Anticoccidial composition comprising coumaric acid and use thereof

Abstract

To provide the use of coumaric acid and / or its salts for the prevention, improvement, and / or treatment of coccidiosis.SOLUTION: The present application relates to an anticoccidial composition comprising coumaric acid and use thereof. A composition comprising coumaric acid and / or a salt thereof, according to an embodiment, has excellent effects of inhibiting the cell penetration of protozoa that can induce coccidiosis and / or inhibiting the proliferation of the protozoa in cells, has excellent effects of preventing, ameliorating and treating coccidiosis in vivo, and remarkably reduces oocyst excretion in feces, thus being able to reduce secondary coccidiosis infection.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0029733 dated March 5, 2021, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference.

[0002] The present invention relates to an anticoccidial composition containing coumaric acid and / or a salt thereof and use thereof. [Background technology]

[0003] Coccidiosis is an intestinal disease caused by Eimeria, a protozoan parasite belonging to the phylum Apicomplexan. Infection with coccidiosis causes symptoms such as digestive disorders, diarrhea, and weight loss, and can even cause the death of livestock, causing a significant economic impact on farmers around the world (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).

[0004] In recent years, many researchers have developed anticoccidial drugs such as ionophores or chemically synthetic compounds that can inhibit the formation of oocyst cell walls or the asexual and sexual reproduction of protozoa as therapeutic agents for treating coccidiosis. However, side effects such as the emergence of drug-resistant protozoa have occurred due to the long-term use of a shuttle program that alternates between ionophores and chemically synthetic compounds.

[0005] In particular, antibiotics that have accumulated in animals due to misuse and overuse of antibiotics have become a serious problem when humans ingest them through meat, and there is a trend toward banning antibiotics in countries around the world due to the problem of antibiotic residues in livestock products. Therefore, there is an urgent need to develop and research alternatives to existing anticoccidiostats, which have side effects such as the emergence of drug-resistant strains and the problem of antibiotic residues in the body. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Publication No. 2003-0091589 Summary of the Invention [Problem to be solved by the invention]

[0007] An embodiment of the present invention provides the use of coumaric acid and / or its salts for the prevention, amelioration, and / or treatment of coccidiosis.

[0008] Another embodiment of the present invention provides a feed composition for preventing or ameliorating coccidiosis, comprising coumaric acid and / or a salt thereof as an active ingredient.

[0009] Yet another embodiment of the present invention provides a pharmaceutical composition for preventing or treating coccidiosis, comprising coumaric acid and / or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0010] Yet another embodiment of the present invention provides the use of coumaric acid and / or its salts for the manufacture of a composition (e.g., feed composition, pharmaceutical composition) for the prevention, amelioration, and / or treatment of coccidiosis.

[0011] Yet another embodiment of the present invention provides a method for preventing, ameliorating, and / or treating coccidiosis, comprising administering the composition (e.g., the feed composition, the pharmaceutical composition, and / or the antiprotozoan composition) to an animal other than a human.

[0012] Yet another embodiment of the present invention provides the use of coumaric acid and / or its salts for use in antiprotozoal treatment of Eimeria parasites (e.g. killing Eimeria parasites; and / or inhibiting cell invasion and / or proliferation of Eimeria parasites).

[0013] Yet another embodiment of the present invention provides an antiprotozoal composition against Eimeria protozoa, comprising coumaric acid and / or a salt thereof as an active ingredient.

[0014] Yet another embodiment of the present invention provides the use of coumaric acid and / or its salts for the preparation of an antiprotozoal composition against Eimeria genus protozoa.

[0015] Yet another embodiment of the present invention provides a method for preventing, ameliorating, and / or treating coccidiosis or a method for controlling Eimeria protozoa, comprising the step of administering coumaric acid and / or a salt thereof to an animal (e.g., a subject in need of protozoan control, or an animal other than human). In this specification, protozoan control comprehensively means antiprotozoan action, such as, but not limited to, killing Eimeria protozoa and / or inhibiting cell invasion and / or proliferation of Eimeria protozoa.

[0016] In one embodiment of the present invention, the coumaric acid has the structure of Formula 1: [ka] [Means for solving the problem]

[0017] The composition according to the present invention containing coumaric acid and / or a salt thereof has excellent anticoccidial activity and / or antiprotozoal activity against protozoa that induce coccidiosis.

[0018] In the present invention, excellent anticoccidial efficacy (activity, effect) may mean one or more (e.g., one or more, two or more, three or more, four or more, or all five) selected from the group consisting of the following (1) to (5): (1) a higher anticoccidial index (ACI) than a control group; (2) when administered to an animal individual in which coccidiosis has been induced, a reduction in mortality rate, lesion score, and / or oocyst discharge in feces compared to a control group; (3) suppression of weight loss due to induction of coccidiosis; (4) a higher insecticidal activity against protozoa that induce coccidiosis compared to a control group; and (5) a higher inhibitory effect on cell invasion by protozoa that induce coccidiosis and / or proliferation of the protozoa within the cells compared to a control group.

[0019] In the present invention, the control group refers to a negative control group (a group that is not treated with any agent or a group that is treated with water and / or a buffer) and / or a positive control group that includes an existing known anticoccidial agent (e.g., diclazuril, gallic acid, and / or salinomycin).

[0020] A composition according to one embodiment may have one or more properties (e.g., one or more, two or more, three or more, four or more, five or more, or all six) selected from the group consisting of the following (1) to (6), which properties may be superior to a control group: (1) superior anticoccidial activity; (2) superior antiprotozoal effect against protozoa that induce coccidiosis; (3) superior acid resistance; (4) superior heat resistance; (5) superior in vivo stability and / or safety; and (6) superior effect in improving weight gain.

[0021] The composition according to one embodiment has excellent acid resistance and / or heat resistance, and when administered to the body, can maintain excellent anticoccidial activity for a long period of time, has excellent in-vivo stability, and can maintain excellent anticoccidial activity even in environments with various temperatures and / or pH ranges, can be applied to various products (e.g., feed additives), and has excellent storage stability.

[0022] According to one embodiment, when administered to the body, the composition is not absorbed by tissues and organs other than the intestine (e.g., blood, liver, kidneys, and / or spleen), and has a low residual amount in the body, resulting in excellent in vivo safety.

[0023] In one embodiment, being excellent in improving weight gain may mean being excellent in the effect of increasing the weight of an individual when administered to the individual, and in one example, the weight gain may mean the weight gain per day, and the individual may be an individual in which coccidiosis has been induced.

[0024] The composition according to one embodiment exhibits anticoccidial activity equivalent to or better than existing known anticoccidial agents (e.g., sulfa drugs such as sulfaquinoxaline, sulfachloropyrazine, and sulfamethazine, polyether ionophore antibiotics such as salinomycin and monensin sodium, amprolium, diclazuril, and / or toltrazuril), while containing coumaric acid and / or a salt thereof as an active ingredient, which is a substance that naturally participates in metabolism during the metabolic process, and thus does not induce side effects or drug resistance, and is not absorbed into the body, so it may be safe even when used for a long period of time. The composition according to one embodiment does not cause resistance or remain in the body, and can be used as a substance for preventing, ameliorating, and / or treating coccidiosis in not only meat chickens but also layer chickens. The composition according to one embodiment can significantly reduce the amount of oocysts discharged from individuals infected with protozoa that induce anticoccidial activity, and can reduce livestock contamination and / or secondary infection rates.

