toxin by cholera toxin-producing Vibrio cholerae. ISA1 and ISA2 most effectively suppressed the production of O1 ElTor type toxin. ISA3 also showed an inhibitory effect on the growth of O1 ElTor type cholera, suggesting that both the toxin production inhibitory effect of ISA and the inhibitory effect of ISA itself were at work.

[0058] Furthermore, as shown in Figure 7, when we investigated the relationship between the cholera toxin production inhibitory effect and compound concentration for ISA1 and ISA2, we found that there was a clear dependence on compound concentration.

[0059] (4) Measurement of transcription levels of cholera toxin production-related genes The bacteria grown on agar plates were suspended in 3 mL of YEP broth and incubated overnight at 30°C. The following day, 180 μL of YEP broth, 20 μL of the cultured bacteria, and 2 μL of DMSO or each compound were mixed and incubated overnight at 30°C. The next day, the bacteria were collected by centrifugation, and the precipitate was suspended in 200 μL of Tris-EDTA buffer (TE buffer). RNA extraction was performed using the Monarch Total RNA Miniprep Kit (BioLabs NEW ENGLAND). The obtained RNA was subjected to a reverse transcription reaction using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO CO., LTD.) to obtain cDNA. The composition and conditions of the reaction mixture followed the instructions.

[0060] The obtained cDNA was diluted in TE buffer to a concentration of 1 ng / μL. 1 μL of cDNA, 4 pmol each of primers, and 5 μL of POWER Track SYBR Green Master mix (Thermo Fisher Scientific) were mixed together, and the total volume was diluted to 10 μL with water. Real-time PCR was then performed using 45 PCR cycles: 10 minutes at 95°C, followed by 10 seconds at 95°C and 30 seconds at 60°C. The results are shown in Figure 8.

[0061] For PCR, the following primers are used. (For target gene amplification) ctxA-F:(5'-GGAGGGAAGAGCCGTGGAT-3')(Sequence ID 1) ctxA-R:(5'-CATCGATGATCTTGGAGCATTC-3')(Sequence ID 2) toxT-F:(5'-TGATGATCTTGATGCTATGGAGAAA-3')(Sequence ID 3) toxT-R:(5'-TCATCCGATTCGTTCTTAATTCAC-3')(Sequence ID 4) Internal control recA-F:(5'-CAATTTGGTAAAGGCTCCATCAT-3')(Sequence ID 5) recA-R:(5'-CCGGTCGAAATGGTTTCTACA-3')(Sequence ID 6)

[0062] As shown in Figure 8, ISA1 was found to repress the transcription of the ctxA gene, which is involved in cholera toxin production, and ISA2 was found to repress the transcription of the toxT gene, which is a regulatory gene in the ToxR regulon.

[0063] (5) Cytotoxicity test Cytotoxicity to eukaryotic cells was evaluated by measuring lactose dehydrogerace (LDH) production in the presence of indole compounds using the Cytox 96 Non-radiactive cytotoxicity assay kit (Promega). 0.1% TritonX-100 was used as the positive control. Eukaryotic cells used included the MRC5 cell line (a normal fibroblast cell line derived from human fetal lung), the A549 cell line (derived from human lung cancer), and the CACO-2 cell line (derived from human colon cancer). The results are shown in Figure 9.

[0064] As shown in Figure 9, neither ISA1 nor ISA2 showed toxicity to eukaryotic cells.

[0065] From the above, it can be said that the compound represented by formula (1), which includes ISA1, ISA2, and ISA3, can suppress the production of cholera toxin by cholera toxin-producing Vibrio cholerae. [Sequence Listing Free Text]

[0066] Sequence IDs 1-6: Primers

Claims

1. A cholera toxin production inhibitor comprising a compound represented by the following formula (1). 【Chemistry 1】 (In formula (1), R 1 , R 2 , R 4 , R 5 , R 6 , and R 7 each independently represents a hydrogen atom, a hydroxyl group, or an alkoxy group having an alkyl group with 1 to 4 carbon atoms, and R 3 represents a hydrogen atom, a formyl group, or a carboxyl group. However, the case where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 are all hydrogen atoms is excluded.)

2. The compound is, in formula (1), R 1 R represents a hydrogen atom or an alkoxy group having an alkyl group with 1 to 4 carbon atoms. 3 R represents a formyl group or a carboxyl group. 2 , R 4 , R 5 , R 6 and R 7 The cholera toxin production inhibitor according to claim 1, wherein the hydrogen atom represents a hydrogen atom or an alkoxy group having an alkyl group having 1 to 4 carbon atoms.

3. The compound is, in formula (1), R 2 , R 4 , R 5 , R 6 and R 7 However, the cholera toxin production inhibitor according to claim 2, which represents a hydrogen atom.

4. The cholera toxin production inhibitor according to claim 3, wherein the compound is any one represented by the following formulas (1a), (1b), and (1c). 【Chemistry 2】

5. A cholera toxin production inhibitor containing kale or a part thereof, or an extract thereof.

6. The production inhibitor according to claim 5, wherein the extract is an extract of the root.

7. A composition for the treatment or prevention of cholera, comprising a cholera toxin production inhibitor according to any one of claims 1 to 6.

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