Anti-coccidial compositions comprising mangosteen and uses thereof

Abstract

The present application relates to an anticoccidial composition comprising Garcinia mangostana and uses thereof. According to embodiments, the composition comprising Garcinia mangostana has an excellent effect of directly killing protozoa that can induce coccidiosis, has an effect of inhibiting penetration of cells by the protozoa, and / or has an effect of inhibiting proliferation of the protozoa in cells, and has an excellent effect on prevention, alleviation, and treatment of coccidiosis in vivo.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0125245, filed on September 25, 2020, and the entire contents disclosed in the corresponding Korean patent application are incorporated in this specification.

[0003] This application relates to an anticoccidial composition comprising mangosteen and its use therein. Background Technology

[0004] Coccidiosis is an intestinal-associated disease caused by a protozoan parasite called *Eimeria*, belonging to the phylum *Apicomplexan*. Infection with coccidiosis causes digestive disorders, diarrhea, and weight loss, and can also lead to livestock death. Therefore, coccidiosis has a significant economic impact on farms 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).

[0005] In recent years, many researchers have developed anticoccidial agents as therapeutics for coccidiosis. These agents, such as ionocarriers or chemically synthesized compounds, can prevent the formation of oocyst cell walls or asexual and sexual reproduction in protozoa. However, due to the long-term use of shuttle programs that alternately treat the resulting ionocarriers and chemically synthesized compounds, side effects such as the emergence of drug-resistant protozoa have occurred.

[0006] In particular, the accumulation of antibiotics in animals due to misuse and overuse is a serious problem, as humans ingest antibiotics through meat. Consequently, many countries around the world are banning the use of antibiotics due to antibiotic residues in livestock products. Therefore, there is an urgent need to develop and research alternatives to conventional anticoccidial agents, which exhibit side effects such as the emergence of resistant strains and internal residues.

[0007] Existing technology

[0008] (Patent Document 1) US Patent Publication No. 2008-0160000 Summary of the Invention

[0009] Technical issues

[0010] One embodiment of this application provides a feed composition for the prevention or relief of coccidiosis, comprising mangosteen.

[0011] Another embodiment of this application provides a pharmaceutical composition for the prevention or relief of coccidiosis, comprising mangosteen.

[0012] Other embodiments of this application provide an antiprotozoan composition against Eimeria protozoa, comprising mangosteen.

[0013] Other embodiments of this application provide a method for preventing, alleviating, or treating coccidiosis, including the step of administering the composition to an animal.

[0014] Other embodiments of this application provide the use of mangosteen in the preparation of compositions (e.g., feed compositions, pharmaceutical compositions) for the prevention, relief and / or treatment of coccidiosis or in the preparation of antiprotozoan compositions; the use of mangosteen for the prevention, relief and / or treatment of coccidiosis; and / or the use of mangosteen for antiprotozoan activities against Eimeria protozoa (e.g., killing Eimeria protozoa; and / or inhibiting cell invasion and / or proliferation of Eimeria protozoa).

[0015] Technical solution

[0016] According to one embodiment, a composition containing mangosteen can exhibit excellent anticoccidial activity and / or antiprotozoan activity against protozoa that induce coccidiosis.

[0017] In this application, superior anticoccidiosis efficacy (activity, effect) may refer to at least one selected from the group consisting of (1) to (5) below (e.g., any one, two or more, three or more, or all of them):

[0018] (1) Higher anticoccosis index (ACI) compared with the control group;

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

[0020] (3) It inhibits weight loss by inducing coccidiosis;

[0021] (4) Compared with the control group, it showed higher insecticidal activity against protozoa that induce coccidiosis; and

[0022] (5) Compared with the control group, the protozoa that induce coccidiosis showed a greater inhibitory effect on cell invasion and / or a greater inhibitory effect on cell proliferation of the protozoa.

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

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

[0025] (1) Excellent anticoccidial activity;

[0026] (2) Excellent anti-antiprotozoan activity against protozoa that induce coccidiosis;

[0027] (3) Excellent acid resistance;

[0028] (4) Excellent heat resistance;

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

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

[0031] The composition according to one embodiment has excellent acid and / or heat resistance, thereby maintaining excellent anticoccidial activity for a long time when administered to the body, and has in vivo stability, thereby maintaining excellent anticoccidial activity even in environments with a variety of temperatures and / or a variety of pH ranges, and is suitable for a variety of products (e.g., feed additives), and its storage stability can be excellent.

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

[0033] In one embodiment, the superior weight gain improvement effect can refer to the superior increase in the subject's weight when the subject is administered the drug, and in one embodiment, the weight gain can refer to daily weight gain, and the subject can be a subject in which coccidiosis has been induced in their body.

[0034] The composition according to one embodiment exhibits anticoccidioidomycin activity equivalent to or higher than conventionally known anticoccidial agents (e.g., sulfonamides such as sulfaquinoxaline, sulfachlorpyrifos and sulfadiazine, polyether ionocarrier antibiotics such as salinomycin and monensin sodium, amprolium, diclazuril, gallic acid and / or toltrazuril) without causing side effects or drug resistance, and can be safe for prolonged use because the composition contains a low-toxicity natural product (mangosteen).

[0035] In this application, "prevention" refers to all actions that inhibit or delay the development of a disease by administering a composition according to one embodiment; "treatment" refers to all actions that improve or beneficially alter the symptoms of a suspected and diseased subject by administering a composition according to one embodiment; and "relief" refers to all actions that reduce parameters related to the condition of treating a disease by administering a composition according to one embodiment, such as, at least, the severity of symptoms. The disease may refer to coccidiosis.

[0036] On the one hand, a feed composition containing mangosteen can be provided for the prevention or mitigation of coccidiosis.

[0037] According to one embodiment, the composition may contain mangosteen as an active ingredient exhibiting anticoccidial activity, and the mangosteen may be the fruit of the mangosteen plant. Mangosteen (Garcinia mangostana L.) is a tall, evergreen tree belonging to the Staphyleaceae family of the Soapberry order. Native to Malaysia, it produces fruits slightly larger than a flat ping-pong ball. Mangosteen fruit contains the representative antioxidant xanthones, and is therefore known to have excellent antioxidant properties. It is also known to have long been used in Thailand and Southeast Asia as a folk remedy for treating wounds such as inflammation or cuts.

[0038] According to one embodiment, mangosteen can be the pericarp (the outer pericarp of the mangosteen fruit), mangosteen pulp, or a combination thereof (pericarp and pulp), and according to one embodiment, compositions containing mangosteen pericarp can have excellent anticoccidial activity. Mangosteen pericarp is a part discarded due to abnormal ingestion, and compositions utilizing this mangosteen pericarp according to one embodiment have advantages in terms of the reuse of insoluble resources, environmental protection, and / or economic feasibility.

