JP7361439B2 - Pest control formulations and soil treatment methods - Google Patents

Description

The present invention relates to preparations for controlling Cryptosporidium containing an isothiocyanic acid compound or the like as an active ingredient, a soil treatment method for controlling Cryptosporidium using these preparations, a therapeutic agent for Cryptosporidiosis, and the like.

Cryptosporidium is a protozoan that belongs to the sporozoan class and is widely distributed around the world. For example, Cryptosporidium parvum (scientific name: Cryptosporidium parvum) has been shown to have a high infection rate as an intestinal parasitic protozoan in cattle and pigs, and has also been reported to be detected in dogs and cats.

Cryptosporidium is an obligate cell-parasitic protozoan that develops through the following life cycle. When Clostosporidium oocysts are excreted with feces and ingested into the host body, the oocysts excyst in the intestinal tract and the four sporozoites inside the oocysts are released. Sporozoites invade epithelial cells in the intestine, form insect cysts, and reproduce asexually within the cysts to proliferate. Proliferated sporozoites repeatedly release to the outside of the cell and invade into the cell, and further proliferate. After that, when it enters the sexual reproductive phase, it differentiates into either male gametocytes or female gametocytes, and these zygotes develop into oocysts, which are released and excreted outside the host body along with feces.

Cryptosporidium oocysts are known to be extremely resistant to chlorine. Therefore, if a water source is contaminated with oocysts released with livestock feces, etc., Cryptosporidium oocysts will be mixed into tap water, which can cause cryptosporidiosis in humans, which has diarrhea as the main symptom. may occur in groups. Cryptosporidiosis in humans usually resolves spontaneously within one to two weeks, but it can lead to death in patients with immunodeficiency. Severe cases of human cryptosporidiosis are treated with drugs such as paromomycin and nitazoxanide.

Additionally, Cryptosporidium is widely prevalent in livestock such as cows and pigs. In particular, calves under one month of age are susceptible to infection due to contamination with oocysts in the rearing environment, exhibit symptoms of watery diarrhea, and may even die due to secondary infection, resulting in large economic losses. On the other hand, few effective preventive or therapeutic measures against cryptosporidiosis in calves have been established.

As a countermeasure against Cryptosporidium, for example, Patent Document 1 describes a cryptosporidiosis control agent containing cashew nut shell oil, and Patent Document 2 describes a cryptosporidiosis prevention agent containing an iron compound as an active ingredient. - Each preventive agent is listed.

In addition, as a matter related to the present invention, Patent Document 3 discloses a method for preventing and treating hairy wart disease by administering allyl isothiocyanate to the limbs and hooves of ungulates.
Japanese Patent Application Publication No. 2015-30717 Japanese Patent Application Publication No. 2010-13408 Patent No. 5740062

As mentioned above, while Cryptosporidium is widespread, effective control measures are required from the viewpoints of public health and livestock economy. Therefore, an object of the present invention is to provide a highly effective means for controlling Cryptosporidium.

The present inventors have newly discovered that isothiocyanic acid compounds such as allyl isothiocyanate significantly suppress the infectivity of Cryptosporidium.

［式I中、Ｒ_１は炭素原子数2～8のアルケニル基、炭素原子数1～6の低級アルキル基、炭素原子数6～12のアリール基、炭素原子数7～20のアラルキル基、又は炭素原子数3～6のシクロアルキル基を表す。］ Therefore, the present invention provides a preparation for controlling Cryptosporidium containing a compound represented by the following general formula (I) as an active ingredient.

[In formula I, R ₁ is an alkenyl group having 2 to 8 carbon atoms, a lower alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 12 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, or Represents a cycloalkyl group having 3 to 6 carbon atoms. ]

This compound has the effect of inhibiting the ability of Cryptosporidium to invade host epithelial cells and suppressing its infection. Therefore, for example, by spraying Cryptosporidium control preparations into livestock farms, such as cattle and pig barns, and the soil in pastures, it is possible to prevent the proliferation and spread of Cryptosporidium protozoa and the occurrence of cryptosporidiosis. - Possibility of effectively controlling exacerbation, propagation, and spread. It may also be effective as a prophylactic or therapeutic agent for cryptosporidiosis.