[0025] In the present invention, "prevention" refers to any action that inhibits or delays the onset of a disease by administering a composition according to an embodiment, "treatment" refers to any action that improves or favorably changes the symptoms of a suspected or affected individual by administering a composition according to an embodiment, and "amelioration" refers to any action that at least reduces a parameter related to the state in which the disease is treated, e.g., the severity of symptoms, by administering a composition according to an embodiment. The disease may refer to coccidiosis.

[0026] One embodiment may provide a feed composition for preventing or ameliorating coccidiosis, comprising coumaric acid and / or a salt thereof (coumaric acid, a salt of coumaric acid, or a combination thereof).

[0027] The "coumaric acid" is a type of hydroxycinnamic acid, which is a hydroxy derivative of cinnamic acid. In one example, the coumaric acid may be one or more selected from the group consisting of p-coumaric acid, m-coumaric acid, and o-coumaric acid.

[0028] In one example, the coumaric acid may be in the trans form and / or the cis form. For example, the p-coumaric acid may be trans-p-coumaric acid and / or cis-p-coumaric acid, the m-coumaric acid may be trans-m-coumaric acid and / or cis-m-coumaric acid, and the o-coumaric acid may be trans-o-coumaric acid and / or cis-o-coumaric acid.

[0029] The p-coumaric acid is "4-hydroxycinnamic acid", "p-hydroxycinnamic acid", or "2-(4-hydroxyphenyl)acrylic acid", and C 9 H 8 O 3 In one example, the p-coumaric acid can be represented by the following formula 1 or formula 2, or a mixture thereof (a compound represented by formula 1 and a compound represented by formula 2). In one example, the Cas number of the p-coumaric acid is Cas No. 501-98-4, Cas No. 7400-08-0, and / or Cas No. 4501-31-9.

[0030] [ka] [ka]

[0031] The m-coumaric acid is "3-hydroxycinnamic acid", "m-hydroxycinnamic acid" or "3-(3-hydroxyphenyl)acrylic acid", 9 H 8 O 3 In one example, the m-coumaric acid can be represented by the following formula 3 or formula 4, or a mixture thereof (a compound represented by formula 3 and a compound represented by formula 4). In one example, the Cas number of the m-coumaric acid is Cas No. 14755-02-3, Cas No. 25429-38-3, and / or Cas No. 588-30-7.

[0032] [ka] [ka]

[0033] The o-coumaric acid is "2-hydroxycinnamic acid" or "2-hydroxycinnamate", C 9 H 8 O 3 In one example, the o-coumaric acid may be represented by the following formula 5 or 6, or a mixture thereof (a compound represented by formula 5 and a compound represented by formula 6). In one example, the Cas number of the o-coumaric acid is Cas No. 614-60-8 and / or Cas No. 495-79-4.

[0034] [ka] [ka]

[0035] The coumaric acid and / or its salts (salts of the coumaric acid) may be obtained by extraction and isolation from natural sources (e.g., plants) and / or bacterial strains, produced by conventional organic synthesis methods, or obtained from manufacturers in the art, but are not limited to these.

[0036] In the present invention, "salt of coumaric acid" refers to a physiologically acceptable salt among salts that are substances in which a cation and an anion are bound by electrostatic attraction, and "pharmaceutical acceptable salt" refers to a salt in a form that can be used medicamentally, such as metal salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. In one example, the metal salts include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, etc.; salts with organic bases include salts with triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.; salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.; salts with organic acids include 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.; salts with basic amino acids include salts with arginine, lysine, ornithine, etc.; salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc.

[0037] In the present invention, "coccidiosis" refers to a disease caused by coccidiosis protozoa (protozoa capable of inducing coccidiosis, for example, Eimeria coccidiosis protozoa) that infects the cytoplasm of the submucosal tissue of the gastrointestinal epithelium, destroying the epithelium and causing enteritis. It is a protozoal disease that causes economic damage to chicken farms by causing watery stools, diarrhea, and bloody stools, reducing the rate of weight gain, and extending the shipping age. Coccidiosis occurs not only in chickens but also in birds, fish, reptiles, and mammals. Specifically, coccidiosis can infect cattle, rabbits, goats, dogs, cats, and experimental animals such as mice and rats, and can cause fatal damage to poultry such as chickens, ducks, geese, turkeys, quails, and pheasants. In one example, the coccidiosis may include acute coccidiosis, subacute coccidiosis, and chronic coccidiosis, etc. The acute coccidiosis may present bloody stool, loss of vitality, and anemia within 48 hours after infection, and the infected individual may die, the subacute coccidiosis may present bloody diarrhea and / or anemia symptoms after infection, and the chronic coccidiosis may present watery stool and / or weight loss symptoms after diarrhea for 1 to 2 days after infection.

[0038] Coccidia protozoan oocysts (cysts, parasite eggs) become infectious when they mature into sporulated oocysts under high humidity and temperature conditions, and after a certain life cycle in the individual's body, they are easily transmitted if the oocysts are excreted in the feces, and the oocyst life cycle is repeated. Coccidia protozoan oocysts (cysts) are known to be highly resistant to the external environment, and the cyst wall is composed of two layers, an inner and outer layer. The outer layer of the cyst wall is a gelatinous substance that is highly resistant to physical external pressure, while the inner layer is rich in nuclear protein and is highly resistant to chemical stimuli, such as disinfectants. Oocysts of Coccidia species can contain four sporocysts, each of which can contain two sporozoites. After infecting animals in the form of oocysts, the sporocysts and sporozoites are released and grow intracellularly, and the sporozoites that have undergone sexual and / or asexual reproduction form oocysts and are excreted in feces. In one example, sporozoites are used interchangeably with protozoa, and the sporozoites (protozoa) can cause lesions.

[0039] According to one example, the coccidiosis may be induced by the protozoan Eimeria sp. In one example, the Eimeria protozoa is Eimeria acervulina (gizzard coccospore), Eimeria tenella (chicken cecal coccospore), Eimeria maxima (maxima coccospore), Eimeria necatrix (necatr coccospore), Eimeria brunetti (brunetti coccospore), Eimeria hagani (Eimeria hagani coccospore), Eimeria mitis (Eimeria mitis coccospore), Eimeria praecox (Eimeria mivati ​​(mice pigeon coccospore), Eimeria aurati (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 duodenalis, Eimeria fraterculae, Eimeria gallopavonis, Eimeria innocua, Eimeria meleagridismeleagridis, Eimeria meleagrimitis, Eimeria phasiani, Eimeria procera, Eimeria purpureicephali, Eimeria ahsata, Eimeria alabamensis, Eimeria alijevi, Eimeria aspheronica, Eimeria arloingi, Eimeria arundeli, Eimeria bakuensis, Eimeria bovis, Eimeria cameli, Eimeria caprina 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 felisi ferrisi, Eimeria flavescens, Eimeria gallatii, Eimeria granulosa, Eimeria hirsihirci, Eimeria intestinalis, Eimeria irresidua, Eimeria intricata, Eimeria jolchijevi, Eimeria krijgsmanni, Eimeria larimerensis, Eimeria macusaniensis, Eimeria magna, Eimeria marconii, Eimeria media, Eimeria melanuri, Eimeria myoxi, Eimeria nagpurensis, Eimeria neeshulji 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 punctate, Eimeria roobroucki, Eimeria saudiensis, Eimeria sealanderi, Eimeria separataThe bacterial strain may be one or more species (e.g., one or more, two or more, three or more, or four or more species) selected from the group consisting of Eimeria separata, Eimeria stiedae, Eimeria ursini, Eimeria vermiformis, Eimeria weybridgensis, Eimeria wobati, and Eimeria zuernii.

