Coccidiostat composition containing violacein and its uses

Abstract

The present invention relates to: an anticoccidial composition comprising violacein, a violacein derivative and / or a salt thereof; and its uses. According to one embodiment, the composition comprising violacein has excellent effects of directly killing protozoa that can induce coccidiosis, inhibiting cell penetration of protozoa and / or inhibiting intracellular proliferation of protozoa, and preventing, alleviating and treating coccidiosis in vivo.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0125246, filed on September 25, 2020, and the entire contents disclosed in the corresponding Korean patent application document are incorporated as part of this specification.

[0003] This application relates to an anticoccidial composition containing violacein, violacein derivatives, and salts thereof, and uses thereof. Background Art

[0004] Coccidiosis is an intestinal-related disease caused by protozoan parasites belonging to the phylum Apicomplexa called Eimeria, and when infected with coccidiosis, symptoms such as digestive system disorders, diarrhea, and weight loss occur, and in addition, it can cause the death of livestock. Therefore, it has a significant economic impact on farms worldwide (Williams RB. A compartmentalised model for the estimation of the cost of coccidiosis to the world’s chicken production industry, Int J Parasitol. 1999; 29(8): 1209-1229).

[0005] In the past few years, many researchers have developed anticoccidials as therapeutic agents for treating coccidiosis, such as ionophores or chemically synthesized compounds, which can prevent the formation of the oocyst cell wall of protozoa or asexual and sexual reproduction. However, due to the long-term use of shuttle programs that alternately treat ionophores and chemically synthesized compounds, side effects such as the emergence of drug-resistant protozoa have occurred.

[0006] In particular, the accumulation of antibiotics in animals due to the misuse and abuse of antibiotics is a serious problem because humans consume antibiotics through meat. Therefore, due to the problem of antibiotic residues in livestock products, many countries in the world are banning the use of antibiotics. Therefore, there is an urgent need to develop and study alternatives to conventional anticoccidial drugs that exhibit side effects such as the emergence of drug-resistant strains and internal residues.

[0007] Accordingly, the inventors of the present application have completed the present invention by confirming that violacein, violacein derivatives, and salts thereof have excellent anticoccidial activity and antiprotozoal activity against protozoa exhibiting coccidiosis.

[0008] Prior art

[0009] (Patent Document 1) US Patent Publication No. 2008-0160000 Summary of the invention

[0010] Technical problem

[0011] One embodiment of the present application provides a composition for preventing or alleviating coccidiosis, comprising at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof as an active ingredient.

[0012] Another embodiment of the present application provides a pharmaceutical composition for preventing or treating coccidiosis, comprising at least one selected from the group consisting of violacein, violacein derivatives, and pharmaceutically acceptable salts thereof as an active ingredient.

[0013] Other embodiments of the present invention provide an antiprotozoal composition against protozoa of the genus Eimeria sp., comprising at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof as an active ingredient.

[0014] Other embodiments of the present application provide a method for preventing, alleviating, or treating coccidiosis, comprising the step of administering the composition to an animal (e.g., an animal other than a human).

[0015] Other embodiments of the present application provide the use of at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof in the preparation of a composition (pharmaceutical composition) for preventing, alleviating, and / or treating coccidiosis or in the preparation of an antiprotozoal composition; the use of at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof for preventing, alleviating, and / or treating coccidiosis; and / or the use of at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof for antiprotozoal (e.g., killing protozoa of the genus Eimeria; and / or inhibiting cell invasion and / or proliferation of protozoa of the genus Eimeria) against protozoa of the genus Eimeria sp.

[0016] Technical solution

[0017] In the present application, excellent anticoccidial efficacy (activity, effect) may refer to at least one (e.g., any one, two or more, three or more, or all) selected from the group consisting of the following (1) to (5):

[0018] (1) A higher anticoccidial index (ACI) compared to the control group;

[0019] (2) When administered to coccidiosis-induced animal subjects, a reduced mortality rate, a reduced lesion score (e.g., appendiceal lesion score), and / or a reduced fecal oocyst excretion compared to the control group;

[0020] (3) Inhibition of weight loss induced by coccidiosis;

[0021] (4) Higher insecticidal activity against the protozoa that induce coccidiosis compared to the control group; and

[0022] (5) Higher inhibition of cell invasion by the protozoa that induce coccidiosis and / or higher inhibition of the proliferation of the protozoa in cells compared to the control group.

[0023] In the present application, the control group may refer to a negative control group (a group without any treatment or a group treated with water and / or buffer) and / or a positive control group containing a conventionally known anticoccidial agent (e.g., diclazuril, salinomycin, and / or gallic acid).

[0024] The composition according to one embodiment may have at least one (e.g., one or more, two or more, three or more, four or more, five or more, or all six) of the characteristics selected from the group consisting of the following (1) to (6), and its characteristics may be more excellent than those of the control group:

[0025] (1) Excellent anticoccidial activity;

[0026] (2) Excellent antiprotozoal effect against the protozoa that induce coccidiosis;

[0027] (3) Excellent acid resistance;

[0028] (4) Excellent heat resistance;

[0029] (5) Excellent in vivo stability and / or safety; and

[0030] (6) Excellent effect of improving weight gain.

[0031] The composition according to one embodiment has excellent acid resistance and / or heat resistance, so that when administered to the body, it maintains excellent anticoccidial activity for a long time, and has in vivo stability, so that even in an environment with various temperatures and / or various pH ranges, it can maintain excellent anticoccidial activity, and can be applied to various products, and the storage stability can be excellent.

[0032] A composition according to one embodiment, when administered in vivo, is not absorbed by other tissues and organs (e.g., blood, liver, kidney, and / or spleen, etc.) except for the intestine, thus having low residues in the body, and therefore the composition can have excellent safety in vivo.

[0033] In one embodiment, the excellent weight gain improvement effect can refer to the excellent effect of increasing the weight of a subject when the composition is administered to the subject, and in one embodiment, the increase in weight can refer to the daily weight gain, and the subject can be a subject in which coccidiosis is induced in vivo.

[0034] The composition according to one embodiment exhibits coccidiostatic activity equivalent to or higher than that of conventionally known coccidiostats (e.g., sulfonamides such as sulfamethazine, sulfachloropyrazine, and sulfamerazine, polyether ionophore antibiotics such as salinomycin and monensin sodium, amprolium, diclazuril, gallic acid, and / or toltrazuril), and can be safely used for a long time because the composition is not absorbed in the body.

[0035] In the present application, "prevention" refers to all actions of suppressing or delaying the development of a disease by the administration of a composition according to one embodiment, "treatment" refers to all actions of improving or beneficially changing the symptoms of a subject suspected of having and suffering from the disease by the administration of a composition according to one embodiment, and "mitigation" refers to all actions of reducing a parameter related to the condition of treating a disease by the administration of a composition according to one embodiment, e.g., at least the degree of symptoms. The disease can refer to coccidiosis.

[0036] The composition according to one embodiment (e.g., a pharmaceutical composition and / or an antiprotozoal composition) may contain at least one (e.g., any one, two or more, three or more, four or more, or all) selected from the group consisting of violacein, violacein derivatives, and salts thereof (or pharmaceutically acceptable salts) as the active ingredient of the composition according to one embodiment.

[0037] On the one hand, a composition for preventing or mitigating coccidiosis can be provided, which contains at least one (e.g., one or more, two or more, three or more, four or more, or all) selected from the group consisting of violacein, violacein derivatives, and salts thereof (salts of violacein or salts of violacein derivatives).

[0038] Violacein is a purple pigment named "3-(2-(5-hydroxyindol-3-yl)-5-oxo-2-pyrrolin-4-ylidene)-2-indolinone" and can have the general formula of Formula 1 below and / or the molecular formula C 20 H 13 N3O3, and can be Cas No. 548-54-9, UNII-QJH0DSQ3SG and / or BRN 0049923. In one embodiment, violacein can be (3Z)-violacein.

[0039] Formula 1

[0040]

[0041] In one embodiment, derivatives of violacein can be at least one (e.g., one or more, two or more, three or more, or all) selected from the group consisting of deoxyviolacein, proviolacein, and oxyviolacein.