According to the present invention, the occurrence and exacerbation of cryptosporidiosis and the propagation and spread of its pathogen can be effectively suppressed.

<About the Cryptosporidium control preparation according to the present invention>
The present invention includes all Cryptosporidium control preparations containing a compound represented by the following general formula (I) as an active ingredient. Here, "Cryptosporidium control" means the control of Cryptosporidium, that is, suppressing the infectivity and proliferation power of Cryptosporidium, and "Cryptosporidium control preparation" is used for the control of Cryptosporidium. In other words, it means a preparation for suppressing the infectivity, proliferation, etc. of Cryptosporidium (the same applies hereinafter).

In the above general formula I, R ₁ is an alkenyl group having 2 to 8 carbon atoms, a lower alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 12 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, Or represents a cycloalkyl group having 3 to 6 carbon atoms.

The alkenyl group having 2 to 8 carbon atoms in R ₁ may be a linear or branched unsaturated hydrocarbon group having 2 to 8 carbon atoms and having at least one double bond. Examples of such groups include vinyl, 1-propenyl, allyl (2-propenyl), butenyl, pentenyl, hexenyl, heptenyl, and octenyl groups.

The lower alkyl group having 1 to 6 carbon atoms in R ₁ may be a linear or branched lower alkyl group having 1 to 6 carbon atoms. Examples of such groups include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, n-hexyl group, etc. can be mentioned.

Examples of the aryl group having 6 to 12 carbon atoms in R ₁ include phenyl group, o-tolyl group, m-tolyl group, p-tolyl group, xylyl group (including all positional isomers thereof), and trimethylphenyl group. (including all positional isomers thereof), tetramethylphenyl group (including all positional isomers thereof), 1-naphthyl group, 2-naphthyl group, biphenyl group, etc.

The aralkyl group having 7 to 18 carbon atoms in R ₁ may be one in which one hydrogen atom of the above lower alkyl group is substituted with the above aryl group, that is, an aryl C1-C6 alkyl group. Such groups include, for example, benzyl group, phenethyl group, phenylpropyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, 1-naphthylethyl group, 2-naphthylethyl group, methylnaphthyl group (its positional isomers ), dimethylnaphthyl group (including all positional isomers thereof), biphenylmethyl group, biphenylethyl group, etc.

Examples of the cycloalkyl group having 3 to 6 carbon atoms in R ₁ include cyclopropyl group, cyclobutyl group, cyclopentyl group, and cyclohexyl group.

Among these, in the above formula I, it is preferable that R ₁ is an alkenyl group having 2 to 6 carbon atoms, and that R ₁ is an allyl group, that is, the compound is allyl isothiocyanate (allylisisocyanate). thiocyanate) is most preferred.

The preparation for controlling Cryptosporidium according to the present invention can adopt a wide variety of known dosage forms, such as solid forms (for example, powders, granules, tablets, etc.) and liquid forms (for example, liquid preparations, suspension agents, etc.). For example, a solid preparation may be dissolved in a solvent to prepare a pesticidal solution before use, or a liquid preparation may be prepared in advance and diluted appropriately at the time of use to prevent pests. May be used as a solution.

Any known solvent can be used as appropriate for dissolving the compound according to the present invention. For example, an appropriate one may be selected from water, vegetable oil, liquid animal oil, phosphate buffer, carbonate buffer, physiological saline, alcohol, propylene glycol, polyethylene glycol, methylcellulose, acetone, DMSO, and the like.

In addition, depending on the purpose, use, dosage form, etc., excipients, disintegrants, binders, lubricants, coating agents, stabilizers, solid carriers, solubilizing agents, buffers, isotonizing agents, surfactants, etc. Agents, preservatives, antibacterial agents, antioxidants, pH adjusters, dispersants, fragrances, colorants, thickeners, and the like may be added as appropriate.

