Agent for inhibiting bacterial flagellar motility

Abstract

To provide a composition and method for inhibiting bacterial flagellar motility.SOLUTION: The present invention provides an agent for inhibiting bacterial flagellar motility that contains rhatannin as an active ingredient, and a pharmaceutical composition that contains the same, as well as a method for inhibiting bacterial flagellar motility which includes contacting bacteria present in an ex vivo environment with rhatannin.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to compositions and methods for inhibiting bacterial flagellar motility. [Background technology]

[0002] Inactivating bacteria is useful in various fields such as environmental hygiene, medicine, veterinary medicine, and agriculture including not only cultivation but also livestock and fisheries. In other words, antibacterial agents generally have high industrial utility.

[0003] Most conventional antibacterial drugs work by targeting vital functions or structures of bacteria, such as metabolism, protein synthesis, DNA synthesis, and cell walls, and inhibiting bacterial growth and survival. Bacteria can acquire various traits through frequent mutation or foreign gene acquisition. In an environment where antibacterial drugs are present, bacteria that have acquired resistance to antibacterial drugs may survive and spread preferentially under strong selective pressure. With the emergence and increase of drug-resistant bacteria becoming a problem, it is not easy to find new target molecules and candidate drugs, and the development costs are not commensurate with the profits, so the development of antibacterial drugs has stagnated worldwide.

[0004] Many plant materials containing antibacterial activity are also known. As an example, Non-Patent Document 1 describes that rhubarb, a species of the Polygonaceae family, has antibacterial activity, and describes that its mechanism of action is to inhibit bacterial growth by destroying the bacterial cell wall structure and changing the permeability of the cell membrane. Plants of the genus Rheum are also known as the raw material for the herbal medicine "Da-huang." Non-Patent Document 2 describes that administration of Dai-huang in a model infection experiment significantly reduced the colonization of Enterohemorrhagic Escherichia coli (EHEC) and increased the survival rate of infected mice. Rhubarb is thought to be composed of a complex collection of a very large number of trace components, rather than one component being the major component (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Xiang et al., Chin Med (2020) 15:88 [Non-Patent Document 2] Amran et al., Microbial Pathogenesis 65 (2013) 57-62 [Non-Patent Document 3] Chen et al. Chinese Medicine (2022) 17:50 Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure provides compositions and methods for inhibiting bacterial flagellar motility. [Means for solving the problem]

[0007] The present inventors have surprisingly discovered that a known substance called rhatannin can inhibit the flagellar movement of bacteria without killing them. This discovery has made it possible to provide a completely new type of antibacterial agent that acts by targeting flagella. The present disclosure includes the following embodiments. [1] A bacterial flagellar motility inhibitor containing rattannin as an active ingredient. [2] The bacterial flagellar motility inhibitor according to [1], which is a solid preparation having a ratannin content of 10% by mass or more, or an aqueous preparation having a ratannin content of 10% by mass or more in the aqueous component. [3] The bacterial flagellar motility inhibitor according to [1] or [2], comprising a plant extract containing the rattannin, wherein the content of the plant extract components that are not rattannins per 1 part by mass of the rattannin is 0 part by mass or more and 9 parts by mass or less. [4] The bacterial flagellar motility inhibitor according to any one of [1] to [3], wherein the bacterial flagellar motility inhibitor is an aqueous preparation, and a water-soluble concentration of the tannin in the aqueous preparation is 0.1 mg / ml or more. [5] The bacterial flagellar motility inhibitor according to any one of [1] to [4], wherein the bacterial flagellar motility inhibitor contains at least 20 mg of tannin. [6] A pharmaceutical composition comprising the bacterial flagellar motility inhibitor according to any one of [1] to [5]. [7] The pharmaceutical composition described in [6], which is an oral administration formulation. [8] The pharmaceutical composition according to [6] or [7], wherein the content of radonnin per single dosage is 20 mg or more. [9] The pharmaceutical composition according to any one of [6] to [8], for preventing or treating a bacterial infection by inhibiting bacterial flagellar movement.

[10] A method for inhibiting bacterial flagellar movement, comprising contacting bacteria present in an in vitro environment with Latin tannin.

