JP7565597B2 - Bacteriostatic Composition - Google Patents

Description

The present invention relates to a bacteriostatic composition containing tryptanthrin for use against Campylobacter bacteria that cause food poisoning.

Food poisoning caused by Campylobacter bacteria, especially Campylobacter jejuni (C. jejuni) or Campylobacter coli (C. coli), is the most common bacterial food poisoning not only in Japan but also worldwide. In particular, because C. jejuni and C. coli are frequently carried in the intestines of livestock, there have been many cases of food poisoning caused by contamination of meat, especially chicken, with these bacteria when shipped from slaughterhouses and poultry processing plants. For this reason, there is a strong demand for effective measures to reduce Campylobacter infection in livestock, especially chickens, and contamination of chicken carcasses with these bacteria at poultry processing plants.

As measures against Campylobacter infection in livestock, the following have been proposed: a vaccine containing an antiserum raised against a non-flagellated Campylobacter strain (Patent Document 1); a Campylobacter growth inhibitor in poultry or livestock containing Clostridium butyricum as an active ingredient (Patent Document 2); drinking water containing lactic acid bacteria and oligosaccharides for controlling Campylobacter infection in chickens (Patent Document 3); and feed for controlling Campylobacter infection in chickens containing mulberry leaves or a mixture of mulberry leaves and oligosaccharides (Patent Document 4).

Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione) is a type of indoloquinazoline alkaloid contained in indigo-bearing plants (hereafter referred to as indigo grass), which are the raw material for the blue pigment indigo. Tryptanthrin has antibacterial activity against Bacillus subtilis, Escherichia coli, methicillin-resistant Staphylococcus aureus, Plasmodium falciparum, Leishmania donovani, Trypanosoma brucei, Toxoplasma gondii, Helicobacter pylori, etc., and is considered to be the scientific basis for the antibacterial action of indigo grass (Non-Patent Document 1). It has also been reported that tryptanthrin and indigo grass extracts containing it also have antibacterial activity against Campylobacter rectus, Streptococcus mutans, and other oral bacteria (Patent Document 5). However, the physiological activity of tryptanthrin against C. jejuni and C. coli, which are considered to be the main causes of food poisoning, is unknown.

Takeo Sasaki, Tohoku Pharmaceutical University Research Journal, No. 62, 25-37 (2015)

Patent Publication No. 2000-351735 Patent Publication No. 04-154727 Patent Publication 2012-180304 Patent Publication No. 2012-125225 WO2008/062861

The present invention aims to provide a new means for inhibiting the growth of Campylobacter bacteria, particularly C. jejuni and C. coli, which cause food poisoning.

The present inventors have discovered that tryptanthrin and indigo plants containing it have high bacteriostatic activity against C. jejuni and C. coli, and have completed the following invention.

(1) A bacteriostatic composition against Campylobacter jejuni or Campylobacter coli, containing tryptanthrin.
(2) The composition according to (1), which contains indigo grass or an extract of indigo grass.
(3) The composition according to (2), wherein the indigo plant is Indigo Root.
(4) The composition according to any one of (1) to (3), which is in the form of a feed or drink for a non-human animal.
(5) The composition according to (4), wherein the non-human animal is a cow, a pig or a poultry.
(6) The composition according to (4) or (5), wherein the non-human animal is a chicken.
(7) A method for bacteriostasis of Campylobacter jejuni or Campylobacter coli in a non-human animal, comprising administering to the non-human animal a composition containing tryptanthrin.
(8) The method according to (7), wherein the non-human animal is a cow, a pig or a poultry.
(9) The method according to (7) or (8), wherein the non-human animal is a chicken.

The bacteriostatic composition of the present invention can selectively and strongly inhibit the growth of Campylobacter bacteria that cause food poisoning without undesirably affecting the intestinal flora of animals.

The following description of the present invention may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. In this specification, the upper and lower limit values of each numerical range can be combined in any way. In addition, in this specification, a numerical range expressed using "~" or "-" means a range that includes the numerical values at both ends as the upper and lower limits, unless otherwise specified.

The present invention provides a bacteriostatic composition for C. jejuni or C. coli that contains tryptanthrin.

Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione) is a compound represented by the following formula (I).