[0039] The composition according to one embodiment can have superior anticoccidial activity compared to the individual components contained in mangosteen (e.g., xanthones, alpha-mangostin, epicatechin, and / or quercetin).

[0040] In one embodiment, mangosteen may be at least one selected from the group consisting of mangosteen raw materials, dried materials, powdered materials and extracts (e.g., one or more, two or more, three or more or all).

[0041] Raw mangosteen can refer to unprocessed mangosteen (e.g., dried, ground into powder, and / or extracted).

[0042] Mangosteen dried material can be obtained by drying mangosteen raw materials, and can be obtained by methods such as vacuum drying, fluidized bed drying, room temperature drying and / or freeze drying.

[0043] Mangosteen powder can be obtained by grinding and / or pulverizing raw mangosteen and / or dried mangosteen material, and can be obtained using a pulverizer.

[0044] In this application, "extract" refers to a preparation obtained by pressing a herb containing a suitable extract and concentrating the extract by evaporation. It can be an extract obtained through a drying extraction process, a diluted solution or concentrate of the extract, or a dried product obtained from its preparation materials and / or purification materials. Mangosteen raw material can be prepared using commonly known extraction, separation, and purification methods in the art. Specifically, extraction methods such as boiling water extraction, hot water extraction, floral fragrance extraction, reflux cooling extraction, or ultrasonic extraction can be used.

[0045] In one embodiment, the mangosteen extract can be commercially available or prepared using extraction methods and solvents known in the pharmaceutical or food industries. For example, conventional extraction methods may include solvent extraction, ultrasonic extraction, filtration, and / or reflux extraction. In one embodiment, the mangosteen extract can be prepared by extraction with an extraction solvent or by fractionation by adding a fractionating solvent to an extract prepared by extraction with an extraction solvent. For example, the mangosteen extract can be extracted using an extraction solvent selected from the group consisting of water, straight-chain or branched alcohols having 1 to 4 carbon atoms, propylene glycol, butanediol, glycerol, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, and mixtures thereof. Specifically, the alcohol having 1 to 4 carbon atoms may be ethanol, methanol, isopropanol, propanol, butanol, and / or tert-butanol. Extraction can be performed by adding an extraction solvent in amounts ranging from 1 to 10 times the weight of the dried mangosteen, specifically 2 to 3 times the weight of the dried mangosteen. The extraction temperature can be 30°C–100°C, 50°C–90°C, or 60°C–80°C. Furthermore, the extraction time can be 10 to 48 hours, 12 to 30 hours, or 18 to 24 hours. Additionally, extraction can be repeated 1 to 5 times, 2 times, or 4 times.

[0046] In one embodiment, mangosteen can be an extract of mangosteen pericarp, for example, an ethanol extract of mangosteen pericarp, wherein the ethanol can be 50%–100% (v / v), 60%–100% (v / v), 65%–100% (v / v), 70%–100% (v / v), 75%–100% (v / v), 80%–100% (v / v), 50%–95% (v / v), 60%–95% (v / v), 65%–95% (v / v), 70%–95% (v / v), 75%–95% (v / v), 80%–95% (v / v), 50%–90% (v / v), 60%–90% (v / v), or 65%–90% (v / v). (v / v), 70% to 90% (v / v), 75% to 90% (v / v), 80% to 90% (v / v), 50% to 85% (v / v), 60 %~85%(v / v), 65%~85%(v / v), 70%~85%(v / v), 75%~85%(v / v), 80%~85%(v / v), 50% to 80% (v / v), 60% to 80% (v / v), 65% to 80% (v / v), 70% to 80% (v / v), 75% to 8 0% (v / v), 50% to 75% (v / v), 60% to 75% (v / v), 65% to 75% (v / v) or 70% to 75% (v / v).

[0047] In this application, "coccidiosis" refers to a disease caused by coccidia (protozoa that can induce coccidiosis, such as Eimeria sp.) parasitizing the cytoplasm of the submucosal tissue of the digestive tract epithelium and damaging the epithelium, thus causing enteritis. It is also a protozoan disease that causes economic losses due to weight gain and regression caused by soft stools, diarrhea, and bloody stools, leading to prolonged market age in broiler farms. Coccidiosis can occur not only in broilers but also in birds and mammals. Specifically, it can infect cattle, rabbits, goats, dogs, cats, and laboratory animals such as mice and rats. Particularly, it can cause fatal harm to poultry such as chickens. In one embodiment, coccidiosis may include acute coccidiosis, subacute coccidiosis, and chronic coccidiosis. Acute coccidiosis can manifest as bloody stools, energy loss, and anemia within 48 hours of infection, and infected subjects may die. Subacute coccidiosis can manifest as hemorrhagic diarrhea and / or anemia after infection. Chronic coccidiosis can manifest as loose stools and / or weight loss after diarrhea within 1 to 2 days after infection.

[0048] When the oocysts (cysts) of coccidial protozoan species mature into sporulated oocysts under high humidity and high temperature, the oocysts become infectious. They are easily transmitted and their life cycle is repeatedly repeated when excreted in feces after a certain life cycle within the body of a subject. It is known that the oocytes (cysts) of coccidial protozoan species are highly resistant to the external environment, with the cyst wall consisting of an inner and outer layer. The outer layer of the cyst wall is a gelatinous substance that strongly resists external physical stress, while the inner layer is rich in nucleoproteins, thus strongly resisting chemical irritants such as disinfectants. The oocysts of coccidial protozoan species can contain four sporangia, each containing two sporozoites. After infecting an animal in the form of oocysts, these sporozoites are released as sporangia and sporozoites, proliferate within the cell, and, through sexual and / or asexual reproduction, form oocysts, which are then extracted in feces. In one embodiment, the sporozoite can be used in the same sense as the protozoan, and the sporozoite (protozoan) can cause lesions.