[0040] A composition according to one embodiment has excellent preventive, ameliorative, and / or therapeutic effects against coccidiosis induced by one or more (e.g., one or more, two or more, three or more, or four or more) types of protozoa selected from the group consisting of Eimeria protozoa listed in Table 1 below, and the Eimeria protozoa listed in Table 1 below may each induce coccidiosis in the animals listed in Table 1.

[0041] [Table 1] TIFF2025003521000010.tif234168TIFF2025003521000011.tif221168TIFF2025003521000012.tif230168TIFF2025003521000013.tif97168

[0042] In one example, the composition may be effective in preventing, ameliorating, and / or treating coccidiosis induced by E. tenella, E. acervulina, and / or E. maxima.

[0043] In one example, the prevention or amelioration of coccidiosis can mean one or more (e.g., one or more, two or more, three or more, or all four) selected from the group consisting of the following (1) to (4), and may be, for example, a reduction, inhibition, and / or increase in one or more selected from the group consisting of the following (1) to (4) compared to a control group (a negative control group and / or a positive control group): (1) a reduction in one or more selected from the group consisting of lesion score (e.g., cecal lesion score), fecal oocyst excretion, and mortality rate; (2) inhibition of weight loss due to coccidiosis; (3) an increase in the anticoccidial index (ACI); and (4) a reduction in cellular invasion by Eimeria protozoa, proliferation of the protozoa within cells, or all of these.

[0044] In one example, the lesion scoring method for determining the lesion score is performed with reference to Johnson JK & Reid WM (1970) literature (Joyce Johnson, 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 graded 0 to 4. In one example, the lesion score may refer to the lesion scores measured in the cecum, duodenum, and / or jejunum, and is calculated as the sum of the respective lesion scores measured in each organ (cecum, duodenum, and / or jejunum).

[0045] In one example, the fecal oocyst output is measured by collecting feces excreted from an individual and measuring the output using a microscope or a counting chamber (eg, a McMaster chamber), or the like.

[0046] In one example, the mortality rate may refer to the mortality rate of animals in which coccidiosis has been induced, and post-mortem examination may be performed to exclude the number of animals that died from causes other than coccidiosis.

[0047] In one example, individuals in which coccidiosis has been induced may lose more weight than individuals in which coccidiosis has not been induced, and a composition according to one example may inhibit weight loss due to coccidiosis induction.

[0048] In one example, the anticoccidial comprehensive index is calculated according to the following Equation 1, where the lesion score is calculated as described above.

[0049] (Formula 1) Anticoccidial Comprehensive Index (ACI) = (Survival rate after challenge (%)) + (Daily weight gain (%) relative to the negative control group) - (Lesion score x 10) - (Fecal oocyst excretion index)

[0050] The challenge inoculation may refer to administration of a protozoan capable of inducing coccidiosis (e.g., oral inoculation, etc.). In one example, the survival rate is measured 5-10 days, 7-10 days, 8-10 days, 7-9 days, 7-8 days, or 7 days after the challenge inoculation, and the survival rate may be measured by performing a post-mortem autopsy to exclude the number of individuals who died from causes other than coccidiosis.

[0051] In the above formula 1, the weight gain relative to the negative control group may be a value calculated as a percentage based on the value of the negative control group (eg, a negative control group not infected with coccidiosis protozoa).

[0052] The lesion score in Equation 1 is as described above.

[0053] In Equation 1, the fecal oocyst excretion index may be a value calculated as a percentage based on the value of a negative control group (e.g., a negative control group infected with protozoa), and quantified as 0 if the calculated value is between 0% and less than 1%, 5 if the calculated value is between 1% and less than 26%, 10 if the calculated value is between 26% and less than 51%, 20 if the calculated value is between 51% and less than 76%, and 40 if the calculated value is between 76% and 100%.

[0054] In one example, the coumaric acid and / or salts thereof are present in the feed composition at 1 w / w% or less, less than 1 w / w%, -1 w / w% or less, 5×10 -2 w / w% or less, 2.5×10 -2 Less than w / w%, 2×10 -2 w / w% or less, 1.25×10 -2 w / w% or less, 10 -2 w / w% or less, 9×10 -3 w / w% or less, 8×10 -3 w / w% or less, 6.25×10 -3 w / w% or less, 7×10 -3 w / w% or less, 6×10 -3 w / w% or less, 5×10 -3 w / w% or less, 4×10 -3 w / w% or less, 10 -7 w / w% or more, 10 -6 w / w% or more, 10 -5 w / w% or more, 10 -4 w / w% or more, 5×10 -4 w / w% or more, 10 -3 w / w% or more, 1.5×10 -3 w / w% or more, 2×10 -3 w / w% or more, 3×10 -3 w / w% or more, 4×10 -3 w / w% or more, 5×10 -3 w / w% or more, 6.25×10 -3 w / w% or less, 10 -7 ~1w / w%, 10 -7 ~10 -1 w / w%, 10 -7 ~5×10 -2 w / w%, 10 -7 ~10 -2 w / w%, 10 -7 ~5×10 -3 w / w%, 10 -7 ~4×10 -3 w / w%, 10 -7 ~10 -3 w / w%, 10 -7 ~5×10 -4 w / w%, 10 -7 ~10 -4 w / w%, 10 -7 ~10 -5in / in%、10 -6 ~1w / %、10 -6 ~10 -1 in / in%、10 -6 ~5×10 -2 in / in%、10 -6 ~10 -2 in / in%、10 -6 ~5×10 -3 in / in%、10 -6 ~4×10 -3 in / in%、10 -6 ~10 -3 in / in%、10 -6 ~5×10 -4 in / in%、10 -6 ~10 -4 in / in%、10 -6 ~10 -5 in / in%、10 -5 ~1w / w%、10 -5 ~10 -1 in / in%、10 -5 ~5×10 -2 in / in%、10 -5 ~10 -2 in / in%、10 -5 ~5×10 -3 in / in%、10 -5 ~4×10 -3 in / in%、10 -5 ~10 -3 in / in%、10 -5 ~5×10 -4 in / in、10 -5 ~10 -4 in / in%、10 -4 ~1w / w%、10 -4 ~10 -1 in / in%、10 -4 ~5×10 -2 in / in%、10 -4 ~10 -2 in / in%、10 -4 ~5×10 -3 in / in%、10 -4 ~4×10 -3 in / in%、10 -4 ~10 -3 in / in%、10 -4 ~5×10 -4 in / in%、10 -3 ~1w / w%、10 -3 ~10-1 w / w%, 10 -3 ~5×10 -2 w / w%, 10 -3 ~10 -2 w / w%, 10 -3 ~5×10 -3 w / w%, 10 -3 ~4×10 -3 w / w%, 10 -3 ~2×10 -3 w / w%, or 10 -3 ~1.5×10 -3 In one example, the feed composition may be a feed (e.g., a compound feed and / or a plain feed to be ultimately ingested by an animal) containing the active ingredient in the above range based on the total weight.