Both C. jejuni and C. coli are major causative bacteria of Campylobacter food poisoning in Japan, and have been reported to be genetically diverse. Tryptanthrin can selectively exert high bacteriostatic activity against various genotype strains of C. jejuni and C. coli. Therefore, a composition containing tryptanthrin can be used for bacteriostasis of C. jejuni and C. coli, and is considered to be able to exert selective bacteriostatic activity with little effect on the intestinal flora when ingested by humans and non-human animals, particularly livestock. In the present invention, a bacteriostatic composition means a composition that inhibits the growth of the target bacteria, i.e., for bacteriostasis, and bacteriostatic activity is used interchangeably with growth inhibitory activity or antibacterial activity.

In one embodiment, the bacteriostatic composition contains tryptanthrin-containing indigo plant or an extract of indigo plant. The indigo plant used in the present invention may be any plant containing tryptanthrin, and examples thereof include Polygonum tinctorium (also called indigo plant) of the Polygonaceae family, Strobilanthes cusia (Strobilanthes cusia) of the Acanthaceae family, Indigofera suffruticosa and Indigofera tinctoria (Indigofera tinctoria) of the Leguminosae family, Isatis tinctoria var. yezoensis, Isatis indigotica, and Isatis tinctoria (Isatis tinctoria) of the Brassicaceae family, and Marsdenia tinctoria (Marsdenia tinctoria) of the Asclepiadaceae family. The indigo plant preferably used in this invention is the indigo plant, Tadeolia japonica.

The indigo plant used in the present invention may be a wild species or a cultivated variety. Cultivated varieties of Tadeo that are preferably used in the present invention include the white flower and red flower varieties of Kogamiko, Akakame Kosenbon, Aokame Kosenbon, Senbon, Osenbon, Miyagi, Matsue, Hiroshima Kannabe, Konba, Nakatsubaki, and Hyakukan. There are no limitations on the cultivation method of the indigo plant, and it may be cultivated by a method conventionally used for cultivating indigo plant, or it may be naturally growing indigo plant.

Although it is possible to use the entire indigo plant, it is preferable to use the leaf part, which contains a lot of tryptanthrin. It is also known that the tryptanthrin content of indigo increases when dried, so it is preferable to use dried indigo. Drying can be performed by sun drying, hot air drying, or other methods known to those skilled in the art.

The indigo grass contained in the bacteriostatic composition is preferably finely divided by shredding, crushing, grinding, etc., and is preferably in the form of a powder.

The indigo plant extract used in the present invention can be prepared by extracting the indigo plant, which is preferably dried, with a solvent. Solvents used in the extraction process include water, lower alcohols (e.g., methanol, ethanol), polyhydric alcohols (e.g., propylene glycol, 1,3-butylene glycol), etc., which can be used alone or in a suitable mixture of two or more. Preferred solvents for extraction from indigo plant include water, lower alcohols, or water and lower alcohols, with ethanol being particularly preferred as the lower alcohol.

The extraction process can be carried out by a general method for producing plant extracts, including contacting indigo grass with a solvent. The amount of solvent used, extraction time, extraction temperature, and other extraction conditions can be appropriately set by those skilled in the art. For example, when using water or a mixed solvent of water with a high water content and a lower alcohol as the extraction solvent, it is preferable to set the extraction temperature higher than room temperature, for example, 60°C or higher, 80°C or higher, or 90°C or higher, in order to increase the extraction efficiency. In addition, additives such as surfactants may be appropriately added to the extraction solvent as long as they do not impair the bacteriostatic activity in the present invention. After extraction, the extract can be recovered by a general method such as filtration, centrifugation, or decantation.

The indigo plant extract obtained by the above extraction process can be contained in the bacteriostatic composition of the present invention in the form of the extract itself, or in the form of a concentrate or dried product of the extract. There are no limitations on the method of concentrating and drying the extract, and it can be carried out by methods known to those skilled in the art, such as heating and freeze-drying.

In addition, in the present invention, commercially available indigo plant extracts, such as tea made from indigo plant, can also be used.