[0042] Deoxyviolacein is named "3-[(4E)-2-(1H-indol-3-yl)-5-oxo-2-pyrrolin-4-ylidene)indolin-2-one" and can have the general formula of Formula 2 below and / or the molecular formula C 20 H 13 N3O2, and can be SCHEMBL15948572, CHEBI:131915, Q27225296 and / or Cas No. 5839-61-2.

[0043] Formula 2

[0044]

[0045] Proviolacein is named "5-(5-hydroxy-1H-indol-3-yl)-3-(1H-indol-3-yl)-2H-pyrrol-2-one" and can have the general formula of Formula 3 below and / or the molecular formula C 20 H 13 N3O2, and can be SCHEMBL16430767, CHEBI:131916 and / or Q27225297.

[0046] Formula 3

[0047]

[0048] Violacein is named "5-hydroxy-3-[2-hydroxy-5-(5-hydroxy-1H-indol-3-yl)-1H-pyrrol-3-yl]indol-2-one", and may be the general formula of the following Chemical Formula 4 and / or C 20 H 13 N3O4.

[0049] Chemical Formula 4

[0050]

[0051] According to a specific embodiment of the present invention, violacein, a derivative of violacein, and / or a salt thereof may be obtained by extraction and separation from natural products and / or strains, or prepared by conventional organic synthesis methods, but not limited thereto. For example, it may be directly isolated from species belonging to the genus Chromobacterium, Collimonas, Duganella, Iodobacter, Janthinobacterium, Microbulbifer, Pseudoaltermonas, Escherichia, or Corynebacterium, or obtained from manufacturers in the art, but not limited thereto.

[0052] The "salt of violacein" or "salt of violacein derivative" in the present application may refer to a physiologically acceptable salt among salts of substances in which cations and anions are combined by electrostatic attraction. The "pharmaceutically acceptable salt" may refer to a salt in a pharmaceutically usable form. For example, the salt may be at least one selected from the group consisting of metal salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. In one embodiment, the metal salt may be at least one selected from the group consisting of alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, barium salt, etc.), aluminum salts, etc.; the salt with an organic base may be at least one selected from the group consisting of salts with triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.; the salt with an inorganic acid may be at least one selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.; the salt with an organic acid may be at least one selected from the group consisting of salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; the salt with a basic amino acid may be at least one selected from the group consisting of salts with arginine, lysine, ornithine, etc.; and the salt with an acidic amino acid may be at least one selected from the group consisting of salts with aspartic acid, glutamic acid, etc.

[0053] In the present application, "coccidiosis" is a disease caused by coccidian protozoa (protozoa that can induce coccidiosis, such as Eimeria sp. coccidian protozoa) parasitizing in the cytoplasm of the submucosa of the digestive tract epithelium and destroying the epithelium, resulting in enteritis, and is a protozoal disease that causes economic losses due to soft stools, diarrhea, and bloody stools, leading to weight gain degradation and an extended slaughter age in broiler farms. Coccidiosis can occur not only in broilers but also in birds and mammals. Specifically, coccidiosis can infect cattle, rabbits, goats, dogs, cats, mice and rats that are experimental animals, etc. In particular, it can cause fatal harm to poultry such as chickens. In one embodiment, coccidiosis may include acute coccidiosis, subacute coccidiosis, and chronic coccidiosis, etc. Acute coccidiosis can exhibit bloody stools, energy loss, and anemia within 48 hours after infection, and the infected subjects may die. Subacute coccidiosis can exhibit hemorrhagic diarrhea and / or anemia symptoms after infection. Chronic coccidiosis can exhibit symptoms of soft stools and / or weight loss after diarrhea within 1 to 2 days after infection.

[0054] When the oocysts (cysts, eggs) of coccidian protozoan species mature into sporulated oocysts at high humidity and high temperature, the oocysts are infectious, and when the oocysts are excreted with feces after a certain life cycle in the body of a subject, the oocysts are easily transmitted and the life cycle of the oocysts repeats continuously. It is known that the oocytes (cysts) of coccidian protozoan species have high resistance to the external environment, and the cyst wall consists of two layers, an inner layer and an outer layer. The outer layer of the cyst wall is a gelatinous substance that strongly resists external physical pressure, and the inner layer is rich in nucleoproteins, so it can strongly resist chemical stimuli such as disinfectants. The oocysts of coccidian protozoan species can contain 4 sporocysts, and each sporocyst can contain 2 sporozoites. After they infect animals in the form of oocysts, they are released in the form of sporocysts and sporozoites, proliferate intracellularly, and the sporozoites that have undergone sexual reproduction and / or asexual reproduction can form oocysts and are excreted in feces. In one embodiment, the sporozoites can be used in the same meaning as the protozoa, and the sporozoites (protozoa) can cause lesions.

[0055] According to one embodiment, coccidiosis can be caused by protozoa of the genus Eimeria. In one embodiment, the protozoa of the genus Eimeria can be at least one selected from the group consisting of: Eimeria acervulina, Eimeria tenella, Eimeria maxima, Eimeria necatrix, Eimeria brunetti, Eimeria hagani, Eimeria mitis, Eimeria praecox, Eimeria mivati, Eimeria aurati, Eimeria baueri, Eimeria lepidosirenis, Eimeria leucisci, Eimeria rutile, Eimeria vanasi, Eimeria amphisbaeniarum, Eimeria witchery, Eimeria yemenensae, Eimeria adenoeides, Eimeria colchici, Eimeria curvata, Eimeria dispersa, Eimeria duodenalis, Eimeria fraterculae, Eimeria gallopavonis, Eimeria innocua, Eimeria meleagridis, Eimeria meleagrimitis, Eimeria phasiani, Eimeria procera, Eimeria purpureicephali, Eimeria ahsata, Eimeria alabamensis, Eimeria arieliEimeria alijevi, Eimeria asperonica, Eimeria arloingi, Eimeria arundeli, Eimeria bakuensis, Eimeria bovis, Eimeria cameli, Eimeria caprina, Eimeria caprovina, Eimeria christenseni, Eimeria clethrionomyis, Eimeria coecicola, Eimeria contorta, Eimeria couesii, Eimeria crandallis, Eimeria dammahensis, Eimeria dowleri, Eimeria exigua, Eimeria falciformis, Eimeria farasanii, Eimeria ferrisi, Eimeria flavescens, Eimeria gallatii, Eimeria granulosa, Eimeria hirci, Eimeria intestinalis, Eimeria irresidua, Eimeria intricata, Eimeria jolchijevi, Eimeria krijgsmanni, Eimeria larimerensis, Eimeria macusaniensis, Eimeria magna, Eimeria marconii, Eimeria media, Eimeria melanuri, Eimeria mississippiensisEimeria acervulina, Eimeria adenoeides, Eimeria ahsata, Eimeria alabamensis, Eimeria alijevi, Eimeria americana, Eimeria anatis, Eimeria anseris, Eimeria arloingi, Eimeria asymmetrica, Eimeria auburnensis, Eimeria bovis, Eimeria brunetti, Eimeria canadiensis, Eimeria caprina, Eimeria cerdonis, Eimeria christenseni, Eimeria columbarum, Eimeria coecicola, Eimeria conjunctiva, Eimeria crandallis, Eimeria cylindrica, Eimeria debliecki, Eimeria dispersa, Eimeria ellipsoidalis, Eimeria falciformis, Eimeria fionnuisce, Eimeria gallopavonis, Eimeria gilruthi, Eimeria granifera, Eimeria hagani, Eimeria houghtonensis, Eimeria intricata, Eimeria irresidua, Eimeria labbeana, Eimeria leuckarti, Eimeria maxima, Eimeria meleagrimitis, Eimeria mivati, Eimeria necatrix, Eimeria nieschulzi, Eimeria ninakohlyakimovae, Eimeria ovinoidalis, Eimeria pallida, Eimeria palustris, Eimeria papillata, Eimeria perforans, Eimeria phocae, Eimeria pileata, Eimeria pipistrellus, Eimeria piriformis, Eimeria prionotemni, Eimeria procyonis, Eimeria punctata, Eimeria roobroucki, Eimeria saudensis, Eimeria sealanderi, Eimeria separata, Eimeria stiedae, Eimeria ursini, Eimeria vermiformis, Eimeria weybridgensis, Eimeria wobati, and Eimeria zuernii.

[0056] A composition according to one embodiment can have excellent effects in preventing, alleviating, and / or treating coccidiosis caused by at least one protozoan selected from the group consisting of protozoa described in Table 1 below, and the protozoa described in Table 1 below can cause coccidiosis in the animals described in Table 1, respectively.