[0049] According to one embodiment, coccidiosis can be caused by protozoa of the genus *Eimeria*. In one embodiment, the protozoa of the genus *Eimeria* can be at least one selected from the group consisting of: *Eimeria acervulina*, *Eimeria tenella*, *Eimeria maxima*, *Eimeria necatrix*, *Eimeria brunetti*, *Eimeria hagani*, *Eimeria mitis*, *Eimeria praecox*, *Eimeria mivati*, *Eimeria aurati*, *Eimeria baueri*, *Eimeria lepidosirenis*, and *Eimeria lepidosirenis*. Eimeria leucisci, Eimeria rutile, Eimeria vanasi, Eimeria amphisbaeniarum, Eimeria witchery, Eimeria yemenensae, Eimeria adenoeides, Eimeria colchici, Eimeria curvata, Eimeria dispersa, Eimeria duodenalis, Eimeria fraterculae, Eimeria gallopavonis, Eimeria innocua, Eimeria meleagridis, Eimeria meleagridis, Eimeria meleagridis Eimeria meleagrimitis, Eimeria phasiani, Eimeria procera, Eimeria purpureicephali, Eimeria ahsata, Eimeria alabamensis, Eimeria alièviiEimeria alijevi, Eimeria aspheronica, Eimeria arloingi, Eimeria arundeli, Eimeria bakuensis, Eimeria bovis, Eimeria cameli, Eimeria caprina, Eimeria caprovina, Eimeria christenseni, Eimeria clethrionomyis, Eimeria coecicola, Eimeria contorta, Eimeria couesii, Eimeria crandallis, Eimeria dammar Eimeria dowleri, Eimeria exigua, Eimeria falciformis, Eimeria farasanii, Eimeria ferrisi, Eimeria flavescens, Eimeria gallatii, Eimeria granulosa, Eimeria hirci, Eimeria intestinalis, Eimeria irresidua, Eimeria intricata, Eimeria jolchijevi, Eimeria krijgsmanni, Eimeria larimersonensis Eimeria larimerensis, Eimeria macusaniensis, Eimeria magna, Eimeria marconii, Eimeria media, Eimeria melanuri, Eimeria misiEimeria myoxi, Eimeria agpurensis, Eimeria nieschulzi, Eimeria ninakohlyakimovae, Eimeria ovinoidalis, Eimeria pallida, Eimeria palustris, Eimeria papillata, Eimeria perforans, Eimeria phocae, Eimeria pileata, Eimeria pipistrellus, Eimeria piriformis, Eimeria prionotemni, Eimeria prosioni The species include *Eimeria procyonis*, *Eimeria punctate*, *Eimeria roobroucki*, *Eimeria saudiensis*, *Eimeria sealanderi*, *Eimeria separata*, *Eimeria stiedae*, *Eimeria ursini*, *Eimeria vermiformis*, *Eimeria weybridgensis*, *Eimeria wobati*, and *Eimeria zuernii*.

[0050] The composition according to one embodiment can have excellent effects in preventing, alleviating and / or treating coccidiosis caused by at least one (e.g., one or more, two or more, three or more or four or more) protozoa selected from the group consisting of the Eimeria genus protozoa described in Table 1 below, which can cause coccidiosis in the animals described in Table 1 respectively.

[0051] Table 1

[0052]

[0053]

[0054]

[0055]

[0056] The composition according to one embodiment can be highly effective in preventing, alleviating and / or treating coccidiosis caused by Eimeria tenella, Eimeria spp. and / or Eimeria gianti.

[0057] In one implementation, prevention or mitigation of coccidiosis may refer to at least one of the following groups (e.g., any one, two or more, three or more, or all of them) selected from (1) to (4) below, and for example, compared with the control group (negative control group and / or positive control group), at least one of the following groups selected from (1) to (4) below may be reduced, suppressed, and / or increased:

[0058] (1) Reduce at least one of the groups consisting of disease score (e.g., appendiceal disease score), fecal oocyst excretion and mortality;

[0059] (2) Inhibit weight loss caused by coccidiosis;

[0060] (3) Increase the anticoccidial index (ACI); and

[0061] (4) Reduce cell invasion of Eimeria protozoa, proliferation of protozoa in cells, or both.

[0062] In one embodiment, the lesion scoring method for determining the lesion score can be based on the literature of Johnson JK and Reid WM (1970) (Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens, Experimental parasitology, 1970), and the lesion score can be from 0 to 4 degrees. In one embodiment, the lesion score can refer to the lesion score measured in the appendix, duodenum, and / or jejunum, and it can be calculated by summing the lesion scores measured in each organ (appendix, duodenum, and / or jejunum).

[0063] In one implementation, the amount of fecal ovules extracted can be measured by collecting feces from the subject and using a microscope or a counting chamber (e.g., a McMaster chamber).

[0064] In one implementation, the mortality rate may refer to the mortality rate of animal subjects who have been induced with coccidiosis, and the number of subjects who died from causes other than coccidiosis can be excluded by performing an autopsy.

[0065] In one embodiment, the weight of a subject induced with coccidiosis may be less than that of a subject without coccidiosis, and the composition according to one embodiment can inhibit the weight loss caused by coccidiosis induction.

[0066] In one embodiment, the anticoccidial index can be calculated according to Equation 1 below, and in Equation 1, the lesion score can be calculated according to the aforementioned method.

[0067] (Equation 1)

[0068] Anticoccidial Index (ACI) = (Survival rate after challenge inoculation (%)) + (Daily weight gain compared to negative control group (%)) - (Lesion score × 10) - (Fecal oocyst excretion index)

[0069] The challenge inoculation can refer to the administration (e.g., oral inoculation) of a protozoan capable of inducing coccidiosis. In one embodiment, the survival rate can be the survival rate measured on days 5–10, 7–10, 8–10, 7–9, 7–8, or 7 after the challenge inoculation, and can be measured by excluding the number of subjects who died from causes other than coccidiosis through autopsy.

[0070] The weight gain in Equation 1 above compared to the negative control group can be a value calculated as a percentage based on the values ​​of the negative control group (e.g., a negative control group of uninfected protozoa).

[0071] The lesion score in Equation 1 above is as described above.

[0072] In Equation 1 above, the percentage is calculated based on the values ​​of the negative control group (e.g., the negative control group of infected protozoa). When the calculated value is above 0% and below 1%, the fecal oocyst excretion index can be 0; when the calculated value is above 1% and below 26%, the fecal oocyst excretion index can be 5; when the calculated value is above 26% and below 51%, the fecal oocyst excretion index can be 10; when the calculated value is above 51% and below 76%, the fecal oocyst excretion index can be 20; and when the calculated value is above 76% and below 100%, the fecal oocyst excretion index can be 40.

