Anticoccidial composition containing ginkgo biloba leaves and its use

Abstract

The present application relates to an anticoccidial composition containing ginkgo biloba and its use. In one example, a composition containing ginkgo biloba has excellent effects of directly killing sporozoites that can induce coccidiosis, inhibiting the invasion of the sporozoites into cells, and / or inhibiting the proliferation of the sporozoites within cells, and is therefore effective in preventing, ameliorating, and treating coccidiosis in vivo.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0125244, dated September 25, 2020, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present application relates to an anticoccidial composition containing ginkgo biloba and its use. [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 lead to the death of livestock, causing a significant economic impact on farmers worldwide (Williams RB. A compartmentalized 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 agents, such as ionophores or chemically synthetic compounds, that can inhibit the formation of oocyst cell walls or the asexual and sexual reproduction of the protozoan as therapeutic agents for treating coccidiosis. However, long-term use of a shuttle program that alternates between ionophores and chemically synthetic compounds has led to side effects, such as the emergence of drug-resistant protozoan species.

[0005] In particular, antibiotics accumulated in animals due to misuse and overuse have become a serious problem as they are ingested by humans through meat, and countries around the world are moving toward banning antibiotic administration due to the problem of antibiotic residues in livestock products. Therefore, there is an urgent need to develop and research alternatives to existing anticoccidial drugs, which have side effects such as the emergence of drug-resistant strains and problems with residues in the body. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Publication No. 2008-0160000 Summary of the Invention [Problem to be solved by the invention]

[0007] One example of the present application provides a feed composition for preventing or ameliorating coccidiosis, which comprises, as an active ingredient, one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof.

[0008] Another example of the present application provides a feed composition for preventing or ameliorating coccidiosis, comprising ginkgo biloba leaves.

[0009] Yet another example of the present application provides a pharmaceutical composition for preventing or treating coccidiosis, comprising as an active ingredient one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and pharmaceutically acceptable salts thereof.

[0010] Yet another example of the present application provides a pharmaceutical composition for preventing or treating coccidiosis, comprising ginkgo biloba leaves.

[0011] Yet another example of the present application provides a composition for antiprotozoal use against Eimeria protozoa, comprising as an active ingredient one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof.

[0012] Yet another example of the present application provides an antiprotozoal composition against Eimeria protozoa, comprising ginkgo biloba leaves.

[0013] Yet another example of the present application provides a method for preventing, ameliorating, or treating coccidiosis, comprising administering the composition to a non-human animal.

[0014] Further examples of the present application include use of one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the manufacture of a composition (e.g., a feed composition, a pharmaceutical composition) or a composition for anti-protozoan use for the prevention, amelioration, and / or treatment of coccidiosis; use of one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the prevention, amelioration, and / or treatment of coccidiosis; and / or use of one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the prevention, amelioration, and / or treatment of coccidiosis; and / or use of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the prevention, amelioration, and / or treatment of coccidiosis; The present invention provides an antiprotozoal composition for use in the treatment of one or more Eimeria protozoa selected from the group consisting of ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof (e.g., killing Eimeria protozoa; and / or inhibiting cell invasion and / or proliferation of Eimeria protozoa).

[0015] Further examples of the present application provide the use of ginkgo biloba leaves for use in the manufacture of compositions (e.g., feed compositions, pharmaceutical compositions) or anti-protozoal compositions for preventing, ameliorating, and / or treating coccidiosis; the use of ginkgo biloba leaves for use in the prevention, amelioration, and / or treatment of coccidiosis; and / or the use of ginkgo biloba leaves for use in anti-protozoal treatment of Eimeria protozoa (e.g., killing Eimeria protozoa; and / or inhibiting cell invasion and / or proliferation of Eimeria protozoa). [Means for solving the problem]

[0016] One example of the present application provides a feed composition for preventing or ameliorating coccidiosis, which comprises, as an active ingredient, one or more (e.g., any one, two or more, three or more, or all) selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof.

[0017] The ginkgolide A is C 20 H 24 O9 and the compound having the structure of Chemical Formula 1 below may be, for example, CAS No. 15291-75-5, UNII-TAZ2DPR77B, CHEMBL465161, BN52020, DTXSID10873222, and / or HMS2089P12.

[0018] [ka]

[0019] The ginkgolide B is C 20 H 24 O 10and the compound having the structure of Chemical Formula 2 below may be, for example, named ginkgo lactone, and may be CAS No. 15291-77-7, BN52021, and / or AK160212.

[0020] [ka]

[0021] The ginkgolide C is C 20 H 24 O 11 and examples of compounds having the structure of Chemical Formula 3 below include CAS No. 15291-76-6, SCHEMBL16452771, MFCD02094178, ZINC85507063, and / or AKOS025311463.

[0022] [ka]

[0023] The quercetin is C 15 H 10 The compound having the molecular formula of O7 and the structure of Chemical Formula 4 below may be, for example, named Meletin, Sophoretin, and / or Xanthaurine, and may be CAS No. 117-39-5.

[0024] [ka]

[0025] Kaempferol is C 15 H 10The compound having the molecular formula O6 and the structure of Chemical Formula 5 below may be, for example, named Robigenin, Rhamnolutein, Populnetib, Trifolitin, and / or Pelargedenolone, and may have CAS No. 520-18-3.

[0026] [ka]

[0027] The bilobalide is C 15 H 18 O8 and the compound having the structure of Chemical Formula 6 below may be, for example, CAS No. 33570-04-6, UNII-M81D2O8H7U, or CHEBI:3103.

[0028] [ka]

[0029] The compounds selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof can be purchased commercially, extracted and separated from natural products, or produced by conventional organic synthesis methods, but are not limited to these.

[0030] In the present application, the term "salt of a compound" refers to a physiologically acceptable salt among salts, which are substances in which a cation and an anion are bound by electrostatic attraction, and may refer to, for example, a salt acceptable for a feed composition, a pharmaceutically acceptable salt, and / or a salt acceptable for an antiprotozoan composition. For example, the salt may be one or more 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 example, the metal salt may be one or more selected from the group consisting of alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, etc.; the salt with an organic base may be one or more selected from the group consisting of salts with triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.; the salt with an inorganic acid may be one or more selected from the group consisting of salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, etc. The salt with an organic acid may be one or more 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 one or more selected from the group consisting of salts with arginine, lysine, ornithine, etc.; and the salt with an acidic amino acid may be one or more selected from the group consisting of salts with aspartic acid, glutamic acid, etc.

[0031] In the present application, excellent anticoccidial efficacy (activity, effect) may mean one or more (e.g., any one, two or more, three or more, or all) selected from the group consisting of the following (1) to (5): (1) The anticoccidial index (ACI) was higher than that of the control group; (2) administered to animals in which coccidiosis has been induced to reduce mortality, lesion scores (e.g., cecal lesion scores), and / or fecal oocyst output compared to controls; (3) suppressing weight loss due to coccidiosis induction; (4) Higher insecticidal activity against coccidiosis-inducing protozoa compared to the control group; and (5) The inhibitory effect on the cell invasion of coccidiosis-inducing protozoa and / or the proliferation of said protozoa within cells is higher than that of the control group.