[11] The method according to

[10] , wherein the tannin is provided in the form of an inhibitor of bacterial flagellar motility according to any one of [1] to [5]. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the experimental results showing the inhibition of flagellar motility rates of various bacteria by rat tannins. [Diagram 2] FIG. 2 shows the results of an experiment demonstrating the low or lack of bacterial growth inhibitory effect of rattannins. [Diagram 3] FIG. 3 shows the experimental results showing that administration of rattannin significantly improved the survival rate of mice infected with enterohemorrhagic Escherichia coli. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] As is well known to those skilled in the art, typical bacterial motility is driven by flagellar rotation. Bacterial motility is necessary, for example, for pathogenic bacteria to migrate to an infection site and exert their pathogenicity. The present inventors have discovered that Latannin has the effect of inhibiting the flagellar motility of a wide range of bacteria. Latannin appears to act specifically on flagella, inhibiting only the motility of bacteria without killing them (at least directly). When purified Latannin was administered to bacteria swimming by active flagellar motility under a microscope, it was observed that the flagellar motility and swimming of the bacteria that remained alive were very clearly inhibited. The effect was not limited to a specific bacterium, but was confirmed in a wide range of bacterial species.

[0010] La tannin itself has been described previously and is a type of condensed tannin found at least in plants of the genus Rheum. The possibility that la tannin exists in organisms other than Rheum, such as other Polygonaceae plants, is not excluded. Nonaka et al. Chem. Pharm. Bull. 29(10) 2862-2870 (1981) describes the identification, structure, and purification method of la tannin in rhubarb. As described by Nonaka et al., la tannin can be obtained as a soluble substance in the methanol eluate obtained by subjecting the methanol extract obtained by extracting the acetone-insoluble fraction of a rhubarb rhizome sample with methanol, subjecting the aqueous extract obtained by extracting the methanol extract with ethyl acetate-water to Sephadex LH-20 column chromatography to obtain a methanol eluate, and then subjecting the methanol eluate obtained by further subjecting the aqueous extract to Sephadex LH-20 column chromatography. Nonaka et al. have shown by thiolysis that latannin is a condensation polymer of galloylated and non-galloylated (epi)catechin (more specifically, 3-O-galloyl-(-)-epicatechin, (+)-catechin, etc.). In one aspect, the present disclosure provides a bacterial flagellar motility inhibitor comprising latannin as an active ingredient.

[0011] In some embodiments, the bacterial flagellar motility inhibitor comprises a plant extract containing latannin. Plants typically contain water, but the term "extract" as used herein refers to a substance other than water. The plant from which the extract is derived is preferably a Rheum plant, such as rhubarb, but may be another plant. A solvent capable of extracting latannin from a plant can be appropriately found by a person skilled in the art based on his or her ordinary knowledge. The solvent may be, for example, water, methanol, ethanol, or a mixed solvent containing any of these. The plant extract may be an extract that has been at least partially purified by, for example, separation extraction using a combination of two different solvents, column chromatography, adsorption, or a combination thereof. It is preferable that the bacterial flagellar motility inhibitor comprises a plant extract containing latannin, and that the content of components other than latannin (herein referred to as "non-latannin extract components") among the components derived from the same plant extract (plant extract components) per part by mass of latannin is 0 parts by mass or more and 9 parts by mass or less. In other words, it is preferable that the plant extract is purified to a latannin concentration of at least 10%. In the bacterial flagellar motility inhibitor, the content of non-ratannin extract components is more preferably 5 parts by mass or less, more preferably 4 parts by mass or less, more preferably 3 parts by mass or less, even more preferably 2 parts by mass or less, and particularly preferably 1 part by mass or less per 1 part by mass of ratannin. This describes a purified plant extract with increasing purity for ratannin. The plant extract may further have a ratannin purity of 50% or more, 70% or more, or 90% or more by mass, or may even be 100%. The original plant extract may contain a variety of other active ingredients (e.g., active ingredients that kill bacteria), but as the purity for ratannin increases, the relative proportion of flagellar motility inhibitory activity increases. It is also possible to extract and purify ratannin from herbal medicines, such as rhubarb, which is the dried rhizome of the rhubarb plant.