[0057] Table 1

[0058]

[0059]

[0060]

[0061]

[0062] The composition according to one embodiment may have excellent effects on preventing, alleviating, and / or treating coccidiosis caused by Eimeria tenella, Eimeria acervulina, and / or Eimeria maxima.

[0063] In one embodiment, the prevention or alleviation of coccidiosis may refer to at least one (e.g., any one, two or more, three or more, or all) selected from the group consisting of the following (1) to (4), and for example, compared with the control group (negative control group and / or positive control group), at least one selected from the group consisting of the following (1) to (4) may be reduced, inhibited, and / or increased:

[0064] (1) Reducing at least one selected from the group consisting of lesion scores (e.g., appendix lesion scores), fecal oocyst excretion, and mortality;

[0065] (2) Inhibiting weight loss caused by coccidiosis;

[0066] (3) Increasing the anticoccidial index (ACI); and

[0067] (4) Reducing the cell invasion of Eimeria protozoa, the proliferation of protozoa in cells, or both.

[0068] In one embodiment, the lesion scoring method for determining the lesion score may refer to the literature of Johnson JK and Reid WM (1970) (Joyce Johnson and W. Malcolm Reid, Anticoccidial drugs: Lesion scoring techniques in battery and floor - pen experiments with chickens, Experimental parasitology, 1970), and the lesion score may be 0 to 4 degrees. In one embodiment, the lesion score may refer to the lesion score measured in the appendix, duodenum, and / or jejunum, and it may be calculated by the sum of each lesion score measured in each organ (appendix, duodenum, and / or jejunum).

[0069] In one embodiment, the fecal oocyst extraction amount may be measured by collecting the feces excreted by the subject and using a microscope or a counting chamber (e.g., McMaster chamber), etc.

[0070] In one embodiment, the mortality rate may refer to the mortality rate of animal subjects induced with coccidiosis. By performing an autopsy, the number of subjects that died from causes other than coccidiosis can be excluded.

[0071] In one embodiment, the body weight of subjects induced with coccidiosis may be reduced compared to that of subjects not induced with coccidiosis. The composition according to one embodiment may inhibit weight loss by inducing coccidiosis.

[0072] In one embodiment, the anti-coccidiosis index can be calculated according to the following Equation 1, and in Equation 1, the lesion score can be calculated by the aforementioned method.

[0073] (Equation 1)

[0074] Anti-Coccidiosis Index (ACI) = (Survival rate (%) after challenge inoculation) + (Daily weight gain (%) compared to the negative control group) - (Lesion score × 10) - (Fecal oocyst excretion index)

[0075] The challenge inoculation may refer to the administration (e.g., oral inoculation, etc.) of protozoa capable of inducing coccidiosis. In one embodiment, the survival rate can be the survival rate measured on the 5th to 10th day, 7th to 10th day, 8th to 10th day, 7th to 9th day, 7th to 8th day, or 7th day after challenge inoculation, and by performing an autopsy, the number of subjects that died from causes other than coccidiosis can be excluded to measure the survival rate.

[0076] The weight gain compared to the negative control group in Equation 1 above can be a value calculated as a percentage based on the value of the negative control group (e.g., the negative control group not infected with protozoa).

[0077] The lesion score in Equation 1 above is as described above.

[0078] In Equation 1 above, by calculating a percentage based on the value of the negative control group (e.g., the negative control group infected with protozoa), when the calculated result value is at a level of 0% or more and less than 1%, the fecal oocyst excretion index can be the numerical value 0; when the calculated result value is at a level of 1% or more and less than 26%, the fecal oocyst excretion index can be the numerical value 5; when the calculated result value is at a level of 26% or more and less than 51%, the fecal oocyst excretion index can be the numerical value 10; when the calculated result value is at a level of 51% or more and less than 76%, the fecal oocyst excretion index can be the numerical value 20; when the calculated result value is at a level of 76% or more and less than 100%, the fecal oocyst excretion index can be the numerical value 40.

[0079] In one embodiment, when the violacein, violacein derivative, and / or its salt are within the above ranges, their anticoccidial activity can be excellent compared to the case where the violacein, violacein derivative, and / or its salt are outside the above ranges.

[0080] In the present application, "feed" may refer to any natural or artificial diet, meal, etc. for animals to eat, ingest, and digest or suitable therefor, or a component of a meal. In a feed composition according to one embodiment, a concentrated feed and / or a special feed may be further included. The concentrated feed is a by-product obtained by purifying seed fruits and grains, the grains including grains such as wheat, oats, corn, etc., and may be bran, the bran including rice bran, wheat bran, barley bran, etc.; sesame cake, which is a by-product obtained by extracting oil from soybeans, fluid, sesame seeds, flaxseeds, coconut trees, etc.; and residues such as residual starch, which is the main component of starch residues and is the remainder after removing starch from sweet potatoes and potatoes; fish solubles, which are obtained by concentrating fresh liquid substances obtained from fish meal, fish waste, and fish; animal feeds such as dried whey, where whey is the remainder after producing casein from meat meal, blood meal, feather meal, skim milk powder, milk for cheese, skim milk, etc.; yeast, chlorella, and / or seaweed, etc.

[0081] A feed composition according to one embodiment may refer to a feed in a form finally ingested by an animal, a dietary supplement and / or a feed additive capable of being mixed with the feed. The dietary supplement is, for example, a composition containing a preparation that provides a therapeutic agent or a digestive agent to an animal, and may refer to a composition that is not a common source of calories ingested by an organism, i.e., an energy source, but is ingested outside of normal animal feed. The feed additive refers to a substance added to the feed for various effects such as supplementing nutrition and preventing weight loss, increasing the digestibility of fiber in the feed, improving the quality of oil, preventing proliferation disorders and enhancing fertility, preventing summer heat stress, etc. In one embodiment, the feed additive may refer to a substance added for the purpose of preventing, alleviating, or treating coccidiosis.

[0082] On the one hand, a pharmaceutical composition for preventing or treating coccidiosis can be provided, comprising at least one (e.g., any one, two or more, three or more, or four or more) selected from the group consisting of violacein, violacein derivatives, and pharmaceutically acceptable salts thereof. The violacein, violacein derivative, and / or its salt (the salt of violacein or the salt of violacein derivative) are as described above.

[0083] The pharmaceutical composition according to one embodiment can be used as a single preparation, and a mixed preparation can be prepared by further including an approved pharmaceutical composition known to have a prophylactic or therapeutic effect against coccidiosis. The unit dosage form of the drug can be made by adding a pharmaceutically acceptable carrier, excipient or diluent.

[0084] In the present application, "pharmaceutically acceptable" means that it has no significant irritating effect on organisms and does not inhibit the biological activity and properties of the administered active substance. The pharmaceutical composition containing a pharmaceutically acceptable carrier according to one embodiment can have any one of the preparations selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories.

[0085] The pharmaceutical composition can be various oral or parenteral preparations. In the case of preparations, commonly used fillers, extenders, binders, wetting agents, disintegrants, diluents such as surfactants or excipients can be used for preparation.

[0086] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. into one or more compounds. In addition, lubricants such as magnesium stearate, talc, etc. can be used in addition to simple excipients. Liquid preparations for oral administration can include suspensions, internal solutions, emulsions, syrups, etc., as well as various excipients such as wetting agents, sweeteners, flavors, preservatives, etc., in addition to the commonly used simple diluents water and liquid paraffin.

[0087] Preparations for parenteral administration can include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. As non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, etc. and injection esters such as ethyl oleate, etc. can be used. As the base of suppositories, polyethylene glycol fatty acid esters, Tween 61, cocoa butter, laurel fat, glycerogelatin, etc. can be used.

[0088] In one embodiment, the pharmaceutical composition can be formulated into various forms for use according to common methods for various purposes, such as oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., injectable preparations such as sterilized injection solutions, etc., and can be administered orally, or administered through various routes including intravenous administration, intraperitoneal administration, subcutaneous administration, rectal administration, topical administration, etc.

[0089] In one embodiment, the pharmaceutical composition may further additionally contain a carrier, excipient or diluent, etc., and examples of suitable carriers, excipients or diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate and mineral oil, etc. In addition, the pharmaceutical composition may further additionally contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, etc.