[0032] In the present application, the control group may refer 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, salinomycin, and / or gallic acid).

[0033] The 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), and the properties may be superior to those of a control group: (1) Excellent anticoccidial activity; (2) Excellent antiprotozoal effect against protozoa that cause coccidiosis; (3) Excellent acid resistance; (4) Excellent heat resistance; (5) Excellent in vivo stability and / or safety; and (6) Excellent effect in improving weight gain.

[0034] The composition according to one example has excellent acid resistance and / or heat resistance, and can maintain excellent anticoccidial activity for a long period of time when administered to the body. It also has excellent in vivo stability, and can maintain excellent anticoccidial activity even in environments with various temperature and / or pH ranges. It can be applied to various products (e.g., feed additives) and has excellent storage stability.

[0035] According to an example embodiment, when administered to the body, the composition is not absorbed into tissues and organs other than the intestine (e.g., blood, liver, kidneys, and / or spleen), leaving little residue in the body, and thus providing excellent in vivo safety.

[0036] In one example, being excellent in improving weight gain may mean being excellent in increasing the weight of an individual when administered to that 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.

[0037] The composition according to one example exhibits anticoccidial activity equivalent to or greater than that of 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, gallic acid, and / or toltrazuril), but does not induce side effects or drug resistance, contains a natural product (ginkgo biloba) with low toxicity, does not remain in the body, and may be safe for long-term use.

[0038] In this application, "prevention" refers to any action that suppresses 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 an individual suspected of or affected by a disease by administering a composition according to an embodiment, and "amelioration" refers to any action that at least reduces a parameter related to the condition of the disease being treated, for example, the severity of symptoms, by administering a composition according to an embodiment. The disease may be coccidiosis.

[0039] In one example, the composition containing one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof may be a composition containing ginkgo leaves, and the ginkgo leaves may be one or more selected from the group consisting of raw ginkgo leaves, dried ginkgo leaves, ground ginkgo leaves, and extracts.

[0040] Another embodiment may provide a feed composition for preventing or ameliorating coccidiosis, comprising ginkgo leaf.

[0041] An example composition may include ginkgo biloba as an ingredient exhibiting anticoccidial activity, and the ginkgo biloba may be one or more types (e.g., any one type, two or more types, three or more types, or all types) selected from the group consisting of raw ginkgo biloba, dried ginkgo biloba, ground ginkgo biloba, and extracts.

[0042] The ginkgo biloba leaf may refer to the leaf of Ginkgo biloba, a plant of the Ginkgoaceae family. Unlike most other gymnosperms, the ginkgo biloba leaf is fan-shaped with a divided middle section. For example, the ginkgo biloba leaf may be a green leaf that emerges in spring and / or a yellow leaf that turns red in autumn. Ginkgo biloba has long been used as a medicine in traditional Chinese medicine, and in modern medicine, ginkgo biloba leaf extract is used as a therapeutic agent for reduced cerebral and peripheral blood flow, sensory and neurological disorders, and impaired memory and cognitive function, and its anti-cancer effects have also been reported.

[0043] In one example, the ginkgo leaves may refer to green leaves before they turn yellow or yellow leaves after they turn yellow.

[0044] The ginkgo biloba raw material may refer to ginkgo biloba leaves that have not been processed (eg, dried, crushed, and / or extracted).

[0045] The dried ginkgo leaf product is obtained by drying the raw ginkgo leaf material, and can be obtained by methods such as reduced pressure drying, vacuum drying, boiling drying, room temperature drying, and / or freeze drying.

[0046] The ground ginkgo leaves may be obtained by crushing and / or grinding the raw ginkgo leaves and / or the dried ginkgo leaves, or may be obtained using a grinder.

[0047] In one example, the ginkgo leaves are dried at 50 to 90° C. for 4 to 24 hours, and then crushed using a crusher to produce ginkgo leaf powder.

[0048] In this application, the term "extract" refers to a preparation prepared by squeezing herbal medicines into a suitable infusion solution and evaporating the infusion solution to concentrate it. It may be an extract obtained by extraction, a diluted or concentrated extract, a dried product obtained by drying the extract, or a crude and / or purified product thereof. The ginkgo biloba extract may be prepared using common extraction, separation, and purification methods known in the art. Specific examples of the extraction method include hot water extraction, hot water extraction, cold maceration extraction, reflux cooling extraction, and ultrasonic extraction.

[0049] In one example, the ginkgo biloba extract can be commercially available or prepared by appropriately selecting an extraction method and extraction solvent commonly known in the pharmaceutical or food industry. For example, the extraction method can include conventional extraction methods such as solvent extraction, ultrasonic extraction, filtration, and / or reflux extraction. In one example, the ginkgo biloba extract can be prepared by extraction with an extraction solvent or by fractionating an extract prepared by extraction with an extraction solvent and adding a fractionation solvent. For example, the ginkgo biloba extract can be extracted using a solvent extraction method using an extraction solvent selected from the group consisting of water, C1-C4 linear or branched alcohols, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, and mixtures thereof. Specifically, the C1-C4 alcohol can be ethanol, methanol, isopropanol, propanol, butanol, and / or tert-butanol. The extraction solvent can be added in an amount 1 to 10 times the amount of dried ginkgo leaves, specifically 2 to 3 times the amount. The extraction temperature can be 30 to 100°C, 50 to 90°C, or 60 to 80°C. The extraction time can be 10 to 48 hours, 12 to 30 hours, or 18 to 24 hours. The extraction can be repeated 1 to 5 times, or 2 to 4 times.

[0050] In this application, "coccidiosis" refers to a protozoan disease caused by coccidiosis protozoa (protozoa capable of inducing coccidiosis, for example, Eimeria coccidiosis) that infects the cytoplasm of the submucosa of the gastrointestinal epithelium, destroying the epithelium and causing enteritis. It is a protozoan disease that causes economic damage to poultry farms by causing watery stools, diarrhea, and bloody stools, reducing weight gain, and extending the slaughter age. Coccidiosis occurs not only in poultry but also in birds and mammals. Specifically, coccidiosis can infect cattle, rabbits, goats, dogs, cats, and laboratory animals such as mice and rats, and can be fatal to poultry such as chickens. In one example, coccidiosis can include acute coccidiosis, subacute coccidiosis, and chronic coccidiosis. Acute coccidiosis causes bloody stools, loss of energy, and anemia within 48 hours of infection, and can lead to the death of infected individuals. Subacute coccidiosis causes bloody diarrhea and / or anemia after infection. Chronic coccidiosis can cause diarrhea for 1 to 2 days followed by watery stools and / or weight loss.

[0051] Oocysts (cysts, parasite eggs) of coccidia species become infectious when they mature into sporulated oocysts under high humidity and temperature conditions. After a certain life cycle within an individual, the oocysts are easily transmitted if they are excreted in the feces, and the oocyst life cycle is repeated. Coccidia oocysts (cysts) are highly resistant to the external environment, and the cyst wall is known to be composed of two layers, an inner and outer. 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 nucleoprotein 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 an animal in the form of oocysts, the sporangia and sporozoites are released and multiply intracellularly. After sexual and / or asexual reproduction, the sporozoites form oocysts and are excreted in feces. In one example, sporozoites are used interchangeably with protozoa, and the sporozoites (protozoa) can cause lesions.