[0012] In some embodiments, regardless of the origin of the la tannin, the bacterial flagellar motility inhibitor is a solid formulation in which the la tannin content relative to the entire formulation is 10% by mass or more, or an aqueous formulation in which the la tannin content in the aqueous component is 10% by mass or more. These contents may be 20% or more, 30% or more, 50% or more, 70% or more, or 90% or more. In certain embodiments, the la tannin content may be 99% or even 100%. The aqueous formulation may be, for example, an aqueous solution or a hydrogel. The la tannin content in the aqueous component of an aqueous solution of 10% by mass or more means that 10% by mass or more of the solute of the aqueous solution is la tannin. A hydrogel includes a state in which an aqueous solution is trapped in a matrix of a water-insoluble substance, and the la tannin content in the aqueous component of a hydrogel of 10% by mass or more means that 10% by mass or more of the solute of the aqueous solution is la tannin. In the solid preparation or in the aqueous preparation, the components other than the tannins may include various excipients, non-tannin extract components, other active compounds, other herbal medicines, or combinations thereof. The excipients may be pharmaceutically acceptable excipients, examples of which include, but are not limited to, lactose, sucrose, glucose, starch, dextrin, cellulose, mannitol, inorganic salts, glycerin, etc. The bacterial flagellar motility inhibitor may include additives known to those skilled in the art, such as preservatives, colorants, flavorings, fragrances, etc. In non-medicinal applications, such as agricultural applications, the variety of excipients and additives that can be used may be further expanded.

[0013] In some embodiments, the bacterial flagellar motility inhibitor is an aqueous preparation, and the aqueous concentration of the tannin in the aqueous preparation is 0.1 mg / ml or more, for example, 0.1 to 25 mg / ml, 0.1 to 10 mg / ml, 0.1 to 2.5 mg / ml, or 0.1 to 1.5 mg / ml. This aqueous concentration may be 0.2 mg / ml or more, 0.5 mg / ml or more, or 1 mg / ml or more.

[0014] In some embodiments, the bacterial flagellar motility inhibitor contains at least 20 mg of tannin. The bacterial flagellar motility inhibitor may contain, for example, 20 to 3000 mg, 50 to 2000 mg, or 100 to 1000 mg of tannin. These bacterial flagellar motility inhibitors may be, for example, a dry solid formulation, but may also be a liquid formulation or an aqueous formulation.

[0015] Since rattannin is a natural component contained in edible plants, it can be characterized by its low cost and safety, which means that it can be obtained without the need for synthesis. Furthermore, flagella are a basic structure that many bacteria, both gram-negative and gram-positive, have in common. The inventors have confirmed that the bacterial flagellar motility inhibitor according to the present disclosure is effective against a wide variety of bacteria, not limited to a specific bacterial species, and the bacterial flagellar motility inhibitor is also characterized by having a broad antibacterial spectrum. Non-limiting examples of bacteria to which the bacterial flagellar motility inhibitor according to the present disclosure can be applied are shown below. Bacteria of the genus Escherichia such as Escherichia coli; Bacteria of the genus Salmonella such as Salmonella enterica and Salmonella bongori; Yersinia Bacteria of the genus Yersinia such as enterocolitica; Bacteria of the genus Citrobacter; Bacteria of the genus Edwardsiella; Bacteria of the genus Enterobacter; Bacteria of the genus Hafnia; Bacteria of the genus Klebsiella; Bacteria of the genus Morganella; Bacteria of the genus Plesiomonas; Bacteria of the genus Proteus; Bacteria of the genus Providencia; Bacteria of the genus Vibrio such as Vibrio cholerae and Vibrio parahaemolyticus; Bacteria of the genus Aeromonas; Bacteria of the genus Haemophilus; Bacteria of the genus Campylobacter; Bacteria of the genus Helicobacter such as Helicobacter pylori; Bacteria of the genus Pseudomonas such as Pseudomonas aeruginosa; Bacteria of the genus Bacillus such as Clostridium botulinum and Clostridium tetani; bacteria of the genus Listeria such as Listeria monocytogenes; bacteria of the genus Enterococcus such as Enterococcus faecalis. In certain embodiments, the bacterium targeted for inhibition of bacterial flagellar motility may be a bacterium other than that of the genus Escherichia, particularly other than Escherichia coli.

[0016] In one aspect, the present disclosure provides a pharmaceutical composition comprising any of the bacterial flagellar motility inhibitors described above. This pharmaceutical composition can be understood as a pharmaceutical composition for inhibiting bacterial flagellar motility. Typically, the pharmaceutical composition of the present disclosure is a preparation for oral administration. Examples of other possible administration routes include, but are not limited to, rectal, oral, nasal, vaginal, intravenous, intradermal, intramuscular, inhalation, and topical administration, as well as administration to the skin with or without trauma.