[0090] In one embodiment, the effective amount of the active ingredient in the pharmaceutical composition (e.g., violacein, violacein derivatives, and their salts) may vary depending on the age, sex, and weight of the patient (subject). Generally speaking, it can be 0.0001 mg / kg to 0.001 mg / kg, 0.0001 mg / kg to 0.003 mg / kg, 0.0001 mg / kg to 0.01 mg / kg, 0.0001 mg / kg to 0.1 mg / kg, 0.0001 mg / kg to 0.3 mg / kg, 0.0001 mg / kg to 1 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 100 mg / kg, 0.0001 mg / kg to 250 mg / kg, 0.0001 mg / kg to 300 mg / kg, 0.0001 mg / kg to 500 mg / kg, 0.0001 mg / kg to 1000 mg / kg, 0.001 mg / kg to 0.01 mg / kg, 0.001 mg / kg to 0.1 mg / kg, 0.001 mg / kg to 0.3 mg / kg, 0.001 mg / kg to 1 mg / kg, 0.001 mg / kg to 10 mg / kg, 0.001 mg / kg to 100 mg / kg, 0.001 mg / kg to 250 mg / kg, 0.001 mg / kg to 300 mg / kg, 0.001 mg / kg to 500 mg / kg, 0.001 mg / kg to 1000 mg / kg, 0.003 mg / kg to 0.01 mg / kg, 0.003 mg / kg to 0.1 mg / kg, 0.003 mg / kg to 0.3 mg / kg, 0.003 mg / kg to 1 mg / kg, 0.003 mg / kg to 10 mg / kg, 0.003 mg / kg to 100 mg / kg, 0.003 mg / kg to 250 mg / kg, 0.003 mg / kg to 300 mg / kg, 0.003 mg / kg to 500 mg / kg, 0.003 mg / kg to 1000 mg / kg, 0.01 mg / kg to 0.1 mg / kg, 0.01 mg / kg to 0.3 mg / kg, 0.01 mg / kg to 1 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 100 mg / kg, 0.01 mg / kg to 250 mg / kg, 0.01 mg / kg to 300 mg / kg, 0.01 mg / kg to 500 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 0.3 mg / kg, 0.1 mg / kg to 1 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 250 mg / kg, 0.1 mg / kg to 300 mg / kg, 0.1 mg / kg to 500 mg / kg, 0.1 mg / kg to 1000 mg / kg, 0.3 mg / kg to 1 mg / kg, 0.3 mg / kg to 10 mg / kg, 0.3 mg / kg to 100 mg / kg, 0.3 mg / kg to 250 mg / kg, 0.3 mg / kg to 300 mg / kg, 0.3 mg / kg to 500 mg / kg, 0.3 mg / kg to 1000 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 250 mg / kg, 1 mg / kg to 300 mg / kg, 1 mg / kg to 500 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 250 mg / kg, 10 mg / kg to 300 mg / kg, 10 mg / kg to 500 mg / kg, 10 mg / kg to 1000 mg / kg, 100 mg / kg to 250 mg / kg, 100 mg / kg to 300 mg / kg, 100 mg / kg to 500 mg / kg, 100 mg / kg to 1000 mg / kg, 250 mg / kg to 500 mg / kg, 300 mg / kg to 500 mg / kg, 250 mg / kg to 1000 mg / kg, 300 mg / kg to 1000 mg / kg, or 500 mg / kg to 1000 mg / kg per kg body weight can be administered daily or every other day, or administered 1 to 3 times a day. However, since it can be increased or decreased according to the administration route, severity of the disease, gender, body weight, age, etc., this dose does not limit the scope of the present application in any way. In one embodiment, when the composition is administered intraperitoneally, the composition can be administered at a concentration of 0.001 mg / kg to 0.3 mg / kg.

[0091] In one embodiment, the dose of the pharmaceutical composition can be within various ranges according to the patient's body weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease, etc.

[0092] In one embodiment, violacein, violacein derivatives, and / or pharmaceutically acceptable salts thereof can be included in the pharmaceutical composition in the following amounts: 1 w / w% or less, less than 1 w / w%, 10 -1 w / w% or less, 5×10 -2 w / w% or less, 2.5×10 -2 w / w% or less, 2×10 -2 w / w% or less, 1.25×10 -2 w / w% or less, 10 -2less than or equal to w / w%, 9×10 -3 less than or equal to w / w%, 8×10 -3 less than or equal to w / w%, 7×10 -3 less than or equal to w / w%, 6×10 -3 less than or equal to w / w%, 5×10 -3 less than or equal to w / w%, 4×10 -3 less than or equal to w / w%, 10 -7 greater than or equal to w / w%, 10 -6 greater than or equal to w / w%, 10 -5 greater than or equal to w / w%, 10 -4 greater than or equal to w / w%, 5×10 -4 greater than or equal to w / w%, 10 -3 greater than or equal to w / w%, 1.5×10 -3 greater than or equal to w / w%, 2×10 -3 greater than or equal to w / w%, 3×10 -3 greater than or equal to w / w%, 4×10 -3 greater than or equal to w / w%, 5×10 -3 greater than or equal to w / w%, 10 -7 w / w% to 1 w / w%, 10 -7 w / w% to 10 -1 w / w%, 10 - 7 w / w% to 5×10 -2 w / w%, 10 -7 w / w% to 10 -2 w / w%, 10 -7 w / w% to 5×10 -3 w / w%, 10 -7 w / w% to 4×10 -3 w / w%, 10 -7 w / w% to 10 -3 w / w%, 10 -7 w / w% to 5×10 -4 w / w%, 10 -7 w / w% to 10 -4 w / w%, 10 -7 w / w% to 10 -5 w / w%, 10 -6 w / w% to 1 w / w%, 10 -6 w / w% to 10 -1 w / w%, 10 -6 w / w% to 5×10 -2 w / w%, 10 - 6 w / w% to 10 -2 w / w%, 10 -6 w / w% to 5×10-3 w / w%, 10 -6 w / w% to 4×10 -3 w / w%, 10 -6 w / w% to 10 - 3 w / w%, 10 -6 w / w% to 5×10 -4 w / w%, 10 -6 w / w% to 10 -4 w / w%, 10 -6 w / w% to 10 -5 w / w%, 10 -5 w / w% to 1 w / w%, 10 -5 w / w% to 10 -1 w / w%, 10 -5 w / w% to 5×10 -2 w / w%, 10 -5 w / w% to 10 -2 w / w%, 10 -5 w / w% to 5×10 -3 w / w%, 10 -5 w / w% to 4×10 -3 w / w%, 10 -5 w / w% to 10 -3 w / w%, 10 -5 w / w% to 5×10 - 4 w / w%, 10 -5 w / w% to 10 -4 w / w%, 10 -4 w / w% to 1 w / w%, 10 -4 w / w% to 10 -1 w / w%, 10 -4 w / w% to 5×10 -2 w / w%, 10 -4 w / w% to 10 -2 w / w%, 10 -4 w / w% to 5×10 -3 w / w%, 10 -4 w / w% to 4×10 -3 w / w%, 10 -4 w / w% to 10 -3 w / w%, 10 -4 w / w% to 5×10 -4 w / w%, 10 -3 w / w% to 1 w / w%, 10 -3 w / w% to 10 -1 w / w%, 10 -3w / w% to 5×10 -2 w / w%, 10 -3 w / w% to 10 -2 w / w%, 10 -3 w / w% to 5×10 -3 w / w%, 10 -3 w / w% to 4×10 -3 w / w%, 10 -3 w / w% to 2×10 -3 w / w% or 10 -3 w / w% to 1.5×10 -3 w / w.