[0052] According to one example, the coccidiosis may be induced by the protozoan Eimeria sp. In one example, the Eimeria protozoa is Eimeria acervulina (gizzard coccosporozoa), Eimeria tenella (chicken cecal coccosporozoa), Eimeria maxima (maxima coccosporozoa), Eimeria necatrix (Eimeria necatrix coccosporozoa), Eimeria brunetti (Eimeria brunetti) (Eimeria hagani) (Eimeria hagani) (Eimeria mitis) (Eimeria mitis) (Eimeria praecox) (Eimeria mivati) (Eimeria aurachi) (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 nyszulji 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 insecticide may be one or more species selected from the group consisting of Eimeria separata, Eimeria stiedae, Eimeria ursini, Eimeria vermiformis, Eimeria weybridgensis, Eimeria wobati, and Eimeria zuernii.

[0053] In one example, the composition has excellent effects in preventing, ameliorating, and / or treating coccidiosis induced by one or more 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.

[0054] [Table 1] TIFF0007783262000008.tif250166TIFF0007783262000009.tif240168TIFF0007783262000010.tif241168TIFF0007783262000011.tif161168

[0055] In one example, the composition may be highly effective in preventing, ameliorating, and / or treating coccidiosis induced by Eimeria tenella, Eimeria acervulina, and / or Eimeria maxima.

[0056] In one example, the prevention or amelioration of coccidiosis may mean one or more (e.g., any one, two or more, three or more, or all) selected from the group consisting of the following (1) to (4), and may be, for example, a decrease, suppression, 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 decrease in one or more of the following: lesion score (e.g., cecal 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, intracellular proliferation of the protozoa, or both.

[0057] In one example, the lesion scoring method for determining the lesion score is 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), and the lesion score may be on a scale of 0 to 4.

[0058] In one example, the lesion score can refer to the lesion scores measured in the cecum, duodenum, and / or coelenteron, and is calculated as the sum of the respective lesion scores measured in each organ (cecum, duodenum, and / or coelenteron).

[0059] In one example, the fecal oocyst output is measured by collecting feces excreted from an individual and measuring the fecal output using a microscope, a counting chamber (eg, a McMaster chamber), or the like.

[0060] 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 have died from causes other than coccidiosis.

[0061] 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.

[0062] In one example, the anticoccidial comprehensive index is calculated using the following Equation 1, and the lesion score in Equation 1 is calculated as described above.

[0063] (Number 1) Anticoccidial Comprehensive Index (ACI) = (survival rate after challenge (%)) + (daily weight gain (%) compared to negative control group) - (lesion score x 10) - (fecal oocyst output index)

[0064] The challenge inoculation may refer to administration (e.g., oral inoculation) of a protozoan capable of inducing coccidiosis. In one example, the survival rate is measured 5 to 10 days, 7 to 10 days, 8 to 10 days, 7 to 9 days, 7 to 8 days, or 7 days after challenge inoculation, and the survival rate can be measured by performing a post-mortem autopsy to exclude the number of individuals who died from causes other than coccidiosis.

[0065] In Equation 1, the weight gain compared to the negative control group may be a value calculated as a percentage based on the value of the negative control group (e.g., a negative control group not infected with the protozoan).

[0066] The lesion score in Equation 1 is as described above.

[0067] In Equation 1, the fecal oocyst excretion index may be a numerical value calculated by calculating a percentage based on the value of a negative control group (e.g., a negative control group infected with protozoa), and the calculated value may be quantified as 0 if the level is 0% or more and less than 1%, 5 if the level is 1% or more and less than 26%, 10 if the level is 26% or more and less than 51%, 20 if the level is 51% or more and less than 76%, and 40 if the level is 76% or more and less than 100%.

[0068] In one example, the active ingredient (one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof) is contained in the feed composition at 1 w / w% or less, less than 1 w / w%, 10 w / w% or less. -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, 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 x 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, 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 -5w / w%、10 -6 ~1w / %、10 -6 ~10 -1 w / w%、10 -6 ~5×10 -2 w / w%、10 -6 ~10 -2 w / w%、10 -6 ~5×10 -3 w / w%、10 -6 ~4×10 -3 w / w%、10 -6 ~10 -3 w / w%、10 -6 ~5×10 -4 w / w%、10 -6 ~10 -4 w / w%、10 -6 ~10 -5 w / w%、10 -5 ~1w / w%、10 -5 ~10 -1 w / w%、10 -5 ~5×10 -2 w / w%、10 -5 ~10 -2 w / w%、10 -5 ~5×10 -3 w / w%、10 -5 ~4×10 -3 w / w%、10 -5 ~10 -3 w / w%、10 -5 ~5×10 -4 in / in、10 -5 ~10 -4 w / w%、10 -4 ~1w / w%、10 -4 ~10 -1 w / w%、10 -4 ~5×10 -2 w / w%、10 -4 ~10 -2 w / w%、10 -4 ~5×10 -3 w / w%、10 -4 ~4×10 -3 w / 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 Contains w / w%.

[0069] In one example, the feed composition may be a feed (e.g., a compound feed and / or a plain feed that is ultimately ingested by an animal) containing the active ingredient in the above range based on the total weight.

[0070] In one example, the active ingredient (one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof) is contained in the feed composition at a concentration of 10,000 ppm or less, 5,000 ppm or less, 2,000 ppm or less, 1,000 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, 80 ppm or less, 9 ... ppm or less, 50 ppm or less, 40 ppm or less, 0.001 ppm or more, 0.01 ppm or more, 0.1 ppm or more, 1 ppm or more, 5 ppm or more, 10 ppm or more, 15 ppm or more, 20 ppm or more, 30 ppm or more, 40 ppm or more, 50 ppm or more, 0.001 to 1000 ppm, 0.001 to 500 ppm, 0.001 to 300 ppm, 0.001 to 200 ppm, 0.001 to 125 ppm, 0.001 to 100 ppm, 0.001 to 90 ppm, 0.001 to 80 ppm, 0.001 to 70 ppm, 0.001 to 60 ppm m, 0.001~50ppm, 0.001~40ppm, 0.001~30ppm, 0.003~1000ppm, 0.003~500ppm, 0.003~300ppm, 0.003~200ppm, 0.003~125ppm, 0.003~100ppm, 0. 003~90ppm, 0.003~80ppm, 0.003~70ppm, 0.003~60ppm, 0.003~50ppm, 0.003~40ppm, 0.003~30ppm, 0.01~1000ppm, 0.01~500ppm, 0.01~300ppm, 0 .01~200ppm, 0.01~125ppm, 0.01~100ppm, 0.01~90ppm, 0.01~80ppm, 0.01~70ppm, 0.01~60ppm, 0.01~50ppm, 0.01~ppm, 0.01~30ppm, 0.1~1000p pm, 0.1~500ppm, 0.1~300ppm, 0.1~200ppm, 0.1~125ppm, 0.1~100ppm, 0.1~90ppm, 0.1~80ppm, 0.1~70ppm, 0.1~60ppm, 0.1~50ppm, 0.1~40ppm, 0.1~30ppm, 1~1000ppm, 1~500ppm, 1~300ppm, 1~200ppm, 1~125ppm, 1~100ppm, 1~90ppm, 1~80ppm, 1~70ppm, 1~60ppm, 1~50ppm, 1~40ppm, 1~30ppm, 3~1000ppm, 3~500ppm, 3~300ppm, 3~200ppm, 3~125ppm, 3~100ppm, 3~90ppm, 3~80ppm, 3~70ppm, 3~60ppm, 3~50ppm, 3~40ppm, 3~30ppm, 5~1000pp It is found in concentrations of 5-500 ppm, 5-300 ppm, 5-200 ppm, 5-125 ppm, 5-100 ppm, 5-90 ppm, 5-80 ppm, 5-70 ppm, 5-60 ppm, 5-50 ppm, 5-40 ppm, 5-30 ppm, 10-1000 ppm, 10-500 ppm, 10-300 ppm, 10-200 ppm, 10-125 ppm, 10-100 ppm, 10-90 ppm, 10-80 ppm, 10-70 ppm, 10-60 ppm, 10-50 ppm, 10-40 ppm, or 10-30 ppm.