Examples of excipients include sugar alcohols (e.g., erythritol, mannitol, xylitol, sorbitol, etc.), saccharides (e.g., white sugar, powdered sugar, lactose, glucose, etc.), cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, etc.). - cyclodextrin, γ-cyclodextrin, hydroxypropyl β-cyclodextrin and sulfobutyl ether β-cyclodextrin sodium), celluloses (such as crystalline cellulose, microcrystalline cellulose, powdered cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl) Cellulose, ethylcellulose, methylcellulose, etc.), starches (e.g. corn starch, potato starch, wheat starch, rice starch, partially pregelatinized starch, pregelatinized starch, etc.), dextrin, maltodextrin, sucrose ester, povidone, hypromellose Phthalate ester, hydroxypropyl methylcellulose acetate succinate, magnesium aluminate metasilicate, synthetic aluminum silicate, light anhydrous silicic acid, calcium silicate, carmellose sodium, hypromellose, gum arabic, sodium alginate, gelatin, pullulan, polyvinyl alcohol, Examples include carboxyvinyl polymer.

Examples of disintegrants include crospovidone, carmellose, carmellose calcium, croscarmellose sodium, low-substituted hydroxypropyl cellulose, sodium starch glycolate, corn starch, potato starch, wheat starch, rice starch, partially pregelatinized starch, α Examples include modified starch, sodium carboxymethyl starch, agar, and gelatin powder.

Examples of binders include hydroxypropylcellulose, carboxymethylcellulose, ethylcellulose, methylcellulose, carmellose sodium, polyvinylpyrrolidone, and the like.

Examples of lubricants include stearic acid, magnesium stearate, calcium stearate, talc, hydrated silicon dioxide, light anhydrous silicic acid, sucrose fatty acid ester, hydrogenated vegetable oil, macrogol, and the like.

Examples of coating agents include white sugar, hydroxypropylcellulose, hydroxypropylmethylcellulose, gelatin, glycerin, sorbitol, ethylcellulose, polyvinylpyrrolidone, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, methyltetaacrylate/methacrylic acid copolymer, aminoalkyl methacrylate. Copolymer E, aminoalkyl methacrylate copolymer RS, cellulose acetate phthalate, methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, and the like.

Examples of stabilizers include fatty acids and their salts, methylparaben, parahydroxybenzoic acid esters (such as propylparaben), alcohols such as chlorobutanol, pendyl alcohol, and phenylethyl alcohol, thimerosal, acetic anhydride, sorbic acid, Sodium bisulfite, L-ascorbic acid, sodium ascorbate, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, tocopherol acetate, gentisic acid, ascorbic acid, benzyl alcohol, tocopherol, gallic acid, gallic acid ester, α-thioglycerol , α-tocopherol, proteins and polysaccharides.

Examples of solid carriers include natural mineral powders (e.g. cationic clay, birophyllite clay, bentonite, montmorillonite, kaolin, clay, talc, chalk, quartz, attapulgite, diatomaceous earth, etc.), synthetic mineral powders (e.g. silicic acid, (alumina, silicate, etc.), and polymeric natural products (eg, crystalline cellulose, cornstarch, gelatin, alginic acid, etc.).

Examples of solubilizing agents that can be used include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate.

As examples of buffers, buffers such as phosphate, acetate, carbonate, citrate-tartrate, trishydroxymethylaminomethane, HEPES, etc. can be used.

Examples of tonicity agents that can be used include sodium chloride, glucose, D-sorbitol, glycerin, and D-mannitol.