[0055] In one example, the coumaric acid and / or salts thereof are present in the feed composition at 1000 ppm or less, 500 ppm or less, 400 ppm or less, 300 ppm or less, 250 ppm or less, 200 ppm or less, 125 ppm or less, less than 125 ppm, 100 ppm or less, 90 ppm or less, 80 ppm or less, 70 ppm or less, 65 ppm or less, 62.5 ppm or less, 60 ppm or less, 50 ppm or less, 0.1 ppm or more, 1 ppm or more, 5 ppm or more, 10 ppm or more, 15ppm or more, 20ppm or more, 30ppm or more, 40ppm or more, 50ppm or more, 62.5ppm or more, 0.1~1000ppm, 0.1~500ppm, 0.1~400ppm, 0.1~300ppm, 0.1~250ppm, 0.1~200ppm, 0.1~125ppm, 0.1~100ppm, 0.1~90ppm, 0.1~80ppm, 0.1~70ppm, 0.1~65ppm, 0.1~60ppm, 0.1~50ppm, 1~1000ppm, 1~500ppm, 1~400ppm, 1~300ppm, 1~250ppm, 1~200ppm, 1~125ppm, 1~100ppm, 1~90ppm, 1~80ppm, 1~70ppm, 1~65ppm, 1~ 60ppm, 1~50ppm, 5~1000ppm, 5~500ppm, 5~400ppm, 5~300ppm, 5~250ppm, 5~200ppm, 5~125ppm, 5~100ppm, 5~90ppm, 5~80ppm, 5~70ppm, 5~65p pm, 5~60ppm, 5~50ppm, 10~1000ppm, 10~500ppm, 10~400ppm, 10~300ppm, 10~250ppm, 10~200ppm, 10~125ppm, 10~100ppm, 10~90ppm, 10~80pp m, 10~70ppm, 10~65ppm, 10~60ppm, 10~50ppm, 20~1000ppm, 20~500ppm, 20~400ppm, 20~300ppm, 20~250ppm, 20~200ppm, 20~125ppm, 20~100p pm, 20~90ppm, 20~80ppm, 20~70ppm, 20~65ppm, 20~60ppm, 20~50ppm, 30~1000ppm, 30~500ppm, 30~400ppm, 30~300ppm, 30~250ppm, 30~200pp m, 30~125ppm, 30~100ppm, 30~90ppm, 30~80ppm, 30~70ppm, 30~65ppm, 30~60ppm, 30~50ppm, 40~1000ppm, 40~500ppm, 40~400ppm, 40~300ppm , 40-250ppm, 40-200ppm, 40-125ppm, 40-100ppm, 40-90ppm, 40-80ppm, 40-70ppm, 40-65ppm, 40-60ppm, 40-50ppm, 50-1000ppm, 50-500ppm, 50-400ppm, 50-300ppm, 50-250ppm, 50-200ppm, 50-125ppm, 50-100ppm, 50-90ppm, 50-80ppm, 50-70ppm, 50-65ppm, or 50-60ppm.

[0056] In the present invention, "feed" can mean any natural or artificial diet, meal, etc., or components of said meal, intended or suitable for eating, ingesting and digesting by an animal. Exemplary feed compositions further include concentrates and / or special feeds. The concentrated feed may be animal feed such as seeds and fruits including grains such as wheat, oats, and corn; brans including rice bran, wheat bran, and barley bran as by-products obtained by refining grains; oil cakes which are by-products obtained by extracting oil from beans, rapeseed, sesame, flaxseed, coconut, and the like; residual starches which are the main components of starch cakes which are the remainders after starch is removed from sweet potatoes, potatoes, and the like; fish meal, fish meal, fish solubles which are concentrated fresh liquids obtained from fish, meat meal, blood meal, feather meal, skim milk powder, cheese from milk, and dried whey which is the residual liquid when producing casein from skim milk; yeast, chlorella, and / or seaweed.

[0057] The feed composition according to an example may refer to a feed in a form that is ultimately ingested by an animal, a dietary supplement that can be added to the feed, and / or a feed additive. The dietary supplement may refer to, for example, a formulation-containing composition that provides a therapeutic agent or digestive agent to an animal, and may refer to a composition that is not a source of caloric intake for a living body, i.e., an energy source, but is ingested in addition to a normal animal feed. The feed additive refers to a substance that is added to a feed for various effects such as supplementing nutrients and preventing weight loss, increasing digestive availability of fiber in the feed, improving milk quality, preventing reproductive disorders and improving conception rate, and preventing high temperature stress in summer. In one example, the feed additive may refer to a substance that is added for the purpose of preventing, improving, or treating coccidiosis.

[0058] In one example, the feed composition may be a feed additive, and when the feed additive according to one example is blended into feed (e.g., formulated feed and / or plain feed that is ultimately ingested by an animal), the feed additive may have a concentration of 0.001% or more, 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.5% or more, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, 0.005% or less, 0.001 to 1%, 0.001 to 0.5%, 0.001 to 0.1%, 0.001 to 0.05%, 0 The compound may be added at a weight amount of 0.001-0.01%, 0.001-0.005%, 0.005-1%, 0.005-0.5%, 0.005-0.1%, 0.005-0.05%, 0.005-0.01%, 0.01-1%, 0.01-0.5%, 0.01-0.1%, 0.01-0.05%, 0.05-1%, 0.05-0.5%, 0.05-0.1%, 0.1-1%, 0.1-0.5%, or 0.5-1% and may be blended with feed ingredients, supplemental feed, supplements, and / or other types of additives other than the active ingredients according to the examples.

[0059] Another embodiment can provide a pharmaceutical composition for preventing or treating coccidiosis comprising coumaric acid and / or a salt thereof, the coumaric acid and / or salt thereof being as described above in relation to the coumaric acid and / or salt thereof contained in the pharmaceutical composition.

[0060] The pharmaceutical composition according to the present invention may be used as a single agent, or may be prepared as a combined preparation by further including a pharmaceutical composition known to have an approved preventive or therapeutic effect against coccidiosis. A pharmaceutical acceptable carrier, excipient, or diluent may be added to the pharmaceutical composition to form a pharmaceutical unit dosage form.

[0061] In the present invention, "pharmaceutical acceptable" means that it does not significantly stimulate the organism and does not inhibit the biological activity and properties of the administered active substance. In one example, the pharmaceutical composition containing a pharmaceutical acceptable carrier may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.

[0062] The pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it may be prepared using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, or surfactant.

[0063] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, syrups, etc., and include various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin.

[0064] Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0065] In one example, the pharmaceutical composition may be formulated and used in various forms, such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injection solutions, in a conventional manner according to the intended purpose, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

[0066] In one example, the pharmaceutical composition may further include a carrier, excipient, or diluent, and suitable examples of the carrier, excipient, or diluent that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. The pharmaceutical composition may further include a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, etc.