[0071] In one example, when the active ingredient (one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof) is contained within the above range, the anticoccidial activity may be superior to when the active ingredient is contained outside the above range.

[0072] In one example, the ginkgo biloba is present in the feed composition at 1 w / w% or less, less than 1 w / w%, 10 -1 Less than w / w%, 5x10 -2 w / w% or less, 2.5x10 -2 Less than w / w%, 2x10 -2 Less than w / w%, 1.25x10 -2 w / w% or less, 10 -2 Less than w / w%, 9x10 -3 Less than w / w%, 8x10 -3 w / w% or less, 6.25x10 -3 Less than w / w%, 7x10 -3 Less than w / w%, 6x10 -3 Less than w / w%, 5x10-3 Less than w / w%, 4x10 -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, 5x10 -4 w / w% or more, 10 -3 w / w% or more, 1.5x10 -3 w / w% or more, 2x10 -3 w / w% or more, 3x10 -3 w / w% or more, 4x10 -3 w / w% or more, 5x10 -3 w / w% or more, 6.25x10 -3 w / w% or more, 10 -7 ~1w / w%, 10 -7 ~10 -1 w / w%, 10 -7 ~5x10 -2 w / w%, 10 -7 ~10 -2 w / w%, 10 -7 ~5x10 -3 w / w%, 10 -7 ~4x10 -3 w / w%, 10 -7 ~10 -3 w / w%, 10 -7 ~5x10 -4 w / w%, 10 -7 ~10 -4 w / w%, 10 -7 ~10 -5 w / w%, 10 -6 ~1w / w%, 10 -6 ~10 -1 w / w%, 10 -6 ~5x10 -2 w / w%, 10 -6 ~10 -2 w / w%, 10 -6 ~5x10 -3 w / w%, 10 -6 ~4x10 -3 w / w%, 10 -6 ~10 -3 w / w%, 10 -6 ~5x10 -4 w / w%, 10 -6 ~10 -4 w / w%, 10-6 ~10 -5 w / w%, 10 -5 ~1 w / w%, 10 -5 ~10 -1 w / w%, 10 -5 ~5x10 -2 w / w%, 10 -5 ~10 -2 w / w%, 10 -5 ~5x10 -3 w / w%, 10 -5 ~4x10 -3 w / w%, 10 -5 ~10 -3 w / w%, 10 -5 ~5x10 -4 w / w%, 10 -5 ~10 -4 w / w%, 10 -4 ~1 w / w%, 10 -4 ~10 -1 w / w%, 10 -4 ~5x10 -2 w / w%, 10 -4 ~10 -2 w / w%, 10 -4 ~5x10 -3 w / w%, 10 -4 ~4x10 -3 w / w%, 10 -4 ~10 -3 w / w%, 10 -4 ~5x10 -4 w / w%, 10 -3 ~1 w / w%, 10 -3 ~10 -1 w / w%, 10 -3 ~5x10 -2 w / w%, 10 -3 ~10 -2 w / w%, 10 -3 ~5x10 -3 w / w%, 10 -3 ~4x10 -3 w / w%, 10 -3 ~2x10 -3 w / w%, or 10 -3 ~1.5x10 -3 is included by w / w%.

[0073] In one example, the feed composition may be a feed (e.g., a compound feed and / or a plain feed that is ultimately ingested by an animal) containing the active ingredient in the above range based on the total weight.

[0074] In one example, the ginkgo biloba is added to the feed composition at a concentration of 10,000 ppm or less, 5,000 ppm or less, 2,000 ppm or less, 1,000 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, 50 ppm or less, 40 ppm or less, 0.001 ppm or more, 0.01 ppm or more, 0.1 ppm or more, 100 ppm or less ... ppm or more, 5 ppm or more, 10 ppm or more, 15 ppm or more, 20 ppm or more, 30 ppm or more, 40 ppm or more, 50 ppm or more, 0.001 to 1000 ppm, 0.001 to 500 ppm, 0.001 to 300 ppm, 0.001 to 200 ppm, 0.001 to 125 ppm, 0.001 to 100 ppm, 0.001 to 90 ppm, 0.001 to 80 ppm, 0.001 to 70 ppm, 0.001 to 60 ppm, 0.001 to 50 ppm, 0.001 to 40 ppm, 0.001 to 30 ppm m, 0.003~1000ppm, 0.003~500ppm, 0.003~300ppm, 0.003~200ppm, 0.003~125ppm, 0.003~100ppm, 0.003~90ppm, 0.003~80ppm, 0.003~ 70ppm, 0.003~60ppm, 0.003~50ppm, 0.003~40ppm, 0.003~30ppm, 0.01~1000ppm, 0.01~500ppm, 0.01~300ppm, 0.01~200ppm, 0.01~125 ppm, 0.01~100ppm, 0.01~90ppm, 0.01~80ppm, 0.01~70ppm, 0.01~60ppm, 0.01~50ppm, 0.01~40ppm, 0.01~30ppm, 0.1~1000ppm, 0.1~50 0ppm, 0.1~300ppm, 0.1~200ppm, 0.1~125ppm, 0.1~100ppm, 0.1~90ppm, 0.1~80ppm, 0.1~70ppm, 0.1~60ppm, 0.1~50ppm, 0.1~40ppm, 0.1~30ppm, 1~1000ppm, 1~500ppm, 1~300ppm, 1~200ppm, 1~125ppm, 1~100ppm, 1~90ppm, 1~80ppm, 1~70ppm, 1~60ppm, 1~50ppm, 1~40ppm, 1~30ppm, 3~1000ppm, 3~500ppm, 3~300ppm, 3~200ppm, 3~125ppm, 3~100ppm, 3~90ppm, 3~80ppm, 3~70ppm, 3~60ppm, 3~50ppm, 3~40ppm, 3~30ppm, 5~1000pp It is found in concentrations of 5-500 ppm, 5-300 ppm, 5-200 ppm, 5-125 ppm, 5-100 ppm, 5-90 ppm, 5-80 ppm, 5-70 ppm, 5-60 ppm, 5-50 ppm, 5-40 ppm, 5-30 ppm, 10-1000 ppm, 10-500 ppm, 10-300 ppm, 10-200 ppm, 10-125 ppm, 10-100 ppm, 10-90 ppm, 10-80 ppm, 10-70 ppm, 10-60 ppm, 10-50 ppm, 10-40 ppm, or 10-30 ppm.