In another embodiment, the bacteriostatic composition contains synthesized tryptanthrin or tryptanthrin purified from a mixture of the above-mentioned indigo herb extract and the like. Tryptanthrin can be synthesized by a method known to those skilled in the art, for example, by thermally decomposing indigo (2,2'-bis(2,3-dihydro-3-oxoindolylidene)), a blue pigment, without a solvent (see, for example, JP 2017-1969, etc.). Purification of tryptanthrin from a mixture can be performed by a method known to those skilled in the art, for example, filtration, centrifugation, solvent fractionation, fractional precipitation, dialysis, various types of chromatography, etc.

There is no limitation on the form of the bacteriostatic composition of the present invention, and it may be in a liquid form, or in a powder, granules, lump or other solid form. In addition, the bacteriostatic composition may contain physiologically active substances other than tryptanthrin (antibacterial agents, growth promoters, etc.), nutritional components (proteins, lipids, minerals, vitamins, etc.), or components such as excipients, binders, disintegrants, lubricants, flavorings, solubilizers, or carriers, so long as they do not interfere with the bacteriostatic activity.

The bacteriostatic composition of the present invention contains an effective amount of tryptanthrin. Here, the effective amount is an amount capable of bacteriostatically inhibiting C. jejuni or C. coli, and is appropriately determined by those skilled in the art depending on factors such as the environment to which the bacteriostatic composition is applied, the amount of C. jejuni or C. coli present in the environment, and the method of application of the bacteriostatic composition. For example, when the bacteriostatic composition is orally administered to chickens, the effective amount of tryptanthrin is, for example, 5 to 2000 μg/day, preferably 10 to 1500 μg/day, more preferably 20 to 1000 μg/day, and particularly preferably 50 to 750 μg/day per chicken.

The bacteriostatic composition of the present invention can be applied to in vitro and in vivo environments where bacteriostasis of C. jejuni or C. coli is required, and in particular, can be applied to animals that carry or may carry C. jejuni or C. coli. Examples of such animals include humans and non-human animals, such as mice, rats, hamsters, dogs, cats, cows, pigs, poultry (chickens, ducks, geese, turkeys, quails, etc.), and monkeys. The bacteriostatic composition can be applied preferably to livestock used for food, such as cows, pigs, and poultry, and particularly preferably to chickens.

In the present invention, application of the bacteriostatic composition means that the bacteriostatic composition is present in an environment where C. jejuni or C. coli is present. The application of the bacteriostatic composition to animals can be carried out by having the animals ingest the bacteriostatic composition. For example, the bacteriostatic composition to be ingested by livestock is preferably in the form of livestock feed or drink containing tryptanthrin, indigo herb, or indigo herb extract. Such feed or drink can be prepared by blending tryptanthrin, indigo herb, or indigo herb extract with general livestock feed or drinking water. By making it in the form of livestock feed or drink, the bacteriostatic composition can be easily ingested without causing stress to livestock, and C. jejuni and C. coli living in the intestinal tract of livestock can be bacteriostatic.

The bacteriostatic composition in the form of livestock feed or drink can be given to livestock throughout the entire rearing period, but from the viewpoint of efficiency, it should be given during the period when bacteriostasis of C. jejuni or C. coli is particularly desired, for example, in the case of meat chickens, from about 3 weeks of age when the appearance of these bacteria begins to be confirmed, to approximately 2 to 3 weeks before shipping.

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

Example 1
- Preparation of tryptanthrin solution Two-fold serial dilutions of tryptanthrin (Fujifilm Wako Pure Chemical Industries) with concentrations ranging from 100 μM to 0.05 μM were prepared using Mueller-Hinton liquid medium (Becton Dickinson).
・Preparation of indigo powder extract 2g of commercially available indigo powder (dried powder of indigo leaves) was added to 100ml of Mueller-Hinton liquid medium (Becton Dickinson), shaken at 100 rpm for 3 hours at room temperature, then centrifuged at 13,000 rpm for 10 minutes and filtered the supernatant through a 0.22μm filter to prepare a 2% indigo extract. The extract was serially diluted 2-fold to prepare dilutions with indigo powder concentrations ranging from 2% to 0.0156%.
- Measurement of minimum inhibitory concentration (MIC)
100μL of tryptanthrin solution or indigo powder extract at each concentration was dispensed into a 96-well plate. After inoculating the test bacteria into the wells to a final concentration of 10 ⁶ CFU/ml, Campylobacter was cultured at 37℃ under microaerobic conditions for 3 days, and other test bacteria were cultured at 37℃ under aerobic conditions for 18 hours. The MIC of tryptanthrin and indigo powder for each test bacteria was measured by visually checking the turbidity of the test solution after culture. The test bacteria used and the MIC of tryptanthrin for each are shown in Table 1. Genotyping of Campylobacter species was performed by Multi-Locus Sequence Typing (MLST) as previously reported (Dingle KE et al., J. Clin Microbiol. 39:14-23 (2001) for C. jejuni, Dingle KE et al., J. Clin Microbiol. 43:340-347 (2005) for C. coli, and van Bergen MA et al., J. Clin Microbiol, 43:5888-98 (2005) for C. fetus).