[0067] In one example, the effective amount of the active ingredient (coumaric acid and / or a salt thereof) in the pharmaceutical composition varies depending on the age, sex, and body weight of the patient (individual), and is generally 0.0001 to 0.001 mg / kg, 0.0001 to 0.01 mg / kg, 0.0001 to 0.1 mg / kg, 0.0001 to 1 mg / kg, 0.0001 to 10 mg / kg, 0.0001 to 100 mg / kg, 0.0001 to 231 mg / kg, 0.0001 to 250 mg / kg, or 100 mg / kg to 250 mg / kg. mg / kg, 0.0001~500mg / kg, 0.0001~1000mg / kg, 0.001~0.01mg / kg, 0.001~0.1mg / kg, 0.001~1mg / kg, 0.001~10mg / kg, 0.0 01~100mg / kg, 0.001~231mg / kg, 0.001~250mg / kg, 0.001~500mg / kg, 0.001~1000mg / kg, 0.01~0.1mg / kg, 0.01~1mg / kg, 0. 01~10mg / kg, 0.01~100mg / kg, 0.01~250mg / kg, 0.01~500mg / kg, 0.01~1000mg / kg, 0.1~1mg / kg, 0.1~10mg / kg, 0.1~100mg / kg, 0.1~231mg / kg, 0.1~250mg / kg, 0.1~500mg / kg, 0.1~1000mg / kg, 1~10mg / kg, 1~100mg / kg, 1~231mg / kg, 1~250mg / kg, 1 to 500 mg / kg, 1 to 1000 mg / kg, 10 to 100 mg / kg, 10 to 231 mg / kg, 10 to 250 mg / kg, 10 to 500 mg / kg, 10 to 1000 mg / kg, 100 to 231 mg / kg, 100 to 250 mg / kg, 100 to 500 mg / kg, 100 to 1000 mg / kg, 250 to 500 mg / kg, or 250 to 1000 mg / kg may be administered daily or every other day, or may be administered in one to three separate doses per day. However, the dosage may be increased or decreased depending on the route of administration, severity of disease, sex, weight, age, etc., and does not limit the scope of the present invention in any way. In one example, when the composition is administered intraperitoneally, it may be administered at a concentration of 0.001 to 250 mg / kg.

[0068] In one example, the dosage of the pharmaceutical composition may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0069] In one example, the coumaric acid and / or salts thereof are present in the pharmaceutical composition at a concentration of 1 w / w% or less, less than 1 w / w%, -1 w / w% or less, 5×10 -2 w / w% or less, 2.5×10 -2 Less than w / w%, 2×10 -2 w / w% or less, 1.25×10 -2 w / w% or less, 10 -2 w / w% or less, 9×10 -3 w / w% or less, 8×10 -3 w / w% or less, 6.25×10 -3 w / w% or less, 7×10 -3 w / w% or less, 6×10 -3 w / w% or less, 5×10 -3 w / w% or less, 4×10 -3 w / w% or less, 10 -7 w / w% or more, 10 -6 w / w% or more, 10 -5 w / w% or more, 10 -4 w / w% or more, 5×10 -4 w / w% or more, 10 -3 w / w% or more, 1.5×10 -3 w / w% or more, 2×10 -3 w / w% or more, 3×10 -3 w / w% or more, 4×10 -3 w / w% or more, 5×10 -3 w / w% or more, 6.25×10 -3 w / w% or less, 10 -7 ~1w / w%, 10 -7 ~10 -1 w / w%, 10 -7 ~5×10 -2 w / w%, 10 -7 ~10 -2 w / w%, 10 -7 ~5×10 -3 w / w%, 10 -7 ~4×10 -3 w / w%, 10 -7 ~10-3 in / in%、10 -7 ~5×10 -4 in / in%、10 -7 ~10 -4 in / in%、10 -7 ~10 -5 in / in%、10 -6 ~1w / %、10 -6 ~10 -1 in / in%、10 -6 ~5×10 -2 in / in%、10 -6 ~10 -2 in / in%、10 -6 ~5×10 -3 in / in%、10 -6 ~4×10 -3 in / in%、10 -6 ~10 -3 in / in%、10 -6 ~5×10 -4 in / in%、10 -6 ~10 -4 in / in%、10 -6 ~10 -5 in / in%、10 -5 ~1w / w%、10 -5 ~10 -1 in / in%、10 -5 ~5×10 -2 in / in%、10 -5 ~10 -2 in / in%、10 -5 ~5×10 -3 in / in%、10 -5 ~4×10 -3 in / in%、10 -5 ~10 -3 in / in%、10 -5 ~5×10 -4 in / in、10 -5 ~10 -4 in / in%、10 -4 ~1w / w%、10 -4 ~10 -1 in / in%、10 -4 ~5×10 -2 in / in%、10 -4 ~10 -2 in / in%、10 -4 ~5×10 -3 in / in%、10 -4 ~4×10 -3w / w%, 10 -4 ~10 -3 w / w%, 10 -4 ~5×10 -4 w / w%, 10 -3 ~1w / w%, 10 -3 ~10 -1 w / w%, 10 -3 ~5×10 -2 w / w%, 10 -3 ~10 -2 w / w%, 10 -3 ~5×10 -3 w / w%, 10 -3 ~4×10 -3 w / w%, 10 -3 ~2×10 -3 w / w%, or 10 -3 ~1.5×10 -3 Included in w / w%.

[0070] In one example, the coumaric acid and / or salts thereof are present in the pharmaceutical composition at 1000 ppm or less, 500 ppm or less, 400 ppm or less, 300 ppm or less, 250 ppm or less, 200 ppm or less, 125 ppm or less, less than 125 ppm, 100 ppm or less, 90 ppm or less, 80 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 62.5 ppm or less, 50 ppm or less, 0.1 ppm or more, 1 ppm or more, 5 ppm or more, 10 ppm or more, 15ppm or more, 20ppm or more, 30ppm or more, 40ppm or more, 50ppm or more, 62.5ppm or more, 0.1~1000ppm, 0.1~500ppm, 0.1~400ppm, 0.1~300ppm, 0.1~250ppm, 0.1~200ppm, 0.1~125ppm, 0.1~100ppm, 0.1~90ppm, 0.1~80ppm, 0.1~70ppm, 0.1~65ppm, 0.1~60ppm, 0.1~50ppm, 1~1000ppm, 1~500ppm, 1~400ppm, 1~300ppm, 1~250ppm, 1~200ppm, 1~125ppm, 1~100ppm, 1~90ppm, 1~80ppm, 1~70ppm, 1~65ppm, 1~ 60ppm, 1~50ppm, 5~1000ppm, 5~500ppm, 5~400ppm, 5~300ppm, 5~250ppm, 5~200ppm, 5~125ppm, 5~100ppm, 5~90ppm, 5~80ppm, 5~70ppm, 5~65p pm, 5~60ppm, 5~50ppm, 10~1000ppm, 10~500ppm, 10~400ppm, 10~300ppm, 10~250ppm, 10~200ppm, 10~125ppm, 10~100ppm, 10~90ppm, 10~80pp m, 10~70ppm, 10~65ppm, 10~60ppm, 10~50ppm, 20~1000ppm, 20~500ppm, 20~400ppm, 20~300ppm, 20~250ppm, 20~200ppm, 20~125ppm, 20~100p pm, 20~90ppm, 20~80ppm, 20~70ppm, 20~65ppm, 20~60ppm, 20~50ppm, 30~1000ppm, 30~500ppm, 30~400ppm, 30~300ppm, 30~250ppm, 30~200pp m, 30~125ppm, 30~100ppm, 30~90ppm, 30~80ppm, 30~70ppm, 30~65ppm, 30~60ppm, 30~50ppm, 40~1000ppm, 40~500ppm, 40~400ppm, 40~300ppm , 40-250ppm, 40-200ppm, 40-125ppm, 40-100ppm, 40-90ppm, 40-80ppm, 40-70ppm, 40-65ppm, 40-60ppm, 40-50ppm, 50-1000ppm, 50-500ppm, 50-400ppm, 50-300ppm, 50-250ppm, 50-200ppm, 50-125ppm, 50-100ppm, 50-90ppm, 50-80ppm, 50-70ppm, 50-65ppm, or 50-60ppm.