[0075] In one example, when the ginkgo biloba is included within the above range, the anticoccidial activity may be superior to that when the ginkgo biloba is included outside the above range.

[0076] In this application, "feed" can mean any natural or artificial diet, meal, etc., or components of said meal, intended or suitable for eating, ingesting, and digestion by an animal. Exemplary feed compositions additionally include concentrates and / or specialized 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, etc.; residual starches which are the main component of starch cakes which are the remainder after starch is removed from sweet potatoes, potatoes, etc.; fish meal, fish meal, fish soluble which is a concentrated fresh liquid obtained from fish; meat meal, blood meal, feather meal, skim milk powder, dried whey which is the residual liquid when producing cheese from milk or casein from skim milk; yeast, chlorella, and / or seaweed.

[0077] The term "feed composition" as used herein refers to a form of feed ultimately ingested by animals, a dietary supplement that can be incorporated into feed, and / or a feed additive. The term "dietary supplement" refers to, for example, a formulation-containing composition that provides a therapeutic agent or digestive aid to animals. It is not typically a source of calorie intake, i.e., energy, but is ingested in addition to regular animal feed. The term "feed additive" refers to a substance added to feed for various purposes, such as supplementing nutrients and preventing weight loss, increasing the digestibility of fiber in feed, improving milk quality, preventing reproductive disorders and conception rates, and preventing high-temperature stress in summer. In one example, the term "feed additive" refers to a substance added to feed for the purpose of preventing, ameliorating, or treating coccidiosis.

[0078] 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., compound feed and / or plain feed that is ultimately ingested by an animal), the amount of the feed additive may be 0.001% (w / w) or more, 0.005% (w / w) or more, 0.01% (w / w) or more, 0.05% (w / w) or more, 0.1% (w / w) or more, based on the total weight of the feed. w) or more, 0.5% (w / w) or more, 1% (w / w) or less, 0.5% (w / w) or less, 0.1% (w / w) or less, 0.05% (w / w) or less, 0.01% (w / w) or less, 0. 005%(w / w) or less, 0.001~1%(w / w), 0.001~0.5%(w / w), 0.001~0.1%(w / w), 0.001~0.05%(w / w), 0.00 1~0.01%(w / w), 0.001~0.005%(w / w), 0.005~1%(w / w), 0.005~0.5%(w / w), 0.005~0.1%(w / w), 0. 005~0.05%(w / w), 0.005~0.01%(w / w), 0.01~1%(w / w), 0.01~0.5%(w / w), 0.01~0.1%(w / w), 0.01 It may be added at a weight concentration of 0.05% (w / w), 0.05-1% (w / w), 0.05-0.5% (w / w), 0.05-0.1% (w / w), 0.1-1% (w / w), 0.1-0.5% (w / w), or 0.5-1% (w / w), and may be blended with feed ingredients, supplementary feed, supplements, and / or other types of additives other than the active ingredient(s) given as examples.

[0079] In one example, when the feed additive is contained within the above range, the anticoccidial activity may be superior to when the feed additive is contained outside the above range.

[0080] One aspect provides a pharmaceutical composition for preventing or treating coccidiosis, comprising as an active ingredient one or more (e.g., any one, two or more, three or more, or all) selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and pharmaceutically acceptable salts thereof.

[0081] Another embodiment can provide a pharmaceutical composition for preventing or treating coccidiosis, comprising ginkgo biloba leaves.

[0082] The pharmaceutical composition according to one embodiment may be used as a single agent, or may be prepared as a combined preparation by additionally including a pharmaceutical composition known to have an approved preventive or therapeutic effect against coccidiosis. A pharmaceutically acceptable carrier, excipient, or diluent may be added to the combined preparation to form a pharmaceutical unit dosage form.

[0083] In this application, "pharmaceutically acceptable" means that it does not significantly stimulate the living body and does not inhibit the biological activity and properties of the administered active substance. For example, the pharmaceutical composition containing a pharmaceutically acceptable carrier may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid contents, oils, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.

[0084] The pharmaceutical composition may be in various oral or parenteral dosage forms, and may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0085] 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, talc, etc. may also be used. Liquid preparations for oral administration include suspensions, liquid preparations, oils, syrups, etc., and include commonly used simple diluents such as water and liquid paraffin, as well as various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc.

[0086] Formulations for parenteral administration include sterile 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, cocoa butter, laurin butter, and glycerogelatin.

[0087] In one example, the pharmaceutical composition can be formulated into various forms, such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injection solutions, by a conventional method according to the intended purpose, and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

[0088] 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. The pharmaceutical composition may also include a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, etc.

[0089] In one example, the effective amount of the active ingredient (e.g., one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and pharmaceutically acceptable salts thereof) or ginkgo biloba 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, or 0.0001 to 0.1 mg / kg body weight. g, 0.0001~1mg / kg, 0.0001~10mg / kg, 0.0001~100mg / kg, 0.0001~250mg / 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, 0.001~100mg / kg, 0.001~250mg / kg, 0.001~500mg / kg, 0.00 1~1000mg / 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, 0.01~100 0mg / kg, 0.1~1mg / kg, 0.1~10mg / kg, 0.1~100mg / kg, 0.1~250mg / kg, 0.1~500mg / kg, 0.1~1000mg / kg, 1~10mg / kg, 1~100mg / k The dosage may be administered daily or every other day, or in one to three divided doses per day, at a dose of 1 to 3 mg / kg, 1 to 250 mg / kg, 1 to 500 mg / kg, 1 to 1,000 mg / kg, 10 to 100 mg / kg, 10 to 250 mg / kg, 10 to 500 mg / kg, 100 to 1,000 mg / kg, 250 to 500 mg / kg, 250 to 1,000 mg / kg, or 500 to 1,000 mg / kg. However, the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, body weight, age, etc., and the scope of the present application is not limited in any way by the dosage. In one example, when the composition is administered intraperitoneally, it may be administered at a concentration of 0.001 to 250 mg / kg.

[0090] In one example, the dosage of the pharmaceutical composition may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, and the like.

[0091] In one example, the concentration of the active ingredient (e.g., one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and pharmaceutically acceptable salts thereof) or ginkgo biloba in the pharmaceutical composition is as described above.

[0092] In one example, the pharmaceutical composition can be administered to a subject through various routes. The administration can refer to 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 any common route as long as it can reach the target tissue. For parenteral administration, topical application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and / or intrathoracic injection can be selected. Furthermore, the composition according to one example may be administered using any device capable of delivering an active ingredient to target cells.