[0093] In one embodiment, violacein, violacein derivatives, and / or pharmaceutically acceptable salts thereof may be included in the pharmaceutical composition at the following concentrations: below 1000 ppm, below 500 ppm, below 400 ppm, below 300 ppm, below 250 ppm, below 200 ppm, below 125 ppm, less than 125 ppm, below 100 ppm, below 90 ppm, below 80 ppm, below 70 ppm, below 65 ppm, below 60 ppm, below 50 ppm, above 0.001 ppm, above 0.01 ppm, above 0.1 ppm, above 1 ppm, above 5 ppm, above 10 ppm, above 15 ppm, above 20 ppm, above 30 ppm, above 40 ppm, above 50 ppm, from 0.001 ppm to 1000 ppm, from 0.001 ppm to 500 ppm, from 0.001 ppm to 300 ppm, from 0.001 ppm to 200 ppm, from 0.001 ppm to 100 ppm, from 0.001 ppm to 90 ppm, from 0.001 ppm to 80 ppm, from 0.001 ppm to 70 ppm, from 0.001 ppm to 60 ppm, from 0.001 ppm to 50 ppm, from 0.001 ppm to 30 ppm, from 0.003 ppm to 1000 ppm, from 0.003 ppm to 500 ppm, from 0.003 ppm to 300 ppm, from 0.003 ppm to 200 ppm, from 0.003 ppm to 100 ppm, from 0.003 ppm to 90 ppm, from 0.003 ppm to 80 ppm, from 0.003 ppm to 70 ppm, from 0.003 ppm to 60 ppm, from 0.003 ppm to 50 ppm, from 0.003 ppm to 30 ppm, from 0.01 ppm to 1000 ppm, from 0.01 ppm to 500 ppm, from 0.01 ppm to 300 ppm, from 0.01 ppm to 200 ppm, from 0.01 ppm to 100 ppm, from 0.01 ppm to 90 ppm, from 0.01 ppm to 80 ppm, from 0.01 ppm to 70 ppm, from 0.01 ppm to 60 ppm, from 0.01 ppm to 50 ppm, from 0.01 ppm to 30 ppm, from 0.1 ppm to 1000 ppm, from 0.1 ppm to 500 ppm, from 0.1 ppm to 300 ppm, from 0.1 ppm to 200 ppm, from 0.1 ppm to 100 ppm, from 0.1 ppm to 90 ppm, from 0.1 ppm to 80 ppm, from 0.1 ppm to 70 ppm, from 0.1 ppm to 60 ppm, from 0.1 ppm to 50 ppm, 0.1 ppm to 30 ppm, 1 ppm to 1000 ppm, 1 ppm to 500 ppm, 1 ppm to 300 ppm, 1 ppm to 200 ppm, 1 ppm to 100 ppm, 1 ppm to 90 ppm, 1 ppm to 80 ppm, 1 ppm to 70 ppm, 1 ppm to 60 ppm, 1 ppm to 50 ppm, 1 ppm to 30 ppm, 3 ppm to 1000 ppm, 3 ppm to 500 ppm, 3 ppm to 300 ppm, 3 ppm to 200 ppm, 3 ppm to 100 ppm, 3 ppm to 90 ppm, 3 ppm to 80 ppm, 3 ppm to 70 ppm, 3 ppm to 60 ppm, 3 ppm to 50 ppm, 3 ppm to 30 ppm, 5 ppm to 1000 ppm, 5 ppm to 500 ppm, 5 ppm to 300 ppm, 5 ppm to 200 ppm, 5 ppm to 100 ppm, 5 ppm to 90 ppm, 5 ppm to 80 ppm, 5 ppm to 70 ppm, 5 ppm to 60 ppm, 5 ppm to 50 ppm, 5 ppm to 30 ppm, 10 ppm to 1000 ppm, 10 ppm to 500 ppm, 10 ppm to 300 ppm, 10 ppm to 200 ppm, 10 ppm to 100 ppm, 10 ppm to 90 ppm, 10 ppm to 80 ppm, 10 ppm to 70 ppm, 10 ppm to 60 ppm, 10 ppm to 50 ppm, 10 ppm to 40 ppm or 10 ppm to 20 ppm.

[0094] In one embodiment, the pharmaceutical composition can be administered to a subject by a variety of routes. Administration can refer to providing a substance to a subject (patient) by any suitable method, and the administration route of the pharmaceutical composition can be oral administration and / or parenteral administration, through all common routes as long as the target tissue is reached. In the case of parenteral administration, topical application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and / or intrathoracic injection can be selected. In addition, the composition according to one embodiment can be administered using any device capable of delivering the active ingredient to the target cells.

[0095] One aspect can provide an antiprotozoal composition against protozoa of the genus Eimeria, comprising at least one (e.g., any one, two or more, three or more, or all) selected from the group consisting of violacein, violacein derivatives, and their salts (salts of violacein, salts of violacein derivatives). Violacein, violacein derivatives, and / or their salts (salts of violacein or salts of violacein derivatives) are as described above.

[0096] In one embodiment, the antiprotozoal activity (action, efficacy) against protozoa of the Eimeria genus is excellent, and it can refer to the following characteristics of (1) and / or (2). For example, compared with the control group (negative control group and / or positive control group), it may exhibit the following characteristics of (1) and / or (2):

[0097] (1) The effect of killing protozoa of the Eimeria genus is excellent; and / or

[0098] (2) Inhibiting the cell invasion of protozoa of the Eimeria genus and / or inhibiting the proliferation of protozoa in cells.

[0099] In one embodiment, violacein, violacein derivatives and / or their salts can be included in the antiprotozoal composition within the above concentration range in the composition and / or pharmaceutical composition. In one embodiment, compared with the case of containing active ingredients outside the concentration range, the composition containing active ingredients within the above concentration range can have excellent antiprotozoal activity.

[0100] On the other hand, a method for preventing, alleviating or treating coccidiosis can be provided, including the step of administering the composition (such as a pharmaceutical composition and / or an antiprotozoal composition) to an animal. In one embodiment, before administering the composition, it may further include confirming (selecting) a subject (patient) in need of preventing, alleviating or treating coccidiosis. The composition and coccidiosis are as described above. According to one embodiment, confirming the subject may include detecting oocysts of protozoa capable of inducing coccidiosis in feces isolated from the subject. In the method for preventing, alleviating or treating coccidiosis according to one embodiment, the administration method, administration route and / or administration dose of the composition are as described above.

[0101] According to one embodiment, the composition can be administered at a pharmaceutically effective dose. In the present application, "pharmaceutically effective dose" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined according to the type of disease, severity, drug activity, sensitivity to the drug, administration time, administration route and excretion ratio, treatment cycle, elements containing drugs used simultaneously, and other elements well-known in the medical field. According to one embodiment, the composition can be administered as a single therapeutic agent or in combination with other anticoccidials, and can be administered simultaneously with, separately from, or sequentially with conventional therapeutic agents, and can be administered once or multiple times. Considering all factors, it is important to administer the minimum amount that can achieve the maximum effect without side effects, and this can be simply determined by those skilled in the art.

[0102] In one embodiment, the subject to which the method for preventing, alleviating or treating coccidiosis is applied refers to an animal that has or may develop coccidiosis, which may be a mammal, including humans, horses, cows, mice, rats, dogs, cats, etc., including birds such as poultry (e.g., breeding chickens, broiler chickens and / or laying hens, etc.), fish, amphibians and / or reptiles, etc.

[0103] In one embodiment, the animal to which the method for preventing, alleviating or treating coccidiosis is applied may be at least one selected from the group consisting of the animals described in Table 1 above (e.g., more than one, more than two, more than three or more than four or all), for example, the animal may be at least one selected from the group consisting of humans, chickens, ducks, geese, turkeys, quails, pheasants, pigeons, parrots, cows, pigs, goats, sheep, horses, antelopes, elands, monkeys, cats, dogs, mice, rats, rabbits, raccoons, squirrels, bats, guinea pigs, camels, llamas, alpacas, wombats, lizards, goldfish, crucian carp, tilapia, barbell fish, lungfish and European whitefish (e.g., more than one, more than two, more than three or more than four or all). In one embodiment, the animal may be an animal other than humans.

[0104] On the other hand, there is provided the use of at least one selected from the group consisting of violacein, violacein derivatives and their salts in the preparation of a composition (e.g., a pharmaceutical composition) or an antiprotozoal composition for preventing, alleviating and / or treating coccidiosis; the use of at least one selected from the group consisting of violacein, violacein derivatives and their salts for preventing, alleviating and / or treating coccidiosis; and / or the use of at least one selected from the group consisting of violacein, violacein derivatives and their salts as an antiprotozoal against protozoa of the genus Eimeria (e.g., killing protozoa of the genus Eimeria; and / or inhibiting the cell invasion and / or proliferation of protozoa of the genus Eimeria). In the above uses, the at least one selected from the group consisting of violacein, violacein derivatives and their salts, coccidiosis, antiprotozoal and protozoa of the genus Eimeria are as described above.