Examples of surfactants include nonionic surfactants (e.g. fatty alcohol ethoxylates, polyoxyethylene alkylphenyl ethers, alkyl glycosides, fatty acid alkanolamides, polyoxyethylene castor oil, hydrogenated castor oil, octyl glucoside, peptyl thio Glucoside, decanoyl-N-methylglucamide, polyoxyethylene dodecyl ether, polyoxyethylene heptamethylhexyl ether, polyoxyethylene isooctyl phenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene fatty acid ester, sucrose fatty acid ester, polyoxyethylene sorbitol ester), anionic surfactants (e.g., sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzenesulfonate, sodium dodecyl-N-sarcosinate, sodium cholate, sodium deoxycholate, Tauro sodium deoxycholate, hydrogenated coconut fatty acid monoglyceride sodium monosulfate, sodium α-olefin sulfonate, sodium N-palmitoylglutamate, sodium N-methyl-N-acyltaurate), cationic surfactants (e.g. lauryltrimethylammonium chloride, stearyltrimethylammonium chloride, benzethonium chloride, cetylpyridinium chloride, benzalkonium chloride, stearyldimethylbenzylammonium chloride, cetyltrimethylammonium bromide, tetradecylammonium bromide, dodecylpyridinium chloride, etc.), amphoteric surfactants (coconut), Oil fatty acid amidopropyl betaine, lauryldimethylaminoacetic acid betaine, lauryldimethylamine oxide, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolium betaine, N-lauryldiaminoethylglycine, N-myristyldiaminoethylglycine, N- Alkyl-1-hydroxyethylimidazoline betaine sodium, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonic acid, palmitoyl lysolecithin, dodecyl-N-betaine, dodecyl-β-alanine, etc.).

Examples of preservatives include parabens (e.g., sodium benzoate, methylparaben, ethylparaben, propylparaben, butylparaben, etc.), cetylpyridinium chloride, alkyldiaminoethylglycine hydrochloride, potassium sorbate, thimerosal, paraoxybenzoic acid esters, Methyl paraoxybenzoate, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and the like can be used.

Examples of antibacterial agents include quaternary ammonium salt-based fungicides (e.g., benzalkonium chloride, benzethonium chloride, etc.), phenol-based fungicides (e.g., 3-methyl-4-isopropylphenol, thymol), and chlorhexidine. , triclosan, cetylpyridinium chloride, zinc gluconate, zinc citrate, plant extracts and essential oils with antibacterial properties (e.g. thyme, lemongrass, citrus, lemon, orange, anise, clove, aniseed, pine, cinnamon, geranium) , rose, mint, lavender, citronella, eucalyptus, peppermint, camphor, ajwain, sandalwood, rosemary, vervain, freegrass, lemongrass, latania, cedar, etc.).

As examples of antioxidants, sulfites, ascorbic acid, etc. can be used.

Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, citric acid, phosphoric acid, boric acid, and sulfuric acid; alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide; Alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, monoethanolamine, diisopropanolamine, etc. can be used. can.

Examples of dispersants that can be used include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polysorbate 80 (TWEEN80).

Examples of fragrances include fragrance ingredients (e.g. 1-carvone, cinnamic aldehyde, orange oil, methyl salicylate, eugenol, menthyl acetate, spilanthol, ethyl acetate, ethyl butyrate, isoamyl acetate, hexanal, hexenal, methyl anthranilate) , ethylmethylphenylglycidate, benzaldehyde, vanillin, ethylvanillin, furaneol, maltol, ethyl maltol, gamma/delta decalactone, gamma/deltaune decalactone, N-ethyl-p-menthane-3-carboxamide, menthyl lactate, ethylene Glycol-1-menthyl carbonate, etc.), natural essential oils (e.g. wintergreen oil, clove oil, thyme oil, sage oil, cardamom oil, rosemary oil, marjoram oil, nutmeg oil, lavender oil, paracress oil, etc.), fruit-based oils Flavoring agents (for example, lemon, orange, grapefruit, apple, banana, strawberry, blueberry, melon, peach, pineapple, grape, muscat, cherry, squash, etc.), peppermint, spearmint, menthol, yogurt, coffee, brandy, etc. can be used. can.

Examples of colorants include caramel pigment, gardenia pigment, anthocyanin pigment, annatto pigment, paprika pigment, safflower pigment, moss pigment, carotene pigment, carotenoid pigment, flavonoid pigment, cochineal pigment, amaranth (red No. 2), erythrosin (red Allura Red AC (Red No. 40), New Coccin (Red No. 102), Phloxin (Red No. 104), Rose Bengal (Red No. 105), Acid Red (Red No. 106), Tartrazine (Yellow No. 4) , Sunset Yellow FCF (Yellow No. 5), Fast Green FCF (Green No. 3), Brilliant Blue FCF (Blue No. 1), Indigo Carmine (Blue No. 2), Blue No. 201, Blue No. 204, Copper Chlorophyll, Copper Chlorophyllin Sodium, mica titanium, titanium oxide, etc. can be used.