[0093] One aspect provides a composition for antiprotozoal use against Eimeria protozoa, comprising as an active ingredient one or more (e.g., any one, two or more, three or more, or all) selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof.

[0094] Another embodiment provides a composition for use against protozoa of the genus Eimeria, which comprises ginkgo biloba leaves. The protozoa of the genus Eimeria are as described above.

[0095] In one example, the superior antiprotozoal activity (effect, efficacy) against Eimeria protozoa may refer to 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) have a high efficacy in killing Eimeria protozoa; and / or (2) Inhibition of the cell invasion effect of Eimeria protozoa and / or the proliferation effect of said protozoa within cells.

[0096] In one example, ginkgo biloba or one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof is included in the antiprotozoal composition in a concentration range as described above for the feed composition and / or pharmaceutical composition. In one example, a composition containing an active ingredient in a concentration range described above may have better antiprotozoal activity than a composition containing an active ingredient in a concentration range outside the above range.

[0097] 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 anti-protozoan composition) to an animal. In one example, the method may further include a step of identifying (screening) an individual (patient) in need of the prevention, amelioration, or treatment of coccidiosis prior to the step of administering the composition. The composition and coccidiosis are as described above. In one example, the step of identifying the individual may include a step of detecting oocysts of a protozoan capable of inducing coccidiosis from feces isolated from the individual.

[0098] 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.

[0099] According to one example, the composition can be administered in a pharmaceutically effective amount. In this application, a "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. According to one example, the composition can be administered as an individual therapeutic agent or in combination with other anticoccidial agents. It can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at the minimum dose without side effects, which can be easily determined by one skilled in the art.

[0100] In one example, the subject to which the method for preventing, ameliorating, or treating coccidiosis is applied refers to an animal that has or may develop 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.

[0101] In one example, the animal to which the method for preventing, ameliorating, or treating coccidiosis is applied may be one or more species selected from the group consisting of animals listed in Table 1, such as one or more species selected from the group consisting of humans, chickens, ducks, geese, turkeys, quails, pheasants, pigeons, parrots, cattle, 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 chub. In one example, the animal may be an animal other than humans.

[0102] Other embodiments include use of one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the manufacture of a composition (e.g., a feed composition, a pharmaceutical composition) or a composition for antiprotozoal use for the prevention, amelioration, and / or treatment of coccidiosis; use of one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the prevention, amelioration, and / or treatment of coccidiosis; and / or use of one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the prevention, amelioration, and / or treatment of coccidiosis; and / or use of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof for the prevention, amelioration, and / or treatment of coccidiosis. The present invention provides an antiprotozoal composition for use in the treatment of one or more Eimeria protozoa selected from the group consisting of ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof (e.g., killing Eimeria protozoa; and / or inhibiting cell invasion and / or proliferation of Eimeria protozoa).

[0103] Other embodiments provide the use of ginkgo biloba leaves for use in the manufacture of compositions (e.g., feed compositions, pharmaceutical compositions) or antiprotozoal compositions for preventing, ameliorating, and / or treating coccidiosis; the use of ginkgo biloba leaves for use in the prevention, amelioration, and / or treatment of coccidiosis; and / or the use of ginkgo biloba leaves for use in antiprotozoal treatment of Eimeria protozoa (e.g., killing Eimeria protozoa; and / or inhibiting cell invasion and / or proliferation of Eimeria protozoa).

[0104] In the above uses, the one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, and salts thereof, ginkgo biloba leaves, coccidiosis, antiprotozoa, and Eimeria protozoa are as described above. [Effects of the Invention]

[0105] In one example, a composition containing ginkgo leaves has excellent effects of directly killing protozoa that can induce coccidiosis, inhibiting the cell invasion of the protozoa, and / or inhibiting the proliferation of the protozoa within cells, and is therefore effective in preventing, ameliorating, and treating coccidiosis in vivo. [Brief explanation of the drawings]

[0106] [Figure 1] The anticoccidial index (ACI) after challenge with Eimeria tenella is shown, following treatment with ginkgo leaf powder and anticoccidial drugs (diclazuril, salinomycin). [Figure 2] The effects of treatment with ginkgo biloba powder and anticoccidial agents (diclazuril, salinomycin, and gallic acid) on the cell invasion and growth of Eimeria tenella are shown. DETAILED DESCRIPTION OF THE INVENTION

[0107] The present invention will be described in more detail below with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0108] Example 1. In vivo anticoccidial activity of ginkgo leaf powder

[0109] Example 1-1. Experimental facility An in vivo anticoccidial efficacy evaluation test was conducted at an animal testing facility in Gyeongsangnam-do, South Korea. One-day-old female Ross chickens were individually weighed and randomly assigned to groups for the experiment. The experimental design and conditions are listed in Table 2.

[0110] [Table 2]

[0111] The farm was managed in accordance with the Korean Poultry Farming Specifications Management Guidelines. Before the start of the study, the cages and farm were cleaned and disinfected. The farm temperature was maintained at 40-41°C and humidity at 40-50%, and was continuously monitored.

[0112] Example 1-2. Preparation of ginkgo leaf powder Ginkgo leaves were obtained from Jeongoksan Farm in Mitan-myeon, Pyeongchang-gun, Gangwon-do, and Jecheon Bank Farm in Susan-myeon, Jecheon-si, Chungcheongbuk-do. Green ginkgo leaves grown between July and September were dried in a 60°C dryer for 10 hours and then crushed in a 100-gauge powder crusher to produce ginkgo leaf powder, which was used in subsequent experiments.

[0113] Examples 1-3. Experimental design The feed used was Daehan Feed A1-First Feed Product, which was self-mixed by adding each ingredient (diclazuril (Yuhan DICLA product from Yuhan Industrial Co., Ltd.), salinomycin (Cheilbio Cheil Salino-60 product), gallic acid (Sigma product), and ginkgo leaf powder prepared in Example 1-2) to the feed at the concentrations listed in Table 3 below. No antibiotics or supplements were used in the general feed or the mixed feed, and no anticoccidial agents were added other than the ingredients. The chickens were given feed ad libitum throughout the experiment. Thirty one-day-old chickens were housed in cages randomly assigned to either the control or test group, and were fed the general feed (A1-First Feed Product) for 7 days, after which they were divided into the control group and the test group and given the prepared mixed feed.

[0114] The formulation of the feed administered to the control group (negative control group or positive control group) and test group, and whether or not coccidiosis was induced by Eimeria tenella, are shown in Table 3 below.

[0115] Coccidiosis was induced by oral gavage challenge of 14-day-old chickens with 90% or more mature (sporulated) Eimeria tenella oocysts (10,000 oocysts per chicken).

[0116] [Table 3]

[0117] Example 1-4. Measurement of anticoccidial activity of ginkgo leaf powder The anticoccidial efficacy of each test group designed in Examples 1 to 3 was expressed as an anticoccidial index (ACI), which was calculated using the following formula 2. The maximum ACI score is 200 points, and a higher ACI score indicates better anticoccidial activity. A score of 120 to 140 points is considered to be effective as an anticoccidial material, a score of 140 to 160 points is considered to be excellent as an anticoccidial material, and a score of 160 points or more is considered to have excellent anticoccidial effect (Luis Miguel De Pablos et al., Anticoccidial activity of maslinic acid against infection with Eimeria tenella in chickens, Parasitol Res, 2010).