[0105] Beneficial effects

[0106] The composition containing violacein, violacein derivatives and their salts according to one embodiment has excellent direct killing effects on protozoa capable of inducing coccidiosis, has an inhibitory effect on the cell invasion of protozoa and / or an inhibitory effect on the proliferation of protozoa in cells, and has excellent preventive, alleviating and therapeutic effects on coccidiosis. Description of the drawings

[0107] Figure 1 Shows the anticoccidial index (ACI) of violacein after a single inoculation of protozoa.

[0108] Figure 2 Shows the anticoccidial index (ACI) of prodigiosin after co-inoculation with three protozoa.

[0109] Figure 3 Shows the inhibitory effect of prodigiosin on the invasion rate of protozoal cells and the inhibitory effect on the proliferation of protozoa within cells.

[0110] Figure 4 Shows the results of the acid tolerance assay of prodigiosin.

[0111] Figure 5 Shows the results of the heat tolerance assay of prodigiosin. Detailed implementation mode

[0112] The present invention will be described in more detail by the following examples, but is not intended to limit the scope by the following examples.

[0113] Example 1. In vivo anticoccidial activity of prodigiosin after single inoculation

[0114] Example 1-1. Experimental facilities and research design

[0115] The evaluation test of in vivo anticoccidial efficacy was carried out in an animal experiment facility in Gyeongsangnam-do, Korea. One-day-old female Ross broilers were individually weighed and randomly grouped for the experiment. The matters and conditions of the experimental design are described in Table 2.

[0116] Table 2

[0117] Category Experimental variable Breeding type Cage Age of free-range broilers 1-day-old Total experimental period 22 days Gender Female Number of broilers per cage 15 birds Number of replicates per treatment group 2 replicates Number of treatment groups 6 groups Total number of broilers 165 broilers Species of protozoa for challenge inoculation Eimeria tenella Number of oocysts for challenge inoculation 10,000 oocysts orally inoculated per broiler

[0118] The breeding farm was managed according to the Korean poultry breeding management guidelines. Before the start of the test, the cages and the breeding farm were cleaned and disinfected. The breeding farm maintained a temperature of 40°C to 41°C and a humidity of 40% to 50%, and was continuously monitored.

[0119] Example 1-2. Experimental design

[0120] The feed used the Korean Feed A1-choi product. Various materials (diclazuril (YUHAN DICLA product of Yuhan Corporation), salinomycin (Cheilsalino-60 product of Cheil Bio), gallic acid (CJ Cheiljedang)) were respectively added to the feed and self-mixed at the concentrations described in Table 3 below. No antibiotics and additives were used in the general feed and the mixed feed, and no coccidiostat was added except for each material. During the entire experiment period, the broiler chickens were fed ad libitum. After randomly placing 15 one-day-old broiler chickens in each cage of the control group or the experimental group and raising them, they were fed the general feed (A1-choi product) for 7 days, and then the above-prepared mixed feed was divided for ingestion by the control group or the experimental group. One cage was used for the uninfected negative control group, and two cages were used for the infected negative control group and the experimental group for the experiment.

[0121] The feed preparations administered to the control group (negative control group or positive control group) and the experimental group, and whether coccidiosis induced by Eimeria tenella are described in Table 3 below.

[0122] On the 14th day of age, the broiler chickens were orally inoculated with 10,000 oocysts (90% sporulated Eimeria tenella oocysts) per chicken to induce coccidiosis.

[0123] Table 3

[0124] Group Treatment Uninfected negative control group General feed Infected negative control group Eimeria tenella infection + ordinary feed Positive control group 1 Eimeria tenella infection + compound feed containing 1 ppm diclazuril Positive control group 2 Eimeria tenella infection + compound feed containing 60 ppm salinomycin Violacein treatment group 1 Eimeria tenella infection + compound feed containing 10 ppm violacein Violacein treatment group 2 Eimeria tenella infection + compound feed containing 60 ppm violacein

[0125] Example 1-3. Determination of the anticoccidial activity of violacein

[0126] The anticoccidial effects of the experimental groups designed in Examples 1-2 above were expressed as the anticoccidial index (ACI), and the anticoccidial index was calculated by the following Equation 2. The ACI score has a full score of 200 points. The higher the ACI score, the more excellent the anticoccidial ability. When the ACI score is 120 points or more and less than 140 points, it is determined that it is effective as an anticoccidial material. When the ACI score is 140 points or more and less than 160 points, it is determined that it is excellent as an anticoccidial material. When the ACI score is 160 points or more, it is determined that the anticoccidial effect is very excellent (Luis Miguel De Pablos et al., Anticoccidial activity of maslinic acid against infection with Eimeria tenella in chickens, Parasitol Res, 2010).

[0127] (Equation 2)

[0128] Anticoccidial Index (ACI) = (Survival rate (%) after challenge inoculation) + (Relative weight gain (RWG, %) compared to the negative control group) - (Lesion score × 10) - (Fecal oocyst excretion index)

[0129] 1) Survival rate: The number of dead individuals was recorded daily, and postmortem examinations were performed to determine the cause of death, excluding individuals that died from causes other than coccidiosis. The survival rate (%) from the time of challenge inoculation to day 8 was used to calculate the anticoccidial index. The measured survival rates are described in Table 4 below.

[0130] 2) Relative weight gain: Before challenge inoculation with protozoa and on day 7 after challenge inoculation, the weight of each cage was measured, and the difference was divided by the number of days to calculate the average daily gain (ADG, g / day). "Relative weight gain (RWG, %) compared to the negative control group" was calculated by dividing the average daily gain (ADG, g / day) of each experimental group by the average daily gain (ADG, g / day) of the uninfected negative control group and then multiplying by 100. This was used in the calculation of the anticoccidial index. The average daily gain (ADG, g / day) measured in each control and experimental group and the relative weight gain (RWG, %) compared to the negative control group are shown in Table 4 below.

[0131] 3) Lesion score: On day 8 after challenge inoculation, four broiler chickens per cage were autopsied, and the intestines were incised and opened. Each coccidial lesion in the appendix region of the broiler chickens was scored. The lesion scoring method was based on the literature of Johnson JK and Reid WM (1970) (Joyce Johnson and W. Malcolm Reid, Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens, Experimental parasitology, 1970). The lesion score ranged from 0 to 4, with 0 corresponding to a normal appendix, 1 indicating mild infection symptoms, 2 indicating moderate infection symptoms, 3 indicating severe infection symptoms, and 4 indicating very severe infection symptoms or death. The lesion index was calculated by multiplying the measured appendix lesion score by 10 and was used in the calculation of the anticoccidial index. The lesion scores and lesion indices measured in each control and experimental group are described in Table 4 below.

[0132] 4) Fecal oocyst excretion: All feces from day 6 to day 8 after challenge inoculation were collected in cages, mixed well, and randomly sampled 3 times, 1 g each time. After floating the oocysts in 1 g of feces with saline, the oocyst excretion was measured using a McMaster chamber, and the results are described in Table 4 below. The oocyst excretion (%) compared to the infected negative control group was calculated by dividing the oocyst excretion of each group by the oocyst excretion of the infected negative control group and multiplying by 100. When the calculated oocyst excretion compared to the infected negative control group was at the level of 0% to less than 1%, the oocyst excretion index was calculated as 0; when at the level of more than 1% to less than 26%, the fecal oocyst excretion index was calculated as 5; when at the level of more than 26% to less than 51%, the fecal oocyst excretion index was calculated as 10; when at the level of more than 51% to less than 76%, the fecal oocyst excretion index was calculated as 20; when at the level of more than 76% to less than 100%, the fecal oocyst excretion index was calculated as 40, and the results are described in Table 4 below, which is used to calculate the anticoccidial index.

[0133] Table 4

[0134]

[0135]

[0136] As described above, the anticoccidial index of each test group measured by Equation 2 above is shown in Table 4 below and Figure 1 in. As shown in Table 4 and Figure 1 below, compared with the uninfected negative control group, the weight gain decreased, and the lesion score and oocyst excretion increased in the infected negative control group of Eimeria tenella. In addition, compared with the infected negative control group, the weight gain and survival rate increased, the lesion score and oocyst excretion decreased, and the anticoccidial index was excellent in all groups treated with prodigiosin after challenge inoculation. In particular, in the treatment group with a compound feed containing 40 ppm of prodigiosin, the oocyst excretion was significantly reduced compared with the positive control groups ingesting salinomycin or diclazuril, thus it can be confirmed that prodigiosin can reduce the infection and secondary infection rates of livestock by coccidiosis. In addition, as a result of Example 7 below, different from the positive control groups, salinomycin or diclazuril, prodigiosin does not remain in organs and blood, so prodigiosin does not require a withdrawal period, and therefore it can be used as a more excellent anticoccidial agent.