[0073] In one embodiment, the mangosteen may be included in the feed composition in the following amounts: less than 1 w / w%, less than 1 w / w%, 10 -1 w / w% or less, 5×10 -2 w / w% or less, 2.5×10 -2 w / w% or less, 2×10 -2 w / w% or less, 1.25×10 -2 w / w% or less, 10 -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, 1.5×10 -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 w / w%~1w / w%, 10 -7 w / w% ~10 -1 w / w%, 10 -7 w / w% ~ 5 × 10 -2 w / w%, 10 -7 w / w% ~10 -2 w / w%, 10 -7 w / w% ~ 5 × 10 -3 w / w%, 10 -7 w / w% ~ 4 × 10 -3 w / w%, 10 -7 w / w% ~ 10 -3 w / w%, 10 -7 w / w% ~ 5 × 10 - 4 w / w%, 10 -7 w / w% ~ 10 -4 w / w%, 10 -7 w / w% ~ 10 -5 w / w%, 10-6 in / in%~1in / in%、10 -6 w / w%~10 - 1 in / in%、10 -6 w / w%~5×10 -2 in / in%、10 -6 w / w%~10 -2 in / in%、10 -6 w / w%~5×10 -3 in / in%、10 -6 w / w%~4×10 -3 in / in%、10 -6 w / w%~10 -3 in / in%、10 -6 w / w%~5×10 -4 in / in%、10 -6 w / w%~10 -4 in / in%、10 -6 w / w%~10 -5 in / in%、10 -5 in / in%~1in / in%、10 -5 w / w%~10 -1 in / in%、10 -5 w / w%~5×10 - 2 in / in%、10 -5 w / w%~10 -2 in / in%、10 -5 w / w%~5×10 -3 in / in%、10 -5 w / w%~4×10 -3 in / in%、10 -5 w / w%~10 -3 in / in%、10 -5 w / w%~5×10 -4 in / in%、10 -5 w / w%~10 -4 in / in%、10 -4 in / in%~1in / in%、10 - 4 w / w%~10 -1 in / in%、10 -4 w / w%~5×10 -2 in / in%、10 -4 w / w%~10 -2 in / in%、10 -4 w / w%~5×10 - 3 in / in%、10 -4w / w% ~ 4 × 10 -3 w / w%, 10 -4 w / w% ~ 10 -3 w / w%, 10 -4 w / w% ~ 5 × 10 -4 w / w%, 10 -3 w / w%~1w / w%, 10 -3 w / w% ~ 10 -1 w / w%, 10 -3 w / w% ~ 5 × 10 -2 w / w%, 10 -3 w / w% ~ 10 -2 w / w%, 10 - 3 w / w% ~ 5 × 10 -3 w / w%, 10 -3 w / w% ~ 4 × 10 -3 w / w%, 10 -3 w / w% ~ 2×10 -3 w / w% or 10 -3 w / w% ~ 1.5 × 10 -3 w / w%. In one embodiment, the feed composition may be a feed containing an active ingredient within a range based on total weight (e.g., a mixed feed and / or compound feed ultimately ingested by the animal).

[0074] In one embodiment, the mangosteen (e.g., mangosteen extract) may be included in the feed composition at the following concentrations: less than 1000 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 250 ppm, less than 200 ppm, less than 125 ppm, less than 125 ppm, less than 100 ppm, less than 90 ppm, less than 80 ppm, less than 70 ppm, less than 65 ppm, less than 62.5 ppm, less than 60 ppm, less than 50 ppm, more than 0.1 ppm, more than 1 ppm, more than 5 ppm, more than 10 ppm, more than 15 ppm, more than 20 ppm, more than 30 ppm, more than 40 ppm, and more than 50 ppm. Above, 62.5ppm and above, 0.1ppm~1000ppm, 0.1ppm~500ppm, 0.1ppm~400ppm, 0.1ppm~300ppm, 0.1ppm~250ppm, 0.1ppm~200ppm, 0.1ppm~125ppm, 0.1ppm~100ppm , 0.1ppm~90ppm, 0.1ppm~80ppm, 0.1ppm~70ppm, 0.1ppm~65ppm, 0.1ppm~60ppm, 0.1ppm~50ppm, 1ppm~1000ppm, 1ppm~500ppm, 1ppm~400ppm, 1ppm~300p pm, 1ppm~250ppm, 1ppm~200ppm, 1ppm~125ppm, 1ppm~100ppm, 1ppm~90ppm, 1ppm~80ppm, 1ppm~70ppm, 1ppm~65ppm, 1ppm~60ppm, 1ppm~50ppm, 5ppm~10 00ppm, 5ppm~500ppm, 5ppm~400ppm, 5ppm~300ppm, 5ppm~250ppm, 5ppm~200ppm, 5ppm~125ppm, 5ppm~100ppm, 5ppm~90ppm, 5ppm~80ppm, 5ppm~70ppm, 5 ppm~65ppm, 5ppm~60ppm, 5ppm~50ppm, 10ppm~1000ppm, 10ppm~500ppm, 10ppm~400ppm, 10ppm~300ppm, 10ppm~250ppm, 10ppm~200ppm, 10ppm~125ppm, 10ppm~100ppm, 10ppm~90ppm, 10ppm~80ppm, 10ppm~70ppm, 10ppm~65ppm, 10ppm~60ppm, 10ppm~50ppm, 20ppm~1000ppm, 20ppm~500ppm, 20ppm~400ppm,20ppm~300ppm, 20ppm~250ppm, 20ppm~200ppm, 20ppm~125ppm, 20ppm~100ppm, 20ppm~90ppm, 20ppm ~80ppm, 20ppm~70ppm, 20ppm~65ppm, 20ppm~60ppm, 20ppm~50ppm, 30ppm~1000ppm, 30ppm~500ppm, 30ppm~400ppm, 30ppm~300ppm, 30ppm~250ppm, 30ppm~200ppm, 30ppm~125ppm, 30ppm~100ppm, 30pp m~90ppm, 30ppm~80ppm, 30ppm~70ppm, 30ppm~65ppm, 30ppm~60ppm, 30ppm~50ppm, 40ppm~1000ppm, 40ppm~500ppm, 40ppm~400ppm, 40ppm~300ppm, 40ppm~250ppm, 40ppm~200ppm, 40ppm~125ppm, 40pp m~100ppm, 40ppm~90ppm, 40ppm~80ppm, 40ppm~70ppm, 40ppm~65ppm, 40ppm~60ppm, 40ppm~50ppm, 5 0ppm~1000ppm, 50ppm~500ppm, 50ppm~400ppm, 50ppm~300ppm, 50ppm~250ppm, 50ppm~200ppm, 50pp m~125ppm, 50ppm~100ppm, 50ppm~90ppm, 50ppm~80ppm, 50ppm~70ppm, 50ppm~65ppm or 50ppm~60ppm. ,

[0075] In one embodiment, when mangosteen (e.g., mangosteen extract) is included in the above range, its anticoccidial activity may be superior compared to mangosteen included outside the above range.

[0076] According to one embodiment, mangosteen extract (e.g., mangosteen pericarp extract) (1) exhibits excellent anticoccidial activity when treated at the same concentration and / or (2) has excellent safety due to low toxicity (e.g., the lowest concentration showing higher cytotoxicity) to each individual component contained in the mangosteen extract (e.g., xanthones, α-dextrin, epicatechin, and / or quercetin). Mangosteen (e.g., mangosteen extract) according to one embodiment can safely exhibit excellent anticoccidial activity even when the concentration contained in the composition is higher than the concentration of each individual component contained in mangosteen and / or even with prolonged intake without separate side effects.