[0118] (Number 2) Anticoccidial Comprehensive Index (ACI) = (post-challenge survival rate (%)) + (daily weight gain (RWG, %) compared to the negative control group) - (lesion score x 10) - (fecal oocyst output index).

[0119] 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 from causes other than coccidiosis was excluded. The survival rate (%) up to 8 days after challenge was used to calculate the overall anticoccidial index.

[0120] The survival rates for each control group and test group are shown in Table 4 below.

[0121] [Table 4]

[0122] Furthermore, the mortality rate of ginkgo leaf powder during the 4-week feeding period was 0%, and no lesions were found in the major organs (kidneys, spleen, liver, heart) at autopsy, ensuring safety at the 125 ppm level.

[0123] 2) Daily weight gain: Individuals were weighed in each cage before and 7 days after challenge with the protozoan, and the difference was divided by the number of days to calculate daily weight gain (ADG, g / d). The average daily gain (ADG, 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 "average daily gain relative to the negative control group (RWG, %)." This was used to calculate the overall anticoccidial index.

[0124] The daily weight gain (ADG, g / d) measured for each control group and test group and the daily weight gain (RWG, %) compared to the negative control group are shown in Table 5 below.

[0125] [Table 5]

[0126] 3) Lesion Scoring: Eight days after challenge, four chickens per cage were necropsied, and the intestines were dissected open. The cecal area of ​​each chicken was scored for coccidial lesions. The lesion scoring method was based on 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 measured cecal lesion score was multiplied by 10 to calculate a lesion index, which was used to calculate the overall anticoccidial index.

[0127] The cecal lesion scores measured in each control group and test group are shown in Table 6 below.

[0128] [Table 6]

[0129] 4) Fecal oocyst output: On days 6-8 after challenge, all feces were collected from each cage and mixed evenly. 1g of feces was randomly sampled three times. Oocysts in 1g of feces were floated in saline, and the amount of oocysts output was measured using a McMaster chamber. The results are shown in Table 7.

[0130] [Table 7]

[0131] The oocyst output of each group was divided by the oocyst output of the infected negative control group and multiplied by 100 to calculate the oocyst output (%) relative to the infected negative control group. The oocyst output index was calculated as follows: 0 if the calculated oocyst output relative to 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.

[0132] The anticoccidial comprehensive index for each test group measured by Equation 2 as described above is shown in Table 8. As shown in Table 8, the anticoccidial comprehensive index for the ginkgo leaf powder intake group was higher than that of the positive control groups, diclazuril or salinomycin intake groups, indicating excellent anticoccidial efficacy.

[0133] [Table 8]

[0134] As can be seen from Tables 4 to 7, the infected negative control group challenged with Eimeria tenella developed coccidiosis, had reduced survival rates and weight gain, and had increased lesion index and fecal oocyst output compared to the uninfected negative control group. The salinomycin and diclazuril groups, used as anticoccidial controls, had increased survival rates and weight gain, and reduced lesion index and fecal oocyst output compared to the infected negative control group. The ginkgo biloba powder group had improved survival rates and weight gain, and reduced lesion index and fecal oocyst output compared to the infected negative control group. This was a more effective treatment than salinomycin 60 ppm and diclazuril 1 ppm.

[0135] As described above, the anticoccidial comprehensive index for each test group measured by Equation 2 is shown in Figure 1 and Table 8. As shown in Figure 1 and Table 8, the anticoccidial comprehensive index for the ginkgo leaf powder intake group was higher than that of the positive control groups, diclazuril or salinomycin intake groups, indicating excellent anticoccidial efficacy.

[0136] Example 2. Direct killing effect of ginkgo leaf powder on Eimeria protozoa In this example, direct killing ability of protozoa (sporozoites) was evaluated for three representative Eimeria species (E. tenella, E. acervulina, and E. maxima) known to infect the majority of farms.

[0137] A fixed amount of oocysts from each protozoan was placed in a tube containing glass beads and crushed. To remove the oocyst cell walls and other debris, the inner sporocysts were purified using a Percoll density gradient and washed with PBS. To excyst the inner sporozoites, sporangia from E. tenella, E. acervulina, and E. maxima were treated with a reagent containing sodium taurocholic acid (Sigma Aldrich, USA) and trypsin (Gibco, USA), incubated, and then washed once with PBS to obtain the protozoan.

[0138] Ginkgo biloba powder and the anticoccidial agents salinomycin, diclazuril, and gallic acid (hereinafter referred to as "materials") were reacted with three Eimeria species at various concentrations ranging from 1 to 500 ppm, and live protozoa (sporozoites) were counted under a microscope. The protozoan mortality rate (%) for each material treatment compared to the negative control group treated with PBS was measured, and the minimum concentration that directly killed 50% of the protozoa is shown in Table 9 below.

[0139] [Table 9]

[0140] As shown in Table 9, the gallic acid treatment group did not show a 50% direct killing effect against protozoa capable of inducing coccidiosis up to 500 ppm, and diclazuril also did not show a 50% direct killing effect against E. acervulina and E. maxima up to 500 ppm. The ginkgo leaf powder (green leaf, yellow leaf) treatment group was able to kill E. tenella, E. acervulina, and E. maxima by 50% even at a significantly lower concentration than the other groups, confirming its significantly superior protozoan killing effect.

[0141] Example 3. Inhibitory effect of ginkgo biloba powder on cell invasion and intracellular proliferation of Eimeria protozoa In this example, the MDBK cell line, a representative animal cell line known to be infected and proliferated by Eimeria, was used to examine the ability of ginkgo biloba powder to inhibit protozoan invasion and proliferation within cells.

[0142] Example 3-1. Inhibitory effect of protozoan cell invasion by ginkgo leaf powder treatment MDBK cells (purchased from ATCC) were seeded at 100,000 cells per well into a 24-well plate and incubated at 37°C for 12 hours. Eimeria tenella parasites were cultured in a similar manner to Example 2. 200,000 parasites were added to each well. The cells were then treated with various concentrations (0.5–10 ppm) of each substance (ginkgo biloba powder and the anticoccidial agents salinomycin, diclazuril, and gallic acid) and cultured at 41°C for 24 hours. The negative control group consisted of MDBK cells infected with Eimeria tenella parasites, while the positive control group consisted of cells incubated with salinomycin, diclazuril, or gallic acid solution. The cells were then washed twice with PBS to remove any parasites that had not invaded the cells. After separating the cells from the parasites contained within them by pipetting, DNA was extracted from the cells and subjected to PCR using primers specific to the E. tenella ITS-1 (Internal transcribed spacer-1) gene. The primer sequences used are listed in Table 10 below.

[0143] [Table 10]

[0144] The Ct values ​​before and after washing were compared for each material, corrected for the ΔCt value of the negative control group, and the inhibition rate (%) of protozoan cell invasion by each material treatment was calculated. The results are shown in Figure 2 and Table 11 below.