[0017] In some embodiments, the pharmaceutical composition contains 20 mg or more of latannin per single dose. The total fluid volume of the stomach, small intestine, and large intestine in a fasting human is estimated to be approximately 163 mL on average (Aliment Pharmacol Ther 2005; 22: 971-979). A single dose of 20 mg or more can achieve a concentration in the digestive tract close to 0.1 mg / mL. The content of latannin per single dose can be 20-3000 mg, 50-2000 mg, or 100-1000 mg. These single doses can be repeated multiple times, for example, twice or more per day.

[0018] The pharmaceutical composition of the present disclosure is useful for preventing or treating bacterial infections by inhibiting bacterial flagellar movement. An example demonstrating this is provided in the Examples section below. The pharmaceutical composition can be ingested or administered before, during, or after consumption of foods suspected of being at risk for bacterial infection, or after the onset of symptoms of bacterial infection.

[0019] Conventional antibacterial drugs, or more generally, antibacterial agents, typically target life-sustaining functions including bacterial metabolism, cell wall or membrane structure, protein synthesis, or nucleic acid synthesis. Therefore, strong selective pressure from the antibacterial agent inevitably results in the survival of resistant bacteria, which tend to expand advantageously in the newly opened niche. In contrast, the drug and antibacterial agent disclosed herein are unique in that they target flagellar motility, which is known to play an important role in the bacterial infection process but is not directly required for life support. The drug and antibacterial agent stop the activity of bacteria without immediately inhibiting their growth or killing them, and therefore are considered to have the advantage of being less selective and less likely to produce resistant bacteria.

[0020] The present disclosure further provides a method for inhibiting bacterial flagellar motility, comprising contacting bacteria present in an in vitro environment, particularly in a human in vivo environment, with a tannin. The tannin may be provided in any of the above-mentioned forms of composition. This method is distinct from administration to the human body.

[0021] The bacterial flagellar motility inhibitor according to the present disclosure is provided in the form of a pharmaceutical and is useful in medicine, and also finds utility in various fields such as veterinary medicine, environmental sanitation, and agriculture, including the livestock and fisheries industries.

[0022] In this specification, a numerical range indicated using the expression "A to B" (or "from A to B" or "between A and B") indicates a range including the stated numerical values ​​A and B as the minimum and maximum values, respectively, and also includes a range excluding the minimum and / or maximum values. In addition, the expression "A to B" is understood to disclose a range of "A or more" or "greater than A" and a range of "B or less" or "less than B". When multiple numerical ranges are described, the upper limit or lower limit of a stated numerical range can be combined with the upper limit or lower limit of another stated numerical range, and the numerical range obtained by such combination is also understood to be disclosed in this application. In this specification, the expression "including A" or "having A" means an object that includes other things besides A in addition to A, unless contrary to the context, and the disclosure of an object that includes only A and does not include other things than A, i.e., an object consisting of A, is also included in the description, and vice versa. Reference to a singular object is understood to encompass disclosure of embodiments in which plural objects are used. EXAMPLES

[0023] 1. Extraction and purification of rattannins Ratannin was extracted and purified based on the method of Nonaka et al. described above. Specifically, 300 ml of methanol was added to 100 g of powder of the herbal medicine Rheum officinalis, which is the dried rhizome of the Rheum genus plant, and the mixture was stirred for 24 hours. After that, the mixture was filtered to remove solids, and the filtrate, the methanol solution, was concentrated to dryness under reduced pressure. The residue was dissolved in a 50% aqueous ethanol solution and applied to Sephadex LH-20 column chromatography. After washing the column with 500 ml of a 50% aqueous ethanol solution and then with 1000 ml of ethanol, the ratannin adsorbed on the column was eluted with methanol. The eluate was concentrated to dryness to obtain purified ratannin. In this way, about 3 g of ratannin was obtained. This ratannin sample was used in the experiments described below.

[0024] 2. Inhibition of bacterial flagellar motility by rat tannins Standard liquid media containing ratannin at concentrations of 0 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1.0 mg / ml were inoculated with overnight cultures of Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium, Vibrio cholerae, and Vibrio parahaemolyticus at a 1 / 100 dilution and cultured. After 3 hours, under a microscope, it was observed that in the ratannin concentration 0 mg / ml (i.e., ratannin-free control) samples, the bacterial individuals of each bacterial species were swimming with uniformly active flagellar movement, whereas in the ratannin-added samples, it was clearly observed that flagellar movement was inhibited and swimming was stagnant in many bacterial individuals of each bacterial species.