[0071] In one embodiment, the pharmaceutical composition can be administered to a subject through various routes. The administration can mean providing a predetermined substance to an individual (patient) in any suitable manner, and the administration route of the pharmaceutical composition can be oral and / or parenteral administration through all common routes as long as it can reach the target tissue. When administering parenterally, topical application on 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 may be administered using any device that can deliver the active ingredient to the target cell.

[0072] In yet another embodiment, a composition for antiprotozoa against Eimeria protozoa, comprising coumaric acid and / or a salt thereof, can be provided. In yet another embodiment, a method for preventing, ameliorating, and / or treating coccidiosis or a method for controlling Eimeria protozoa, comprising the step of administering coumaric acid and / or a salt thereof to an animal (e.g., a subject in need of protozoa control, or an animal other than humans), can be provided. In this specification, protozoa control comprehensively means antiprotozoa action, such as, but not limited to, killing Eimeria protozoa and / or inhibiting cell invasion and / or proliferation of Eimeria protozoa. The coumaric acid, salts of coumaric acid, and / or Eimeria protozoa have been described above.

[0073] In one example, superior antiprotozoal activity (effect, efficacy) against the Eimeria protozoa may mean the following characteristics (1) and / or (2), and may, for example, exhibit the following characteristics (1) and / or (2) compared to a control group (negative control group and / or positive control group): (1) superior effect in killing Eimeria protozoa; and / or (2) inhibition of the cell invasion effect of Eimeria protozoa and / or the proliferation effect of the protozoa within the cells.

[0074] In one embodiment, the coumaric acid and / or salts of coumaric acid are included in the antiprotozoal composition in a concentration range as described above for the feed composition and / or pharmaceutical composition. In one embodiment, a composition containing the active ingredient in the above-mentioned concentration range may have better antiprotozoal activity than a composition containing the active ingredient in a concentration range outside the above-mentioned range.

[0075] Yet another embodiment may provide a method for preventing, ameliorating, or treating coccidiosis, comprising administering the composition (e.g., the feed composition, the feed additive, the pharmaceutical composition, and / or the antiprotozoan composition) to an animal (e.g., an animal other than humans). In one example, the method may further comprise identifying (selecting) an individual (patient) in need of preventing, ameliorating, or treating coccidiosis prior to administering the composition. The composition and coccidiosis are as described above. According to one example, identifying the individual may comprise detecting oocysts of a protozoan capable of inducing coccidiosis from feces isolated from the individual. In one example of the method for preventing, ameliorating, or treating coccidiosis, the administration method, administration route, and / or dosage of the composition are as described above.

[0076] According to one example, the composition may be administered in a pharma- tically effective amount. In the present invention, a "pharma- tically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of an effective dose may be determined depending on factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, co-used drugs, and other factors well known in the medical field. According to one example, the composition may be administered as an individual therapeutic agent or in combination with other anticoccidial agents, and may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, or may be administered in single or multiple doses. It is important to administer an amount that provides maximum effect at a minimum amount without side effects, taking into account all of the above factors, which can be easily determined by one skilled in the art.

[0077] In one example, the subject to which the method for preventing, ameliorating, or treating coccidiosis is applied refers to an animal that is or may be affected by coccidiosis, and the animal may be a mammal, including humans, horses, cows, mice, rats, dogs, cats, etc., a bird, including poultry (e.g., breeding chickens, meat chickens, and / or laying hens), a fish, an amphibian, and / or a reptile, etc.

[0078] In one example, the animal to which the method for preventing, ameliorating, or treating coccidiosis is applied may be one or more species (e.g., one or more species, two or more species, or three or more species) selected from the group consisting of animals listed in Table 1, for example, one or more species (e.g., one or more species, two or more species, or three or more species) selected from the group consisting of humans, chickens, ducks, geese, turkeys, quails, pheasants, pigeons, parrots, cows, pigs, goats, sheep, horses, antelopes, oryx, monkeys, cats, dogs, mice, rats, rabbits, raccoon dogs, squirrels, bats, guinea pigs, camels, lizards, alpacas, wombats, lizards, goldfish, crucian carp, tilapia, barbels, lungfish, and European chubs. In one example, the animal may be an animal other than humans.

[0079] That is, the present invention provides the following. (I) A feed composition for preventing or ameliorating coccidiosis, comprising coumaric acid or a salt thereof as an active ingredient. (II) The feed composition according to (I) above, wherein the coccidiosis is induced by a protozoan of the genus Eimeria. (III) The feed composition according to (I) above, wherein the coumaric acid is one or more selected from the group consisting of p-coumaric acid, m-coumaric acid, and o-coumaric acid. (IV) The feed composition of (I) above, wherein the prevention or amelioration of coccidiosis is one or more selected from the group consisting of the following (1) to (4): (1) reduction in one or more selected from the group consisting of lesion score, fecal oocyst excretion, and mortality rate; (2) inhibition of weight loss due to coccidiosis; (3) increase in anticoccidial index (ACI); and (4) reduction in cellular invasion by Eimeria protozoa, proliferation of the protozoa within cells, or all of these. (V) The feed composition according to (I) above, wherein the feed composition contains the active ingredient at a concentration of 1% (w / w) or less based on the total weight of the feed composition. (VI) The feed composition according to any one of the above (I) to (IV), which is a feed additive. (VII) A pharmaceutical composition for preventing or treating coccidiosis, comprising coumaric acid or a pharma- ceutical acceptable salt thereof as an active ingredient. (VIII) A composition for use as an antiprotozoan against Eimeria protozoa, comprising coumaric acid or a salt thereof as an active ingredient. (IX) A method for preventing, ameliorating, or treating coccidiosis, comprising the step of administering to an animal other than a human a composition selected from the group consisting of the feed composition of any one of (I) to (V), the pharmaceutical composition of (VII), and the antiprotozoan composition of (VIII). Effect of the Invention

[0080] The composition of the present invention containing coumaric acid and / or a salt thereof has an excellent effect of inhibiting cell invasion by protozoa capable of inducing coccidiosis and / or an excellent effect of inhibiting the proliferation of the protozoa within cells, and is excellent in the prevention, amelioration and treatment of coccidiosis in the body, and can significantly reduce the amount of oocysts discharged in feces, thereby reducing secondary infections of coccidiosis. [Brief description of the drawings]

[0081] [Figure 1] The anticoccidial comprehensive index (ACI) of coumaric acid is shown when inoculated with a protozoan capable of inducing coccidiosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] The present invention will be described in more detail below with reference to examples. However, these are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited to these examples.