[0077] In this application, "feed" may refer to any natural or artificial diet, meal, or component of a meal that is intended for animal consumption, ingestion, digestion, or suitability thereto. In a feed composition according to one embodiment, concentrated feed and / or specialty feed may be further included. Concentrated feed is a byproduct obtained through the purification of seeds, fruits, and grains, including grains such as wheat, oats, and corn, and may be bran, including rice bran, wheat bran, and barley bran; sesame cake, a byproduct obtained from pressing oil from soybeans, liquids, sesame seeds, flax seeds, coconuts, etc.; and residues such as residual starch, the main component of starch residue, which is the remainder after starch removal from sweet potatoes and potatoes; fish-solubles, obtained by concentrating fresh liquids obtained from fishmeal, fish waste, and fish; animal feed, such as dried whey, wherein whey is the remainder after the production of casein from meat meal, blood meal, feather meal, skim milk powder, cheese milk, skim milk, etc.; yeast, chlorella, and / or seaweed, etc.

[0078] According to one embodiment, a feed composition can refer to feed in the form ultimately ingested by animals, dietary supplements and / or feed additives that can be mixed with feed. Dietary supplements are compositions, for example, containing preparations that provide therapeutic or digestive aids to animals, and can refer to compositions that are not a common source of calories, i.e., an energy source, but are ingested outside of normal animal feed. Feed additives are substances added to feed for various purposes, such as supplementing nutrition and preventing weight loss, improving the digestibility of fiber in feed, improving oil quality, preventing reproductive disorders and enhancing fertility, and preventing summer heat stress. In one embodiment, a feed additive can refer to a substance added for the purpose of preventing, alleviating, or treating coccidiosis.

[0079] In one embodiment, the feed composition may be a feed additive, and when the feed additive according to one embodiment is mixed into feed (e.g., mixed feed and / or compound feed ultimately ingested by the animal), the feed additive may be present in amounts 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, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.005%, 0.001% to 1%, 0.001% to 0.5%, 0.001% to 0.1%, 0.001% to 0.05%, 0. The feed additive may be added at a weight of 0.01% to 0.01%, 0.001% to 0.005%, 0.005% to 1%, 0.005% to 0.5%, 0.005% to 0.1%, 0.005% to 0.05%, 0.005% to 0.01%, 0.01% to 1%, 0.01% to 0.5%, 0.01% to 0.1%, 0.01% to 0.05%, 0.05% to 1%, 0.05% to 0.1%, 0.1% to 1%, 0.1% to 0.5%, or 0.5% to 1%, and according to one embodiment, the feed additive may be mixed with feed ingredients, supplements, adjuvants, and / or other types of additives, excluding the active ingredient.

[0080] One approach is to provide a pharmaceutical composition for the prevention, relief, or treatment of coccidiosis, comprising mangosteen, wherein the mangosteen contained in the pharmaceutical composition is as described above. The mangosteen contained in the pharmaceutical composition is as described above.

[0081] The pharmaceutical composition according to one embodiment may comprise mangosteen peel, mangosteen pulp, or a combination thereof (mangosteen peel and pulp).

[0082] According to one embodiment, a pharmaceutical composition may comprise at least one (e.g., one or more, two or more, three or more, or all four) selected from the group consisting of raw materials, dried materials, powdered materials, and extracts of mangosteen.

[0083] A pharmaceutical composition according to one embodiment may comprise an extract of mangosteen peel (e.g., an ethanolic extract of mangosteen peel).

[0084] The pharmaceutical composition according to one embodiment can be used as a single formulation and can also be used as a mixed formulation by further comprising an approved pharmaceutical composition known to have preventive or therapeutic effects on coccidiosis. A unit dosage form of the drug can be prepared by adding a pharmaceutically acceptable carrier, excipient, or diluent.

[0085] In this application, "pharmaceuticalally acceptable" means having no significant irritant effect on organisms and not inhibiting the biological activity and properties of the drug-active substance. A pharmaceutical composition comprising a pharmaceutically acceptable carrier according to one embodiment may have any one of the following formulations selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories.

[0086] The pharmaceutical composition can be a variety of oral or parenteral formulations. In the case of formulations, it can be prepared using commonly used fillers, expanders, binders, wetting agents, disintegrants, diluents such as surfactants or excipients.

[0087] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., into one or more compounds. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc., can be used. Liquid dosage forms for oral administration, besides the commonly used simple diluents water and liquid paraffin, can also contain suspensions, internal solutions, emulsions, syrups, etc., as well as various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc.

[0088] Preparations for parenteral administration can include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a base for suppositories, polyethylene glycol fatty acid esters, Tween 61, cocoa butter, lauryl acetate, and glycerin gelatin can be used.

[0089] In one embodiment, the pharmaceutical composition can be formulated into various forms for use according to common methods for various purposes, such as oral preparations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, injectable preparations like sterile injectable solutions, and can be administered orally or via various routes including intravenous administration, intraperitoneal administration, subcutaneous administration, rectal administration, and local administration.

[0090] In one embodiment, the pharmaceutical composition may further comprise a carrier, excipient, or diluent, and examples of suitable carriers, excipients, or diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc. Furthermore, the pharmaceutical composition may also further comprise fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc.

[0091] In one embodiment, the effective amount of the active ingredient (mangosteen (e.g., mangosteen extract)) in the pharmaceutical composition can vary depending on the patient's (subject's) age, sex, and weight, generally ranging from 0.0001 mg / kg to 0.001 mg / kg, 0.0001 mg / kg to 0.01 mg / kg, 0.0001 mg / kg to 0.1 mg / kg, 0.0001 mg / kg to 1 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0 .0001mg / kg~100mg / kg, 0.0001mg / kg~231mg / kg, 0.0001mg / kg~250mg / kg, 0.0001mg / kg~500mg / kg, 0.00 01mg / kg~1000mg / kg, 0.001mg / kg~0.01mg / kg, 0.001mg / kg~0.1mg / kg, 0.001mg / kg~1mg / kg, 0.001mg / kg ~10mg / kg, 0.001mg / kg~100mg / kg, 0.001mg / kg~231mg / kg, 0.001mg / kg~250mg / kg, 0.001mg / kg~500mg / k g, 0.001mg / kg~1000mg / kg, 0.01mg / kg~0.1mg / kg, 0.01mg / kg~1mg / kg, 0.01mg / kg~10mg / kg, 0.01mg / kg~ 100mg / kg, 0.01mg / kg~250mg / kg, 0.01mg / kg~500mg / kg, 0.01mg / kg~1000mg / kg, 0.1mg / kg~1mg / kg, 0.1m g / kg~10mg / kg, 0.1mg / kg~100mg / kg, 0.1mg / kg~231mg / kg, 0.1mg / kg~250mg / kg, 0.1mg / kg~500mg / kg, 0.1mg / kg~1000mg / kg, 1mg / kg~10mg / kg, 1mg / kg~100mg / kg, 1mg / kg~231mg / kg, 1mg / kg~250mg / kg, 1mg / kg~500mg / kg, 1mg / kg~1000mg / kg, 10mg / kg~100mg / kg, 10mg / kg~231mg / kg, 10mg / kg~250mg / kg, 10mg The dosage can be 10 mg / kg to 500 mg / kg, 10 mg / kg to 1000 mg / kg, 100 mg / kg to 231 mg / kg, 100 mg / kg to 250 mg / kg, 100 mg / kg to 500 mg / kg, 100 mg / kg to 1000 mg / kg, 250 mg / kg to 500 mg / kg, or 250 mg / kg to 1000 mg / kg per kg of body weight, and can be administered daily or every other day, or divided into 1 to 3 daily doses. However, since the dosage can be increased or decreased according to the route of administration, the severity of the disease, sex, weight, age, etc., this dosage does not limit the scope of this application in any way. In one embodiment, when the composition is administered intraperitoneally, the composition can be administered at a concentration of 0.001 mg / kg to 250 mg / kg or 0.001 mg / kg to 231 mg / kg.