[0145] Example 3-2. Inhibitory effect of ginkgo leaf powder treatment on intracellular proliferation of protozoa 100,000 MDBK cells (purchased from ATCC) were dispensed into a 24-well plate and incubated at 37°C for 12 hours. Eimeria tenella protozoa were cultured in a similar manner to Example 2. 200,000 protozoa were added to each well and incubated at 41°C for 24 hours. The cells were washed twice with PBS to remove any unattached protozoa. The cells were treated with various concentrations of ginkgo biloba powder and the anticoccidial agents salinomycin, diclazuril, and gallic acid, and then incubated for an additional 24 hours at 41°C. The negative control group consisted of MDBK cells infected with the protozoa, and the positive control group consisted of a group incubated with Eimeria tenella protozoa in a salinomycin and diclazuril solution. After separating the cells from the protozoa grown within them by pipetting, DNA was extracted from the cells and subjected to PCR using primers specific to the E. tenella ITS-1 (internal transcribed spacer-1) gene. The primer sequences used are listed in Table 10.

[0146] The Ct values ​​of the material-treated group were compared with those of the negative control group, and the inhibition rate (%) of protozoan cell invasion and the inhibition rate (%) of intracellular protozoan proliferation due to material treatment were calculated. The results are shown in Figure 2 and Table 11 below.

[0147] [Table 11]

[0148] As shown in Figure 2 and Table 11, the inhibitory effects of Eimeria tenella cell invasion and intracellular proliferation were examined by material. Ginkgo biloba powder inhibited Eimeria tenella cell invasion and intracellular proliferation by more than 40% at both 5 ppm and 10 ppm. Ginkgo biloba powder at 10 ppm was superior to salinomycin and diclazuril in inhibiting Eimeria tenella cell invasion and intracellular proliferation. Compared to gallic acid, its inhibitory effect on protozoan cell invasion was lower, but its inhibitory effect on intracellular proliferation was significantly greater. While commercially available anticoccidials, diclazuril and gallic acid, only exhibited one of the effects of inhibiting protozoan cell invasion or intracellular proliferation, respectively, salinomycin and ginkgo biloba powder were both effective. Ginkgo biloba powder, in particular, exhibited a more effective inhibitory effect on intracellular protozoan proliferation than salinomycin.

[0149] Example 4. Evaluation of anticoccidial efficacy of each component of ginkgo leaf powder To identify which of the components of ginkgo biloba powder exhibited anticoccidial activity, ginkgolide A, ginkgolide B, ginkgolide C, quercetin, kaempferol, bilobalide, or ginkgo biloba powder was treated and the mortality rate of the Eimeria acervulina protozoan was evaluated. The concentration of each component was selected to be 5 to 500 ppm, and the mortality rate (%) of the protozoan was measured using the same method as in Example 2. The results are shown in Table 12 below.

[0150] [Table 12]

[0151] As can be seen from Table 12, ginkgo leaf powder, similar to the results of Example 2, killed 50% of Eimeria acervulina protozoa at concentrations of 16 ppm or higher and 100% at concentrations of 63 ppm or higher. Ginkgolide B and ginkgolide C demonstrated Eimeria acervulina protozoa killing ability at concentrations of 125 ppm or higher. Ginkgolide A demonstrated Eimeria acervulina protozoa killing ability at concentrations of 31 ppm or higher. Bilobalide and quercetin demonstrated Eimeria acervulina protozoa killing ability at concentrations of 63 ppm or higher, with a mortality rate of 50% or higher at 500 ppm or higher. Kaempferol demonstrated protozoa killing ability at concentrations of 125 ppm or higher, with a mortality rate of 50% or higher at 500 ppm or higher.

[0152] Example 5. Evaluation of acid resistance of ginkgo leaf powder In this example, the acid resistance of ginkgo leaves was evaluated. The ginkgo leaf powder prepared in Examples 1-2 was added with hydrochloric acid (HCl) solution to adjust the pH to 2.0, 2.5, or 3.0, and then allowed to stand at 40°C for 1 hour. Sodium hydroxide (NaOH) solution was then added to neutralize the solution (pH 7.0). 200,000 Eimeria maxima protozoa (sporozoites) were then exposed to various concentrations of ginkgo leaf powder solutions (10-500 ppm) diluted with PBS solution and incubated at 41°C for 4 hours. The protozoan mortality rate (%) was then measured using a method similar to Example 2, and the results are shown in Table 13 below. In Table 13, "protozoa alone" refers to the group not treated with ginkgo leaf powder, and "control" refers to the group treated with ginkgo leaf powder without treatment with hydrochloric acid. As shown in Table 13, it was confirmed that ginkgo leaf powder's killing effect on protozoa that cause coccidiosis was not lost even under strongly acidic conditions.

[0153] [Table 13]

[0154] Example 6. Evaluation of heat resistance of ginkgo leaf powder In this example, the heat resistance of ginkgo biloba was evaluated. The ginkgo biloba powder prepared in Examples 1-2 was exposed to temperatures ranging from 85 to 95°C for 15 minutes, cooled, and then diluted with PBS solution to concentrations of 1, 10, 50, and 100 ppm. 200,000 Eimeria acervulina protozoa were exposed to ginkgo biloba powder at various concentrations ranging from 1 to 100 ppm for 4 hours at 41°C. The protozoa mortality rate (%) was measured using a method similar to that in Example 2, and the results are shown in Table 14. In Table 14, "protozoa alone" refers to a group not treated with ginkgo biloba powder, and "control" refers to a group treated with ginkgo biloba powder but not exposed to high temperatures.

[0155] As shown in Table 14, it was confirmed that ginkgo leaf powder maintained its killing effect on the protozoa that cause coccidiosis even under adverse conditions of high temperature.

[0156] [Table 14]

[0157] From the above description, those 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 the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts thereof.

Claims

1. A feed composition for preventing or ameliorating coccidiosis, comprising, as an active ingredient, one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, kaempferol, bilobalide, and salts thereof.

2. The feed composition according to claim 1, further comprising quercetin or a salt thereof as an active ingredient.

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

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 decrease in one or more selected from the group consisting of lesion score, fecal oocyst output, 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, the proliferation of the protozoa within the 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 5, which is a feed additive. thing.

7. A pharmaceutical composition for preventing or treating coccidiosis, comprising as an active ingredient one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, kaempferol, bilobalide, and pharmaceutically acceptable salts thereof.

8. The pharmaceutical composition according to claim 7, further comprising quercetin or a salt thereof as an active ingredient.

9. A composition for antiprotozoal use against Eimeria protozoa, comprising as an active ingredient one or more selected from the group consisting of ginkgolide A, ginkgolide B, ginkgolide C, kaempferol, bilobalide, and salts thereof.

10. The antiprotozoan composition according to claim 9, further comprising quercetin or a salt thereof as an active ingredient.

11. 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 6; the pharmaceutical composition according to claim 7 or 8; and the composition for antiprotozoan disease according to claim 9 or 10.

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