[0083] Example 1. In vivo anticoccidial activity of coumaric acid

[0084] Example 1-1. Experimental facility and study design In vivo efficacy evaluation studies were conducted in a dedicated chicken broiler animal testing facility in the U.S. One-day-old female Ross chickens were individually weighed and randomly assigned to groups for use in the study.

[0085] The experimental design and conditions are listed in Table 2.

[0086] [Table 2]

[0087] Example 1-2. Experimental design The general feed used was a Daehan Feed A1-First Feed product, which was self-mixed by adding the following ingredients: salinomycin (Cheilbio's Cheil Sarino-60), VantiPEAL (Kemin, Cozante, main substance: Gallic acid), and p-coumaric acid (Cas No. 501-98-4; Sigma) at the concentrations shown in Table 3 below. No antibiotics or supplements were used in the general feed or mixed feed, and no anticoccidial agents were added other than the ingredients. The chickens were allowed to eat the feed ad libitum throughout the experiment period.

[0088] Coccidiosis was induced by orally inoculating (challenging) 21-day-old chickens with sporulated Eimeria tenella oocysts (10,000 oocysts per individual via oral tube inoculation.

[0089] The feed formulations administered to the control group (negative control group or positive control group) and test groups, and the presence or absence of coccidiosis induction by Eimeria tenella, are shown in Table 3 below. One-day-old broiler chickens were housed in cages randomly arranged according to control or test group, with six chickens in each cage. After being fed general feed for 14 days, the chickens were divided into control and test groups and given the compound feed prepared above.

[0090] [Table 3]

[0091] Example 1-3. Measurement of anticoccidial activity of coumaric acid The anticoccidial efficacy of each test group designed in Example 1-2 was expressed as an anticoccidial index (ACI), which was calculated by the following formula 2. The maximum ACI score was 200 points, and the higher the ACI score, the better the anticoccidial activity. If the ACI score was 120 or more and less than 140 points, it was determined that the material was effective as an anticoccidial material, if the ACI score was 140 or more and less than 160 points, it was determined that the material was excellent as an anticoccidial material, and if the ACI score was 160 points or more, it was determined that the anticoccidial effect was excellent (Luis Miguel De Pablos et al., Anticoccidial activity of maslinic acid against infection with Eimeria tenella in chickens, Parasitol Res, 2010).

[0092] (Formula 2) Anticoccidial Comprehensive Index (ACI) = (Survival rate after challenge (%)) + (RWG (%) per day relative to the negative control group) - (Lesion score x 10) - (Fecal oocyst shedding index)

[0093] 1) Survival rate: The number of dead individuals was recorded daily, and post-mortem examinations were performed to determine the cause of death. The number of individuals who died due to causes other than coccidiosis was excluded. The survival rate (%) up to 8 days after challenge was used to calculate the overall anticoccidial index.

[0094] 2) Daily weight gain: The weight of each individual was measured for each cage before and up to 8 days after challenge with the protozoa, and the difference was divided by the number of days to calculate the daily weight gain (ADG, g / d). The daily weight gain (ADG, average daily gain; g / d) of each experimental group was divided by the weight gain of the uninfected negative control group (ADG, g / d), and multiplied by 100 to calculate the "daily weight gain relative to the negative control group (RWG, %)," which was used to calculate the overall anticoccidial index.

[0095] The daily weight gain (ADG, g / d) measured for each control group and test group and the daily weight gain (RWG, %) relative to the negative control group are shown in Table 4 below.

[0096] 3) Lesion scoring: On the 9th day after challenge, 4 chickens were necropsied in each cage, and the intestines were incised and opened. The chickens were scored for coccidial lesions in the cecum. The lesion scoring method was performed with reference to Johnson JK & Reid WM (1970) (Joyce Johnson, W. Malcolm Reid, Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens, Experimental parasitology, 1970). Lesion scores ranged from 0 to 4, with 0 representing a normal cecum, 1 representing mild infection, 2 representing severe infection, 3 representing severe infection, and 4 representing very severe infection or death. The cecal lesion scores measured in each control group and test group are shown in Table 4 below. The measured cecal lesion scores were multiplied by 10 to obtain a lesion index, which was used to calculate the overall anticoccidial index.

[0097] 4) Oocyst excretion in feces: The feces from the 6th to 9th days after challenge were collected by cage and mixed uniformly, and then 1g of each sample was randomly sampled three times. Oocysts in 1g of feces were floated in salt water, and the amount of oocyst excretion was measured using a McMaster chamber. The results are shown in Table 4 below.

[0098] The oocyst excretion rate (%) relative to the infected negative control group was calculated by dividing the oocyst excretion rate for each group by the oocyst excretion rate for the infected negative control group and multiplying the result by 100. The oocyst excretion index was calculated as follows: 0 if the calculated oocyst excretion rate for the infected negative control group was 0% to less than 1%, 5 if 1% to less than 26%, 10 if 26% to less than 51%, 20 if 51% to less than 76%, and 40 if 76% to less than 100%, and this was used to calculate the overall anticoccidial index.

[0099] [Table 4]

[0100] As shown in Table 4, the negative control group (PC) infected with Eimeria tenella developed coccidiosis, reduced body weight gain, and increased lesion score and oocyst (egg) excretion compared to the non-infected negative control group (NC). The salinomycin-treated group, used as the anticoccidiostat control group, showed increased body weight gain and reduced lesion score and oocyst excretion compared to the infected negative control group (PC). The VantiPEARL-treated group, the natural anticoccidiostat control group, also showed increased body weight gain and reduced egg excretion and lesion index compared to the PC group.

[0101] The p-coumaric acid group had increased body weight gain and reduced lesion scores and oocyst excretion compared to the PC group. The anticoccidial comprehensive index was calculated and compared, and the p-coumaric acid 125 ppm group had a superior anticoccidial efficacy of 160 points compared to the positive control groups, Salinomycin (149 points) and VantiPEAL (147 points).

[0102] Example 3. Effect of coumaric acid on cell invasion and proliferation inhibition of Eimeria protozoa In this example, the ability of coumaric acid to inhibit intracellular protozoan invasion and intracellular proliferation was examined using the MDBK cell line, a representative animal cell line known to be infected and proliferated by Eimeria.

[0103] Example 3-1. Acquisition of protozoa A certain amount of oocysts of E. tenella and E. acervulina were crushed in a tube containing glass beads, and then the inner sporocysts were purified by Percoll density gradient to remove the crushed oocyst cell wall and other debris, and washed with PBS solution. For excystation of the inner protozoa, the sporangia of E. acervulina and E. tenella were treated with a reagent containing sodium taurocholic acid (Sigma Aldrich, USA) and trypsin (Gibco, USA), respectively, and incubated, then washed once with PBS solution to obtain the protozoa.