[0025] The proportion of motile bacterial individuals in each sample was measured under a microscope (n=100 or more). The results are shown in Figure 1. A clear decrease in motility was already observed at a ratannin concentration of 0.1 mg / ml, and the bacterial motility rate decreased in a ratannin concentration-dependent manner in all bacterial species. This result illustrates that ratannin has an inhibitory effect on flagellar motility in a variety of bacteria.

[0026] 3. Effect of Ratannins on Bacterial Growth A piece of filter paper soaked with the test substance was placed on a standard agar medium spread with a diluted suspension of the test bacteria for inoculation. The test substances were antibiotics (ampicillin or tetracycline) as a control, or 0.1 to 100 mg / ml of latannin. The test bacteria were the same as those in Figure 1. The E. coli in Figures 1 and 2 was the B2F1 strain of enterohemorrhagic E. coli (EHEC). These agar media were statically cultured under standard conditions to grow the bacteria.

[0027] The results are shown in Figure 2. In this assay, if a test substance inhibits the growth of the test bacteria, an inhibition zone is formed around the corresponding filter paper strip. This is because the test substance diffuses from the filter paper strip during the incubation period and inhibits the growth of the bacteria, resulting in the absence of bacterial layer formation only around the filter paper strip and leaving the agar medium surface exposed. As is clear from Figure 2, a clear inhibition zone was formed around the antibiotic-impregnated filter paper strip as a control (except for ampicillin-resistant P. aeruginosa). In contrast, with ratannin, signs of inhibition zone formation were observed at a high concentration of 100 mg / ml, but there were no or almost no inhibition zones at concentrations below 10 mg / ml. These results demonstrate that ratannin is substantially devoid of activity to inhibit cell growth or cell survival. It is particularly noteworthy that no inhibition of cell growth or survival was observed at concentrations below 1 mg / ml, which were able to provide significant flagellar motility inhibition in the experiment in Figure 1.

[0028] 4. Rattannins protect against bacterial infections A survival curve analysis was performed using a model infection experiment system similar to that described in Non-Patent Document 2. Specifically, a group of four-week-old mice was given drinking water containing streptomycin from three days before infection and fasted for half a day before infection. Then, on day 0, all mice were orally infected with the same amount of streptomycin-resistant enterohemorrhagic Escherichia coli (EHEC) B2F1 strain by intragastric administration. In this test, WT-B2F1, which is more lethal than GFP-B2F1, was used (Non-Patent Document 2). Then, 0.3 mg / g of Rhubarb or the same weight of Raphanus sativus was orally administered to the mice in the form of an aqueous solution once a day from day 1 to day 5. The obtained survival curve is shown in FIG. 3.

[0029] Under these test conditions, the survival rates of both the untreated control group and the Rheum officinalis-treated group fell below 50% by about the 10th day after infection, and all mice in the untreated control group had died by the 16th day. In contrast, the group treated with purified ratannin maintained a 100% survival rate until the end of the 26-day test period. These results demonstrate that ratannins, which have flagellar motility inhibitory properties, may be significantly useful for preventing, treating, and / or preventing the progression of bacterial infections.

Claims

1. A bacterial flagellar motility inhibitor containing rattannin as an active ingredient.

2. The bacterial flagellar movement inhibitor according to claim 1, which is a solid preparation having a tannin content of 10% by mass or more, or an aqueous preparation having a tannin content of 10% by mass or more in the water-soluble component.

3. The bacterial flagellar movement inhibitor according to claim 1, comprising a plant extract containing the rattannin, and the content of the plant extract components that are not rattannin per 1 part by mass of the rattannin is 0 parts by mass or more and 9 parts by mass or less.

4. The bacterial flagellar motility inhibitor according to claim 1, wherein the bacterial flagellar motility inhibitor is an aqueous preparation, and the aqueous preparation has a water-soluble concentration of tannin of 0.1 mg / ml or more.

5. The bacterial flagellar motility inhibitor according to claim 1, wherein the bacterial flagellar motility inhibitor contains at least 20 mg of lattannin.

6. A pharmaceutical composition comprising the bacterial flagellar motility inhibitor according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, which is an oral administration formulation.

8. 7. The pharmaceutical composition according to claim 6, wherein the content of radonnin per single dosage is 20 mg or more.

9. The pharmaceutical composition according to claim 6 for preventing or treating a bacterial infection by inhibiting bacterial flagellar movement.

10. A method for inhibiting bacterial flagellar movement, comprising contacting bacteria present in an in vitro environment with Latin tannin.

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