Examples of thickeners include sodium alginate, propylene glycol alginate ester, locust bean gum, guar gum, sodium caseinate, egg albumin, gelatin agar, starch, modified starch, calcium carboxymethylcellulose, sodium carboxymethylcellulose, carboxyvinyl polymer, methylcellulose, ethylcellulose. , hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carmellose sodium, sodium polyacrylate, polyvinylpyrrolidone, hypromellose, sodium hyaluronate, sodium chondroitin sulfate, sugar alcohols (sorbitol, xylitol, maltitol, lactitol, etc.), polyhydric Alcohol (glycerin, propylene glycol, polyethylene glycol, polyvinyl alcohol, etc.), polyvinylpyrrolidone, sodium starch glycolate, sodium starch phosphate, xanthan gum, carrageenan gum, gum tragacanth, quince seed extract, sodium chondroitin sulfate, etc. can be used.

The above-mentioned compound according to the present invention has an infection-suppressing effect on, for example, Cryptosporidium in general, such as Cryptosporidium parvum.

By using the preparation for controlling Cryptosporidium according to the present invention in an object or area to be controlled, the infectivity of Cryptosporidium existing there can be suppressed and Cryptosporidium can be effectively controlled.

In principle, the control target can be applied to any object, place, or area that is or may be contaminated with Cryptosporidium, and is not particularly limited. For example, livestock breeding facilities (livestock facilities such as livestock barns, livestock insemination facilities, pasture fields, etc.), animal breeding facilities and areas including laboratory animals and breeding animals, water and sewage treatment facilities, reservoirs, medical and nursing care facilities and areas, For the prevention of Cryptosporidium on the floors, walls, entrances and exits of meat production/processing facilities, food production/processing facilities, etc., items installed/arranged within these facilities, and equipment/equipment used in operations performed within these facilities. The present invention can be applied. It is also effective in controlling Cryptosporidium contamination in liquids such as water and sewage, polluted water, and stored water.

For example, a liquid preparation according to the present invention may be prepared in advance or at the time of use, and the object to be controlled may be dipped in the solution, or the object or area to be controlled may be wiped with a cloth impregnated with the solution. Cryptosporidium can be controlled by spraying the control solution on the object or area to be controlled, or by spraying it in the form of a mist with a sprayer, or in the form of foam with a foaming nozzle. Cryptosporidium may be controlled by Further, when the target to be controlled is a liquid, Cryptosporidium may be controlled by, for example, adding the above-mentioned compound to the liquid and making the liquid contain the above-mentioned compound. In addition, for example, a powdered preparation may be sprayed or dispersed as it is on the object or area to be controlled.

For example, when controlling Cryptosporidium in a large area such as a livestock farm, the control solution or powder formulation according to the present invention may be sprayed on the area to be controlled. When controlling Cryptosporidium on the floors and walls of each facility or on objects installed or arranged within the facility, wipe the object or area to be controlled with a cloth impregnated with the control solution of the present invention. Alternatively, the pesticidal solution or powder preparation according to the present invention may be sprayed onto the object/area to be controlled substantially uniformly in the form of mist using a sprayer or the like, or may be sprayed in the form of foam using a foaming nozzle or the like. Alternatively, a step-in tank may be prepared at the entrance/exit of each facility, and the pest control solution according to the present invention may be placed therein to prevent Cryptosporidium from being present under the feet of people entering and exiting the facility. In addition, when performing Cryptosporidium control on instruments, equipment, etc. used in work performed within a facility, those instruments, equipment, etc. may be immersed in the control solution according to the present invention, or those instruments, equipment, etc. The pesticidal solution or powder preparation according to the present invention may be sprayed substantially uniformly in the form of mist, or may be sprayed in the form of foam using a foaming nozzle or the like.