[0104] Example 3-2. Inhibitory effect of protozoan cell invasion and proliferation of intracellular protozoa by coumaric acid treatment Eimeria tenella and Eimeria acervulina protozoa were obtained by the method of Example 3-1. 5Sporozoites were added to wells of MDBK cells (purchased from ATCC) covered with a monolayer, and the cells were treated with each material (p-coumaric acid; sigma, Cas No. 501-98-4, and anticoccidial gallic acid; sigma, Cas No. 149-91-7, salinomycin; Cas No. sigma, 53003-10-4, diclazuril; sigma, Cas No. 101831-37-2, p-coumaric acid, gallic acid, and salinomycin were treated at 10 ppm, and diclazuril was treated at 1 ppm) and cultured at 40°C for 24 hours. The negative control group was MDBK cells infected with the protozoa, and the positive control group was the group incubated with gallic acid, salinomycin, or diclazuril solution together with the protozoa. The cells were then washed twice with PBS solution to remove any protozoa that had not invaded the cells. After separating the cells from the protozoa contained within them by pipetting, DNA was extracted from the cells using a DNA extract kit (Intron Biotechnology) and real-time PCR was performed using primers specific to the E. tenella ITS-1 (Internal transcribed spacer-1) gene or the E. acervulina ACE gene. The sequences of the primers used are listed in Table 5 below.

[0105] [Table 5]

[0106] The Ct values ​​before and after washing were compared for each material, and the fold change (2 -△Ct ) was used to calculate the inhibition rate of protozoan cell invasion by the material treatment (Invasion inhibition or cell invasion inhibition rate; %), and the results are shown in Tables 6 and 7. Table 6 shows the results for Eimeria tenella, and Table 7 shows the results for Eimeria acervulina.

[0107] [Table 6]

[0108] [Table 7]

[0109] Eimeria tenella protozoa were obtained in the same manner as in Example 3-1. 2 × 10 5 A sporozoite was added to a well containing MDBK cells (purchased from ATCC) covered with a monolayer, and after culturing for 24 hours at 40°C, the cells were washed twice with PBS solution to remove any protozoa that were not attached to the cells. The cells were treated with each of the substances (p-coumaric acid, and the anticoccidial agent gallic acid, salinomycin: 10 ppm, diclazuril: 1 ppm) and further cultured for 24 hours at 40°C.

[0110] The negative control group was MDBK cells infected with the protozoa, and the positive control group was a group incubated with salinomycin, gallic acid, or diclazuril solution together with the protozoa. After separating the cells from the protozoa multiplied within the cells by pipetting, DNA was extracted from the cells using a DNA extract kit (Intron Biotechnology) and PCR was performed using primers specific for E. tenella ITS-1. The sequences of the primers used are listed in Table 5 above. The Ct values ​​of the material-treated groups were compared with those of the negative control group to determine the fold change (2 -△Ct ) was calculated to calculate the propagation inhibition rate (%) of intracellular protozoa (sporozoites) due to the material treatment, and the results are shown in Table 6 above.

[0111] As shown in Table 6, diclazuril showed an inhibitory effect on intracellular protozoan proliferation, and gallic acid was effective only on the cell invasion of protozoa, whereas coumaric acid inhibited both the cell invasion of Eimeria tenella and the propagation of intracellular protozoa, with the degree of inhibition being superior to that of the positive control group. Also, as shown in Table 7, the inhibitory effect on cell invasion of Eimeria acervulina in the coumaric acid treatment group was superior to that of all positive control groups (diclazuril, gallic acid, and salinomycin).

[0112] Example 4. Anticoccidial effects of various coumaric acid isomers In this example, we evaluated the ability of various coumaric acid isomers to directly kill the protozoan (sporozoite) of Eimeria tenella, the most common and severe disease in the world, and investigated the ability of various coumaric acid isomers to inhibit intracellular protozoan invasion using the MDBK cell line, a representative animal cell line known to be infected with Eimeria.

[0113] Example 4-1. Direct killing effect on Eimeria protozoa A certain amount of protozoan oocysts were crushed in a tube containing glass beads, and then the inner sporocysts were purified using Percoll density gradient to remove the crushed oocyst cell walls and other debris, and washed with PBS solution. To excyst the inner sporozoites, the sporangia of Eimeria tenella were treated with a reagent containing sodium taurocholic acid (Sigma Aldrich, USA) and trypsin (Gibco, USA), incubated, and washed once with PBS solution to obtain the protozoa.

[0114] 0.1%DMSO, m-coumaric acid (sigma, 14755-02-3), Caffeic acid (sigma, 331-39-5), Ferulic After reacting Eimeria tenella with each of the acids (Sigma, 1135-24-6) at a concentration of 10 ppm, only live protozoa (sporozoites) were counted under a microscope. The mortality rate (%) of the protozoa treated with each material was measured against the negative control group treated with PBS, and the results are shown in Table 8 below.

[0115] [Table 8]

[0116] As shown in Table 8, at the same concentration, m-coumaric acid showed a significantly superior direct killing effect against Eimeria protozoa than caffeic acid and ferulic acid.

[0117] Example 4-2. Confirmation of ability to inhibit intracellular protozoan invasion In the same manner as in Example 3-2, 0.1% DMSO, p-coumaric acid (sigma, Cas No. 501-98-4), m-coumaric acid (sigma, 14755-02-3), and o-coumaric acid (sigma, 614-60-8) were treated at a concentration of 10 ppm each, and the cell invasion inhibition rate against Eimeria tenella protozoa was measured, and the results are shown in Table 9.

[0118] [Table 9]

[0119] As shown in Table 9, it was confirmed that p-coumaric acid, m-coumaric acid, and o-coumaric acid all exhibited inhibitory effects on intracellular protozoan invasion by Eimeria tenella.

[0120] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing its technical ideas or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below rather than the detailed description, and equivalent concepts.

Claims

1. A feed composition for preventing or improving coccidiosis, comprising coumaric acid or a salt thereof as an active ingredient, The feed composition comprises coumaric acid or a salt thereof at a concentration of 10 to 500 ppm.

2. 2. The feed composition of claim 1, wherein the coccidiosis is induced by a protozoan of the genus Eimeria.

3. The feed composition according to claim 1, wherein the coumaric acid is at least one selected from the group consisting of p-coumaric acid, m-coumaric acid, and o-coumaric acid.

4. The feed composition according to claim 1, wherein the prevention or amelioration of coccidiosis is one or more selected from the group consisting of the following (1) to (4): (1) a reduction in one or more selected from the group consisting of lesion score, fecal oocyst excretion, and mortality rate; (2) suppression of weight loss due to coccidiosis; (3) an increase in the anticoccidial index (ACI); and (4) A reduction in cell invasion by Eimeria protozoa, proliferation of the protozoa within cells, or all of these.

5. The feed composition according to claim 1, wherein the feed composition contains the active ingredient at a concentration of 1% (w / w) or less based on the total weight of the feed.

6. The feed composition according to any one of claims 1 to 4, which is a feed additive.

7. A pharmaceutical composition for preventing or treating coccidiosis, comprising coumaric acid or a pharma- ceutical acceptable salt thereof as an active ingredient, The pharmaceutical composition comprises coumaric acid or a salt thereof in a concentration of 10 to 500 ppm.

8. A composition for use against protozoa of the genus Eimeria, comprising coumaric acid or a salt thereof as an active ingredient, The composition for antiprotozoa contains coumaric acid or a salt thereof at a concentration of 10 to 500 ppm.

9. A method for preventing, ameliorating, or treating coccidiosis, comprising the step of administering to an animal other than a human a composition selected from the group consisting of the feed composition according to any one of claims 1 to 5, the pharmaceutical composition according to claim 7, and the antiprotozoan composition according to claim 8.

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