[0092] In one embodiment, the dosage of the pharmaceutical composition can be within a range depending on factors such as the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity.

[0093] In one embodiment, the mangosteen may be included in the pharmaceutical composition in the following amounts: less than 1 w / w%, less than 1 w / w%, 10 -1 w / w% or less, 5×10 -2 w / w% or less, 2.5×10 -2 w / w% or less, 2×10 -2 w / w% or less, 1.25×10 -2 w / w% or less, 10 -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 more than w / w%, 5×10 -4 more than w / w%, 10 -3 more than w / w%, 1.5×10 -3 more than w / w%, 2×10 -3 more than w / w%, 3×10 -3 more than w / w%, 4×10 -3 more than w / w%, 5×10 -3 more than w / w%, 10 -7 w / w% to 1 w / w%, 10 -7 w / w% to 10 -1 w / w%, 10 -7 w / w% to 5×10 -2 w / w%, 10 -7 w / w% to 10 -2 w / w%, 10 -7 w / w% to 5×10 -3 w / w%, 10 -7 w / w% to 4×10 -3 w / w%, 10 -7 w / w% to 10 -3 w / w%, 10 -7 w / w% to 5×10 - 4 w / w%, 10 -7 w / w% to 10 -4 w / w%, 10 -7 w / w% to 10 -5 w / w%, 10 -6 w / w% to 1 w / w%, 10 -6 w / w% to 10 - 1 w / w%, 10 -6 w / w% to 5×10 -2 w / w%, 10 -6 w / w% to 10 -2 w / w%, 10 -6 w / w% to 5×10 -3 w / w%, 10 -6 w / w% to 4×10 -3 w / w%, 10 -6 w / w% to 10 -3 w / w%, 10 -6 w / w% to 5×10 -4 w / w%, 10 -6 w / w% to 10 -4 w / w%, 10 -6 w / w% to 10 -5w / w%, 10 -5 w / w% ~ 1 w / w%, 10 -5 w / w% ~ 10 -1 w / w%, 10 -5 w / w% ~ 5×10 - 2 w / w%, 10 -5 w / w% ~ 10 -2 w / w%, 10 -5 w / w% ~ 5×10 -3 w / w%, 10 -5 w / w% ~ 4×10 -3 w / w%, 10 -5 w / w% ~ 10 -3 w / w%, 10 -5 w / w% ~ 5×10 -4 w / w%, 10 -5 w / w% ~ 10 -4 w / w%, 10 -4 w / w% ~ 1 w / w%, 10 - 4 w / w% ~ 10 -1 w / w%, 10 -4 w / w% ~ 5×10 -2 w / w%, 10 -4 w / w% ~ 10 -2 w / w%, 10 -4 w / w% ~ 5×10 - 3 w / w%, 10 -4 w / w% ~ 4×10 -3 w / w%, 10 -4 w / w% ~ 10 -3 w / w%, 10 -4 w / w% ~ 5×10 -4 w / w%, 10 -3 w / w% ~ 1 w / w%, 10 -3 w / w% ~ 10 -1 w / w%, 10 -3 w / w% ~ 5×10 -2 w / w%, 10 -3 w / w% ~ 10 -2 w / w%, 10 - 3 w / w% ~ 5×10 -3 w / w%, 10 -3 w / w% ~ 4×10 -3 w / w%, 10 -3 w / w% ~ 2×10 -3 w / w% or 10-3 w / w%~1.5×10 -3 in / in%。

[0094] In one embodiment, the mangosteen (e.g., mangosteen extract) may be included in the pharmaceutical composition at the following concentrations: less than 1000 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 250 ppm, less than 200 ppm, less than 125 ppm, less than 125 ppm, less than 100 ppm, less than 90 ppm, less than 80 ppm, less than 70 ppm, less than 65 ppm, less than 60 ppm, less than 62.5 ppm, less than 50 ppm, more than 0.1 ppm, more than 1 ppm, more than 5 ppm, more than 10 ppm, more than 15 ppm, more than 20 ppm, more than 30 ppm, more than 40 ppm, and more than 50 ppm. Above, 62.5ppm and above, 0.1ppm~1000ppm, 0.1ppm~500ppm, 0.1ppm~400ppm, 0.1ppm~300ppm, 0.1ppm~250ppm, 0.1ppm~200ppm, 0.1ppm~125ppm, 0.1ppm~100ppm , 0.1ppm~90ppm, 0.1ppm~80ppm, 0.1ppm~70ppm, 0.1ppm~65ppm, 0.1ppm~60ppm, 0.1ppm~50ppm, 1ppm~1000ppm, 1ppm~500ppm, 1ppm~400ppm, 1ppm~300p pm, 1ppm~250ppm, 1ppm~200ppm, 1ppm~125ppm, 1ppm~100ppm, 1ppm~90ppm, 1ppm~80ppm, 1ppm~70ppm, 1ppm~65ppm, 1ppm~60ppm, 1ppm~50ppm, 5ppm~10 00ppm, 5ppm~500ppm, 5ppm~400ppm, 5ppm~300ppm, 5ppm~250ppm, 5ppm~200ppm, 5ppm~125ppm, 5ppm~100ppm, 5ppm~90ppm, 5ppm~80ppm, 5ppm~70ppm, 5 ppm~65ppm, 5ppm~60ppm, 5ppm~50ppm, 10ppm~1000ppm, 10ppm~500ppm, 10ppm~400ppm, 10ppm~300ppm, 10ppm~250ppm, 10ppm~200ppm, 10ppm~125ppm, 10ppm~100ppm, 10ppm~90ppm, 10ppm~80ppm, 10ppm~70ppm, 10ppm~65ppm, 10ppm~60ppm, 10ppm~50ppm, 20ppm~1000ppm, 20ppm~500ppm, 20ppm~400ppm,20ppm~300ppm, 20ppm~250ppm, 20ppm~200ppm, 20ppm~125ppm, 20ppm~100ppm, 20ppm~90ppm, 20ppm ~80ppm, 20ppm~70ppm, 20ppm~65ppm, 20ppm~60ppm, 20ppm~50ppm, 30ppm~1000ppm, 30ppm~500ppm, 30ppm~400ppm, 30ppm~300ppm, 30ppm~250ppm, 30ppm~200ppm, 30ppm~125ppm, 30ppm~100ppm, 30pp m~90ppm, 30ppm~80ppm, 30ppm~70ppm, 30ppm~65ppm, 30ppm~60ppm, 30ppm~50ppm, 40ppm~1000ppm, 40ppm~500ppm, 40ppm~400ppm, 40ppm~300ppm, 40ppm~250ppm, 40ppm~200ppm, 40ppm~125ppm, 40pp m~100ppm, 40ppm~90ppm, 40ppm~80ppm, 40ppm~70ppm, 40ppm~65ppm, 40ppm~60ppm, 40ppm~50ppm, 5 0ppm~1000ppm, 50ppm~500ppm, 50ppm~400ppm, 50ppm~300ppm, 50ppm~250ppm, 50ppm~200ppm, 50pp m~125ppm, 50ppm~100ppm, 50ppm~90ppm, 50ppm~80ppm, 50ppm~70ppm, 50ppm~65ppm or 50ppm~60ppm. ,