[0137] Example 2. In vivo anticoccidial activity of prodigiosin after combined inoculation

[0138] Example 2-1. Experimental facilities and research design

[0139] An evaluation test of in vivo anticoccidial activity was conducted in an animal experiment facility in Chungcheongnam-do, Korea. One-day-old male Ross broilers were individually weighed and randomly grouped for the experiment. The matters and conditions of the experimental design are described in Table 5.

[0140] Table 5

[0141]

[0142]

[0143] The farms were managed according to the Korean Poultry Farming Management Guidelines. Before the start of the experiment, the cages and farms were cleaned and disinfected. The farms were maintained at a temperature of 40 °C to 41 °C and a humidity of 40% to 50%, and were continuously monitored.

[0144] Example 2-2. Experimental Design

[0145] The feed was mixed in Neobase, and various materials (salinomycin (Cheilbio Cheilsalino-60 product), gallic acid (CJ Cheiljedang)) were separately added to the feed and self-mixed at the concentrations described in Table 6 below. No antibiotics and additives were used in the general feed and the mixed feed, and no coccidiostat was added except for each material. During the entire experiment, the broilers were fed ad libitum. After randomly placing 8 one-day-old broilers in each cage of the control group or the test group and raising them, they were fed the general feed (A1-choi product) for 7 days, and then the above-prepared mixed feed was divided into the control group or the test group for ingestion.

[0146] The feed preparations administered to the control group (negative control group or positive control group) and the test group, and whether coccidiosis was induced by three protozoa are described in Table 6 below.

[0147] Coccidiosis induction was carried out by oral inoculation (challenge inoculation) with a cannula of 10,000, 50,000, 50,000 oocysts (eggs) of Eimeria tenella, Eimeria acervulina, and Eimeria maxima with a maturity of over 90% (sporulation) per individual to 14-day-old broilers.

[0148] Table 6

[0149]

[0150]

[0151] Example 2-3. Determination of the Anticoccidial Activity of Violacein

[0152] The anticoccidial effects of the experimental groups designed in Example 2-2 above are expressed as the anticoccidial index (ACI), and the anticoccidial index is calculated in a method similar to that in Example 1-3 above. However, in this example, the protozoa of Eimeria tenella, Eimeria acervulina, and Eimeria maxima are challenged and inoculated, so the lesion score is calculated by summing each score of the duodenum, jejunum, and appendix respectively, which is different from Example 1-3.

[0153] Table 7

[0154]

[0155] As described above, the anticoccidial indices of each experimental group measured by Equation 2 above are described in Table 7 below and Figure 2 in. As shown in Table 7 and Figure 2 , compared with the uninfected negative control group, the weight gain decreased, and the lesion score and oocyst excretion increased in the negative control group with triple protozoal coinfection. In addition, in all violacein treatment groups, compared with the infected negative control group, the lesion score and oocyst excretion decreased, and the anticoccidial index was excellent. In particular, in the compound feed treatment group containing 15 ppm violacein, compared with the positive control group with salinomycin intake, the lesion score and oocyst excretion were significantly reduced. Thus, it can be confirmed that violacein can reduce the ratio of livestock contaminated by coccidiosis and secondary infection.

[0156] Example 3. Determination of the direct killing effect of violacein on protozoa

[0157] In this example, the direct killing ability of protozoa (sporozoites) against 3 representative Eimeria species (Eimeria tenella, Eimeria acervulina, Eimeria maxima) known to be infected in most farms was evaluated.

[0158] A certain amount of oocysts of each coccidian protozoa were placed in a test tube containing glass beads and crushed, then the crushed oocyst cell walls and other debris were removed, percoll density gradient was used to purify the internal sporocysts, and they were washed with PBS solution. The sporocysts of Eimeria tenella, Eimeria acervulina, and Eimeria maxima were treated with reagents containing sodium taurocholate (Sigma aldrich, USA) and trypsin (Gibco, USA) respectively for excystation of internal sporozoites, and incubated, then washed once with PBS solution to obtain protozoa.

[0159] After violacein was reacted with the anticoccidial agents salinomycin, diclazuril, and gallic acid (hereinafter, materials) at different concentrations of 1 ppm to 500 ppm with three Eimeria protozoa, only live protozoa (sporozoites) were counted by microscopic observation. Then, the mortality rate (%) of the protozoa when treated with each material was measured compared to the negative control group treated with PBS, and the minimum concentration that directly killed 100% of the protozoa is shown in Table 8 below.

[0160] Table 8

[0161]

[0162] As a result of confirming the in vitro direct killing effect on coccidian protozoan species (three species), as shown in Table 8, in the gallic acid treatment group known as an anticoccidial agent, up to 500 ppm did not show a 100% killing effect on protozoa, and in the diclazuril treatment group, up to 500 ppm did not show a 100% sporozoite killing effect on Eimeria tenella and Eimeria maxima. In the violacein treatment group, even at a concentration significantly lower than other groups, it could kill 100% of Eimeria tenella, Eimeria acervulina, and Eimeria maxima, so its protozoan (sporozoite) killing effect was confirmed to be very excellent.

[0163] Example 4. Inhibitory effect of violacein on cell invasion and proliferation of Eimeria protozoa

[0164] In this example, using the representative animal cell MDBK cell line known to cause infection and proliferation of the genus Eimeria, the inhibitory ability of violacein on intracellular protozoan invasion and intracellular protozoan proliferation was studied.

[0165] 100,000 MDBK cells (purchased from ATCC) were aliquoted into 24-well plates and then incubated at a temperature of 37 °C for 12 hours. Protozoa of Eimeria tenella were obtained in a similar manner to the method of Example 3 above. 200,000 protozoa per well were added to the wells with the aliquoted cells, and each material (violacein and the anticoccidial agents salinomycin, diclazuril, and gallic acid) was used to treat the cells at a concentration, and the cells were cultured at a temperature of 41 °C for 24 hours. The negative control group was MDBK cells infected with Eimeria tenella protozoa, and the positive control group refers to the group in which salinomycin, diclazuril, or gallic acid solution was incubated with Eimeria tenella protozoa. Thereafter, in order to remove the protozoa that did not invade the cells, the cells were washed twice with PBS solution. After removing the cells and intracellular protozoa by pipette, DNA was extracted from the cells, and PCR was performed using the Eimeria tenella ITS-1 (Internal Transcribed Spacer-1) gene-specific primers. The sequences of the primers used are described in Table 9 below.

[0166] Table 9

[0167]

[0168] The Ct values of each material before and after washing were compared with the ΔCt values of the negative control group and corrected with the ΔCt values of the negative control group to calculate the inhibition rate (%) of cell invasion of protozoa by the treatment of each material. The results are shown in Figure 3 and Table 10 below.

[0169] 100,000 MDBK cells (purchased from ATCC) were aliquoted into 24-well plates and then incubated at 37 °C for 12 hours. Eimeria tenella protozoa were obtained in a similar manner to the method of Example 3 above. 200,000 protozoa per well were added to the wells with cell aliquots, cultured at 41 °C for 24 hours, and then the cells were washed 2 times with PBS solution to remove the protozoa that did not attach to the cells. Each material (prodigiosin and the anticoccidial drugs salinomycin, diclazuril, and gallic acid) was used to treat the cells at a concentration and further cultured at 41 °C for 24 hours. The negative control group was MDBK cells infected with protozoa, and the positive control group was the group in which salinomycin and diclazuril solutions were incubated with Eimeria tenella protozoa. After removing the cells and intracellular protozoa by pipette, DNA was extracted from the cells and PCR was performed using Eimeria tenella ITS-1 (internal transcribed spacer-1) gene-specific primers. The sequences of the primers used are described in Table 9 above.

[0170] By comparing the Ct values of the material treatment group and the negative control group, the inhibition rate (%) of intracellular protozoa proliferation after material treatment was calculated. The results are shown in Figure 3 and Table 10.