When the Cryptosporidium control preparation according to the present invention is used as a liquid preparation or prepared in a liquid form, the concentration of the compound according to the present invention in the control solution is, for example, a final concentration (concentration at the time of use) of 1 ~50,000mg/L is preferred, 10~20,000mg/L is more preferred, 100~10,000mg/L is even more preferred, and 150~5,000mg/L is preferred. More preferably, it is between 200 and 1,000 mg/L, most preferably between 200 and 1,000 mg/L. When the preparation for controlling Cryptosporidium according to the present invention is used as a solid preparation, the compound according to the present invention preferably contains 1 to 100% by weight, and preferably 3 to 50% by weight. It is more preferable that the content is 5 to 30% by weight, and most preferable that the content is 5 to 30% by weight.

The preparation for controlling Cryptosporidium according to the present invention only needs to contain at least the compound according to the present invention in a predetermined concentration range, and contains components other than the compound effective for controlling Cryptosporidium and/or ingredients other than Cryptosporidium. It may further contain ingredients effective for controlling pathogens.

<About the soil treatment method according to the present invention>
The present invention includes all soil treatment methods for controlling Cryptosporidium using the above-mentioned control preparation.

For example, Cryptosporidium contamination in a livestock farm can be effectively suppressed by spraying a pesticidal preparation containing a compound represented by formula (I) over the entire soil of a livestock farm.

A wide variety of known means can be used for the dispersing means, and there is no particular limitation. For example, Cryptosporidium may be controlled by preparing a liquid preparation according to the present invention in advance or at the time of use, and spraying the control solution over the area to be controlled. Alternatively, for example, a powdered preparation may be prepared and sprayed directly onto the area to be controlled.

<About the prophylactic or therapeutic agent for cryptosporidiosis according to the present invention>
The present invention includes all agents for preventing or treating cryptosporidiosis that contain the above-mentioned compounds as active ingredients.

As mentioned above, this compound has the effect of inhibiting the ability of Cryptosporidium to invade host epithelial cells and suppressing its infection, and can be used as a prophylactic or therapeutic agent for cryptosporidiosis in humans, cattle, pigs, etc. may also be effective.

The prophylactic or therapeutic agent for cryptosporidiosis according to the present invention can be widely used in known dosage forms, such as solid forms (e.g., powders, granules, tablets, etc.) and liquid forms (e.g., solutions, suspensions, etc.). .

For example, when making solid preparations such as powders, granules, and tablets, depending on the purpose, use, dosage form, etc., this drug may contain excipients, disintegrants, binders, lubricants, coating agents, stabilizers, etc. Solid carriers, buffers, tonicity agents, surfactants, preservatives, antibacterial agents, antioxidants, pH adjusters, dispersants, fragrances, colorants, thickeners, soothing agents, etc. may be added as appropriate. You can leave it there. As an example of a soothing agent, for example, benzyl alcohol can be used. For each of the other components, for example, those mentioned above can be widely used.

For example, when making liquid preparations such as solutions and suspensions, depending on the purpose, use, dosage form, etc., in addition to solvents, solubilizing agents, buffering agents, tonicity agents, surfactants, and preservatives may be added. , an antibacterial agent, an antioxidant, a pH adjuster, a coloring agent, a thickener, a soothing agent, etc. may be added as appropriate. For each component, for example, those described above can be widely used.

The drug according to the present invention can be applied to mammals such as humans, livestock (eg, cows, pigs, etc.), dogs, cats, etc.

The administration route is not particularly limited as long as the drug reaches the intestines of the target (patient/affected animal). For example, solid or liquid preparations may be administered orally, or injections, suppositories, pellets, etc. may be administered parenterally. In addition, when applied to cattle, pigs, dogs, cats, etc., it may be administered by including it in feed or the like and ingesting it.