The MICs of tryptanthrin against C. jejuni and C. coli were 0.78-12.5 μM for all strains. On the other hand, the MICs of tryptanthrin against C. fetus were >100 μM except for one strain. In addition, the MICs of tryptanthrin against Escherichia coli, Enterobacter cloacae, Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabili, and Salmonella enterica, which are enterobacteria commonly found in chickens, were all >100 μM.

According to the international publication WO2008/062861, the MIC of tryptanthrin against C. rectus, a type of oral bacteria, was 25 μg/ml (approximately 100 μM). Therefore, it was confirmed that tryptanthrin exhibits selective and high bacteriostatic activity against Campylobacter and other enterobacteria, including C. jejuni and C. coli.

The MICs of indigo powder against C. jejuni and C. coli were 0.5 or 1%, and the MICs of indigo powder against C. fetus were 1 or 2%. On the other hand, the MICs of indigo powder against Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabili, Salmonella enterica, and Enterococcus faecalis were all >2%. This confirmed that tryptanthrin, even in the form of indigo powder, exhibits selective bacteriostatic activity against C. jejuni and C. coli.

Example 3
Twenty-four one-day-old chickens were divided into six groups, and four chickens were placed in each isolator. Tryptanthrin solutions were prepared to final concentrations of 100 μM, 10 μM, and 0 μM, and each concentration of tryptanthrin solution was administered to two groups in drinking water from two days of age through the test period. C. jejuni Cj13 bacterial solution was orally administered to four-day-old chickens using a stomach tube at 10 ⁶ or 10 ³ CFU/chick. Two weeks after administration, the chickens were euthanized with animal isoflurane and the number of bacteria in the cecal contents was measured.

The number of C. jejuni bacteria in 1g of cecal contents of three groups orally administered C. jejuni Cj13 at ¹⁰³ CFU/bird was compared. The number of bacteria in the 10μM and 100μM tryptanthrin groups (average 4.0× ¹⁰⁶ and 2.9× ¹⁰⁶ CFU/g) was significantly lower (p<0.05) than the number in the non-tryptanthrin group (average 2.6× ¹⁰⁸ CFU/g). In the three groups orally administered C. jejuni at ¹⁰⁶ CFU/bird, the number of C. jejuni bacteria in the 100μM tryptanthrin group (average 2.4× ¹⁰⁷ CFU/g) was significantly lower (p<0.05) than the number in the non-tryptanthrin group (average 3.7× ¹⁰⁸ CFU/g). This confirmed that tryptanthrin exhibits bacteriostatic activity against C. jejuni in vivo as well as in vitro.

Claims (9)

A bacteriostatic composition against Campylobacter jejuni or Campylobacter coli, containing tryptanthrin. The composition according to claim 1, which contains indigo grass or an extract of indigo grass. The composition according to claim 2, wherein the indigo plant is tallow tree. The composition according to any one of claims 1 to 3, in the form of a feed or drink for a non-human animal. The composition according to claim 4, wherein the non-human animal is a cow, a pig or a poultry. The composition according to claim 4 or 5, wherein the non-human animal is a chicken. A method for bacteriostatically inhibiting Campylobacter jejuni or Campylobacter coli in a non-human animal, comprising administering to the non-human animal a composition containing tryptanthrin. The method according to claim 7, wherein the non-human animal is a cow, a pig or a poultry. The method according to claim 7 or 8, wherein the non-human animal is a chicken.