[0095] According to one embodiment, mangosteen extract (e.g., mangosteen extract and / or mangosteen pericarp extract) (1) exhibits excellent anticoccidial activity when treated at the same concentration and / or (2) has excellent safety due to low toxicity (e.g., higher minimum concentrations showing cytotoxicity) to each individual component contained in the mangosteen extract (e.g., xanthones, α-dextrin, epicatechin, and / or quercetin). Mangosteen (e.g., mangosteen extract) according to one embodiment can safely exhibit excellent anticoccidial activity even when the concentrations contained in the composition are higher than the concentrations of the various components contained in mangosteen and / or even with prolonged intake, no separate side effects occur.

[0096] In one embodiment, the pharmaceutical composition can be administered to a subject via multiple routes. Administration can refer to providing a substance to a subject (patient) by any suitable method. The route of administration of the pharmaceutical composition can be oral and / or parenteral, through all conventional routes, as long as the target tissue is reached. In the case of parenteral administration, options include external application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and / or intrapleural injection. Furthermore, the composition according to one embodiment can be administered using any device capable of delivering the active ingredient to target cells.

[0097] In other respects, an antiprotozoan composition resistant to Eimeria protozoa may be provided, comprising mangosteen. The aspects of the mangosteen and / or the Eimeria protozoa are as described above.

[0098] An antigenic composition according to one embodiment may comprise mangosteen peel, mangosteen pulp, or a combination thereof (mangosteen peel and pulp).

[0099] An antigenic composition according to one embodiment may comprise at least one (e.g., one or more, two or more, three or more, or all four) selected from the group consisting of raw materials, dried materials, powdered materials, and extracts of mangosteen.

[0100] According to one specific embodiment of the antigenic animal composition, it may contain a mangosteen pericarp extract (e.g., an ethanol extract of mangosteen pericarp).

[0101] In one embodiment, the antiprotozoan activity (effect, efficacy) against Eimeria protozoa is superior, which may refer to the following characteristics (1) and / or (2), for example, it may exhibit the following characteristics (1) and / or (2) compared with control groups (negative control group and / or positive control group):

[0102] (1) It has excellent killing effects on Eimeria protozoa; and / or

[0103] (2) Inhibit the cell invasion of Eimeria protozoa and / or inhibit the proliferation of protozoa in cells.

[0104] In one embodiment, mangosteen (e.g., mangosteen extract) may be included in the feed composition and / or pharmaceutical composition within the aforementioned concentration range. In one embodiment, compositions containing active ingredients within the aforementioned concentration range may exhibit superior antiprobiotic activity compared to compositions containing active ingredients outside the aforementioned concentration range.

[0105] On the other hand, a method for preventing, alleviating, or treating coccidiosis can be provided, comprising the step of administering the composition (e.g., a feed composition, feed additive, pharmaceutical composition, and / or antiprotozoan composition) to an animal. In one embodiment, prior to administering the composition, the method may further include identifying (selecting) a subject (patient) who requires prevention, alleviation, or treatment of coccidiosis. The composition and coccidiosis are as described above. According to one embodiment, identifying the subject may include detecting oocysts of protozoa capable of inducing coccidia in feces isolated from the subject.

[0106] In a method for preventing, alleviating, or treating coccidiosis according to one embodiment, the method of administration, route of administration, and / or dosage of the composition are as described above.

[0107] According to one embodiment, the composition can be administered at a pharmaceutically effective dose. In this application, a "pharmaceutically effective dose" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level can be determined based on the patient's disease type, severity, drug activity, drug sensitivity, timing of administration, route of administration and excretion ratio, treatment duration, concurrently used drug-containing components, and other components well known in the medical field. According to one embodiment, the composition can be administered as a standalone therapeutic agent or in combination with other anticoccidial agents, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered once or multiple times. Taking all factors into account, it is important to administer the amount that achieves the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0108] In one embodiment, the subjects to which the methods for preventing, mitigating, or treating coccidiosis are applied are animals that have or may have coccidiosis. These animals may be mammals, including humans, horses, cattle, mice, rats, dogs, cats, etc., birds, fish, amphibians, and / or reptiles, including poultry (e.g., breeder chickens, broilers, and / or laying hens).

[0109] In one embodiment, the animal used in the method for preventing, alleviating, or treating coccidiosis may be at least one (e.g., more than one, two, or three) selected from the group consisting of the animals described in Table 1 above. For example, the animal may be at least one (e.g., more than one, two, or three) selected from the group consisting of humans, chickens, ducks, geese, turkeys, quails, pheasants, pigeons, parrots, cattle, pigs, goats, sheep, horses, antelopes, eland, monkeys, cats, dogs, mice, rats, rabbits, raccoons, squirrels, bats, guinea pigs, camels, llamas, alpacas, wombats, lizards, goldfish, crucian carp, tilapia, barramundi, lungfish, and European whitefish. In one embodiment, the animal may be an animal other than a human.

[0110] In other aspects, a composition for the prevention, relief, and treatment of coccidiosis using mangosteen is provided.

[0111] Uses of mangosteen in (e.g., feed compositions, pharmaceutical compositions) or antiprotozoan compositions; uses of mangosteen for the prevention, relief, and / or treatment of coccidiosis; and / or uses of mangosteen against antiprotozoa of the genus *Eimeria* (e.g., killing *Eimeria* protozoa; and / or inhibiting *Eimeria* protozoan cell invasion and / or proliferation). In the above uses, mangosteen is as described above, for example, mangosteen can be a raw material, dried material, powdered material, and extract of mangosteen. In the above uses, coccidiosis, antiprotozoa, and *Eimeria* protozoa are as described above.