[0171] As Figure 3 and shown in Table 10 above, diclazuril only showed an effect of inhibiting the proliferation of intracellular protozoa, and gallic acid showed an effect on the intracellular invasion of protozoa. On the other hand, prodigiosin inhibited both the cell invasion and intracellular protozoa proliferation of Eimeria tenella protozoa, and the degree was more excellent than that of the positive control group. In the prodigiosin 1 ppm and 0.5 ppm administration groups, the experimental results related to the inhibitory effect on intracellular protozoa proliferation did not show statistically significant differences.

[0172] Table 10

[0173]

[0174] Example 5. Evaluation of the acid resistance of prodigiosin

[0175] In this example, the acid resistance of violacein will be evaluated. Hydrochloric acid (HCl) solution is added to a 200 ppm violacein solution to adjust it to pH 2, pH 3, and pH 5.5, and then it is left standing for 1 hour at a temperature of 40 °C. Then, it is neutralized (pH 7.0) by adding sodium hydroxide (NaOH) solution, and 200,000 Eimeria maxima protozoa (sporozoites) are exposed to different concentrations of violacein from 0.1 ppm to 500 ppm diluted with PBS solution, and the reaction is carried out at a temperature of 41 °C for 4 hours. Then, the protozoan mortality rate (%) is measured by a method similar to that in Example 3 above. Figure 4 and shown in Table 11. In Table 11, the control group refers to the violacein treatment group that has not been treated under acidic conditions, and the single protozoa refers to the group without violacein treatment.

[0176] As Figure 4 and shown in Table 11, it can be confirmed that violacein does not lose its killing effect on the protozoa that cause coccidiosis even under strong acidic conditions.

[0177] Table 11

[0178]

[0179] As Figure 4 and shown in Table 11, it can be confirmed that in the control group and all experimental groups, the protozoan killing effect of violacein in a concentration-dependent manner is excellent. Even if violacein is placed under acidic conditions of pH 2 - pH 3 for 1 hour and then restored to neutral, the protozoan killing effect of acidic violacein at pH 2 - pH 3 does not appear.

[0180] Example 6. Evaluation of the heat resistance of violacein

[0181] In this example, the heat resistance of violacein is evaluated. Violacein is exposed at a temperature of 85 °C - 95 °C for 10 minutes, then cooled, and diluted with PBS solution to a concentration of 0.1 ppm - 100 ppm. 200,000 Eimeria maxima protozoa are reacted with different concentrations of violacein from 0.1 ppm to 100 ppm at a temperature of 41 °C for 4 hours, and then the protozoan mortality rate (%) is measured by a method similar to that in Example 3 above. Figure 5 and shown in Table 12. In Table 12 below, the control group refers to the violacein solution that has not been treated under high-temperature conditions, and the single protozoa refers to the group without violacein treatment.

[0182] Table 12

[0183]

[0184]

[0185] As shown in Table 12 and Figure 5 as shown, it was confirmed that in the control group and all experimental groups, the protozoa-killing effect of violacein in a concentration-dependent manner was excellent. Even when violacein was placed at high temperatures (80°C to 95°C), the protozoa-killing effect of violacein did not occur at all concentrations.

[0186] Example 7. In Vivo Safety Assessment

[0187] Under the conditions described in Example 1, the safety assessment of violacein was carried out in a breeding facility. 7-day-old broiler chickens were fed a diet containing 50 ppm violacein for 7 days, necropsied at 14 days of age, and feed, cecal contents of the appendix, blood, spleen, liver, and kidneys were collected. For the feed used in the formulation with violacein, the product of Daehanfeed Al-Choi was used.

[0188] The violacein content of each collected tissue sample was tested by the HPLC method. For feed pretreatment, 1 g was weighed for each treatment group and diluted by adding 10 mL of MTBE solution, then sonicated for 1 hour and filtered using a 0.45 μm syringe filter. Then, after evaporating the filtered sample for 1 hour, 1 mL of 100% methanol was added. In addition, it was sonicated for 1 minute, which did not dissolve, and the supernatant was filtered again using a 0.45 μm syringe filter once, and then placed in a vial for analysis. For the HPCL pretreatment of blood and organs, the samples of each treatment group were combined and homogenized 3 times at 4000 bpm for 10 seconds each. Then, the supernatant was collected in a 1.5 mL microtube and frozen at -70°C. Subsequently, after freeze-drying the sample, 50 mg of the sample was weighed and placed in a microcentrifuge tube. 300 μL of MTBE solution was added to the weighed sample, and the microcentrifuge tube itself was placed in a 1.5 mL standard tube and mixed using a vortex mixer for 60 seconds. Further sonicated for 1 hour and centrifuged twice at 5000 rpm. It was confirmed that 200 μL to 300 μL of the solution was collected in a 1.5 mL tube, and the MTBE solution was evaporated. 100 μL of 100% methanol was added and dissolved. If it did not dissolve, it was sonicated for 10 minutes, and 50 μL of the supernatant was collected and placed in a vial for analysis. Then, the pretreated samples were analyzed using a set of LC / MS instruments to measure the residual amount of violacein in the body after feeding, and the results are shown in Table 13 below. In Table 13, ND (not detected) indicates not detected.

[0189] Table 13

[0190]

[0191]

[0192] As shown in Table 13, as a result of analyzing the content of violacein in the feed and the cecal lumen contents, blood, spleen, liver, and kidneys of broiler chickens fed with this feed, violacein was orally ingested by the broiler chickens and then reached the cecum through the stomach. In addition, violacein was not detected in the blood, liver, kidneys, and spleen, thus confirming that violacein does not remain in the body and confirming that violacein only stays in the intestine and can inhibit the production of coccidia. Sequence Listing <110> CJ CheilJedang Corporation <120> Anti-coccidial Composition Containing Violacein and Its Use <130> OPP20211038KR <150> KR 10-2020-0125246 <151> 2020-09-25 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic_Eimeria tenella ITS-1_Forward <400> 1 tggaggggat tatgagagga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic_Eimeria tenella ITS-1_Reverse <400> 2 caagcagcat gtaacggaga 20

Claims

1. A composition for preventing or alleviating coccidiosis, comprising at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof as an active ingredient, wherein, The violacein derivative is at least one selected from the group consisting of deoxyviolacein, violacein precursor, and oxyviolacein, and wherein, the coccidiosis is induced by protozoa of the genus Eimeria sp.

2. The composition according to claim 1, wherein, The prevention or alleviation of coccidiosis is at least one selected from the group consisting of the following (1) to (4): (1) reducing at least one selected from the group consisting of lesion score, fecal oocyst excretion amount, and mortality; (2) inhibiting weight loss caused by coccidiosis; (3) increasing the anti-coccidial index (ACI); and (4) reducing cell invasion of protozoa of the genus Eimeria sp., proliferation of protozoa in cells, or both.

3. A pharmaceutical composition for preventing or treating coccidiosis, comprising at least one selected from the group consisting of violacein, violacein derivatives, and pharmaceutically acceptable salts thereof as an active ingredient, wherein, The violacein derivative is at least one selected from the group consisting of deoxyviolacein, violacein precursor, and oxyviolacein, and wherein, the coccidiosis is induced by protozoa of the genus Eimeria sp.

4. An antiprotozoal composition against protozoa of the genus Eimeria, comprising at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof as an active ingredient, wherein, The violacein derivative is at least one selected from the group consisting of deoxyviolacein, violacein precursor, and oxyviolacein.

5. Use of at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof in the preparation of a pharmaceutical composition for preventing, alleviating, or treating coccidiosis, wherein, The violacein derivative is at least one selected from the group consisting of deoxyviolacein, violacein precursor, and oxyviolacein, and wherein, the coccidiosis is induced by protozoa of the genus Eimeria sp.

6. Use of at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof in the preparation of an antiprotozoal composition against protozoa of the genus Eimeria, wherein, The violacein derivative is at least one selected from the group consisting of deoxyviolacein, violacein precursor, and oxyviolacein.

7. Use of at least one selected from the group consisting of violacein, violacein derivatives, and salts thereof in the preparation of a composition for preventing, alleviating, or treating coccidiosis, wherein, The violacein derivative is at least one selected from the group consisting of deoxyviolacein, violacein precursor, and oxyviolacein, and wherein, the coccidiosis is induced by protozoa of the genus Eimeria sp.

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