The dosage may be appropriately determined depending on the subject, symptoms, administration method, etc., and is not particularly limited. The dosage is, for example, preferably 0.01 to 200 mg/kg (body weight)/day, more preferably 0.05 to 100 mg/kg (body weight)/day, most preferably 0.1 to 30 mg/kg. (weight)/day.

In Example 1, it was verified whether the invasion of Cryptosporidium into host intestinal cells is suppressed by allyl isothiocyanate.

HCT-8 cells (human colon cancer-derived cells) were prepared as host cells. HCT-8 cells were seeded in a 96-well plate and cultured to a confluent state.

Cryptosporidium was prepared as follows. After accumulating Cryptosporidium oocysts by centrifugation, taurocholic acid and trypsin were added, and the mixture was allowed to stand at 37°C for 60 minutes to excyst. The resulting sporozoites were collected and suspended, and used as test materials.

As a test substance, Wasaolo (registered trademark, manufactured by Mitsubishi Chemical Foods Co., Ltd., powder formulation containing 10% by weight of allyl isothiocyanate, hereinafter the same) was dissolved in purified water and used.

Sporozoites (2×10 ⁴ cells/well) and 100 μL of a medium containing allyl isothiocyanate were added to each well in which HCT-8 cells were seeded, and the wells were allowed to stand for 48 hours. The medium is a normal medium containing RPMI-1640 supplemented with 10% FCS, etc., with final concentrations of allyl isothiocyanate of 0.001 μg/mL, 0.01 μg/mL, 0.1 μg/mL, and 1.0 μg/mL, respectively. Wasaolo solution was added and prepared. In addition, in the negative control, Wasaolo solution was not added to the medium, and in the positive control, paromomycin was added to the medium instead of Wasaolo solution at a final concentration of 1.0 μg/mL.

After standing for 48 hours in the presence of allyl isothiocyanate, the sporozoites remaining in the medium (sporozoites that did not invade HCT-8 cells) were removed together with the medium, washed with PBS, and the HCT-8 cells on the bottom were removed. Fixed with methanol. Then, immunostaining was performed using an anti-Cryptosporidium antibody as a primary antibody, and the number of Cryptosporidium that had invaded the HCT-8 cells was counted under a microscope.

The results are shown in Figure 1. FIG. 1 is a graph showing the invasion rate of Cryptosporidium into HCT-8 cells in the presence of allyl isothiocyanate. "Invasion rate into host cells" on the vertical axis in the figure is a bar graph of the invasion rate of Cryptosporidium when the number of invasions of Cryptosporidium into HCT-8 cells in the absence of allyl isothiocyanate is taken as 100%. express something. In the figure, the "N" bar graph represents the negative control (invasion rate of Cryptosporidium into HCT-8 cells in the absence of allyl isothiocyanate, i.e. 100%), and the "P" bar graph represents the positive control (paromomycin 1.0 μg/ (invasion rate of Cryptosporidium into HCT-8 cells in the presence of mL). The bar graphs labeled “1 to 4” in the figure indicate HCT-8 cells when allyl isothiocyanate was added at a final concentration of 0.001 μg/mL, 0.01 μg/mL, 0.1 μg/mL, or 1.0 μg/mL, respectively. represents the invasion rate of Cryptosporidium.

As shown in Figure 1, compared to the invasion rate of Cryptosporidium into host cells in the absence of allyl isothiocyanate (100%), in the presence of allyl isothiocyanate, the invasion rate of the protozoan into host cells was significantly higher and the concentration decreased dependently. Furthermore, in comparison with the positive control (in the presence of paromomycin), the invasion of protozoa into host cells was significantly suppressed in the presence of allyl isothiocyanate. These results showed that allyl isothiocyanate has the effect of inhibiting the ability of Cryptosporidium to invade host epithelial cells and suppressing its infection.

Graph showing the invasion rate of Cryptosporidium into HCT-8 cells in the presence of allyl isothiocyanate in Example 1.

Claims (2)

A preparation for controlling Cryptosporidium in livestock farms, containing allyl isothiocyanate as an active ingredient. A soil treatment method for controlling Cryptosporidium using the pesticidal preparation according to claim 1.

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