[0112] Beneficial effects

[0113] According to one embodiment, a composition containing mangosteen has excellent direct killing effect on protozoa capable of inducing coccidiosis, has cell invasion inhibition effect on protozoa and / or cell proliferation inhibition effect on protozoa, and has excellent preventive, alleviating and therapeutic effects on coccidiosis in vivo. Attached Figure Description

[0114] Figure 1 The study demonstrated the inhibitory effect of mangosteen, xanthones, and anticoccidial agents (diclazuril and salinomycin) on cell invasion rates in protozoa.

[0115] Figure 2 The study demonstrated the inhibitory effect of protozoan treatment with mangosteen, xanthones, and anticoccidial agents (diclazuril and salinomycin) on cell proliferation.

[0116] Figure 3 The results of the acid resistance test for mangosteen extract are shown.

[0117] Figure 4 The results of the heat resistance test for mangosteen extract are shown. Detailed Implementation

[0118] The invention will be described in more detail through the following embodiments, but is not intended to limit the scope of the invention through the following embodiments.

[0119] Example 1. In vivo anticoccidial activity of mangosteen

[0120] Example 1-1. Experimental Facilities

[0121] An in vivo anticoccidial efficacy study was conducted at an animal testing facility in Gyeongsangnam-do, South Korea. One-day-old female Ross broilers were individually weighed and randomly assigned to groups for the experiment. The experimental design considerations and conditions are described in Table 2.

[0122] Table 2

[0123] category Experimental variables Aquaculture type cage Broiler chicken free-range age 1 day old Total experimental period 22 days gender female Number of broilers / cage 20 Number of repetitions / treatment groups 2 repetitions Number of processing groups 5 groups Total number of broiler chickens 200 broiler chickens Challenges in inoculating protozoan species tender Eimeria coccidia Challenge the number of protozoa inoculated 10,000 oocysts orally inoculated into broilers

[0124] The farm was managed according to the Korean Poultry Farming Management Guidelines. The cages and the farm were cleaned and disinfected before the trial began. The farm was maintained at a temperature of 40°C–41°C and a humidity of 40%–50%, and this was continuously monitored.

[0125] Examples 1-2. Preparation of mangosteen extract

[0126] 120 kg of dried and powdered mangosteen (Garcinia mangostana L., source: Heung-illherb.Co.,Ltd.; origin: Changsha, Wangcheng New District, China, Changsha Herbway Biotech of Wangcheng District.Co.,Ltd.; purchased in June 2020) fruit peel was extracted twice by immersion in a constant temperature water bath using 75% ethanol (cereal alcohol, purified ethanol) as the solvent. After extraction, the powder was filtered through filter paper, concentrated under reduced pressure using a rotary vacuum concentrator, and then pulverized to obtain approximately 1 kg of extracted powder for subsequent experiments.

[0127] Examples 1-3. Experimental Design

[0128] Feed A1-choi products from South Korea were used. Various ingredients (salinomycin (Cheil Bio Cheilsalino-60 product), xanthone (Sigma Aldrich X600 product), and mangosteen extract prepared in Examples 1-2 above) were added to the feed separately and self-mixed at the concentrations described in Table 3. No antibiotics or supplements were used in the general feed or mixed feed, and no anticoccidial agents were added except for the various ingredients. Broilers were fed freely throughout the experiment. Twenty one-day-old broilers were randomly placed in cages for each control or experimental group and fed a general feed for 7 days. Then, the prepared mixed feed was divided into control and experimental groups for consumption.

[0129] The feed formulations administered to the control group (negative or positive control group) and the experimental group, as well as whether coccidiosis was induced by Eimeria tenella, are described in Table 3 below. Twenty one-day-old broilers were randomly placed in cages in each experimental group and fed a normal diet for seven days. Then, the formulated feeds as described in Table 3 were distributed to each experimental group and ingested.

[0130] 14-day-old broilers were orally vaccinated with 10,000 oocysts (90% of which are sporozoites of Eimeria tenella) per chicken to induce coccidiosis.

[0131] Table 3

[0132]

[0133] Examples 1-4. Determination of the anticoccidial activity of mangosteen

[0134] The anticoccidial effect of the experimental groups designed in Examples 1-3 above is expressed as the anticoccidial index (ACI), which is calculated using the following Equation 2. The ACI score is out of 200. The higher the ACI score, the better the anticoccidial ability. When the ACI score is above 120 and below 140, it is determined to be effective as an anticoccidial material. When the ACI score is above 140 and below 160, it is determined to be excellent as an anticoccidial material. When the ACI score is above 160, it is determined to have very good anticoccidial effect (Luis Miguel De Pablos et al., Anticoccidial activity of maslinic acid against infection with Eimeria tenella in chickens, Parasitol Res, 2010).

[0135] (Equation 2)

[0136] Anticoccidial Index (ACI) = (Survival rate after challenge inoculation (%)) + (Daily weight gain compared to negative control group (RWG, %)) - (Lesion score × 10) - (Fecal oocyst excretion index)

[0137] 1) Survival rate: The number of dead individuals was recorded daily, and post-mortem examinations were performed to determine the cause of death, excluding individuals who died from causes other than coccidiosis. The survival rate (%) from challenge inoculation to day 8 was used to calculate the anticoccidial index.

[0138] 2) Daily weight gain: The weight of each cage was measured before and on day 7 after protozoan challenge vaccination. The difference was divided by the number of days to calculate the daily weight gain (ADG, g / day). "Daily weight gain compared to the negative control group (RWG, %)" was calculated by dividing the daily weight gain (ADG, mean daily gain; g / day) of each experimental group by the weight gain (ADG, g / day) of the uninfected negative control group and then multiplying by 100. This was used to calculate the anticoccidial index.

[0139] Daily weight gain (ADG, g / day) measured in each control and experimental group, and daily weight gain (RWG, %) compared to the negative control group, are shown in Table 4 below.

[0140] Table 4

[0141]

[0142] 3) Lesion Scoring: On day 8 post-challenge vaccination, necropsy was performed on 4 broilers per cage, and the intestines were cut open. Each coccidial lesion in the appendix region of the broilers was scored. The lesion scoring method can be referred to the literature of Johnson JK and Reid WM (Joyce Johnson and W. Malcolm Reid, Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens, Experimental parasitology, 1970). The lesion score ranged from 0 to 4, with 0 corresponding to a normal appendix, 1 to mild infection, 2 to moderate infection, 3 to severe infection, and 4 to very severe infection or death. The appendix lesion scores measured in each control and experimental group are described in Table 5 below. The measured appendix lesion scores were multiplied by 10 to calculate the anticoccidial index.

[0143] Table 5

[0144] Group Appendicitis score Uninfected negative control group 0 Negative control group of infection 3.1 Salinomycin treatment group 2.7 Oxanthrone treatment group 2.4 Mangosteen extract treatment group 1.9

[0145] 4) Fecal oocyst excretion: All feces from days 6 to 8 of challenge inoculation were collected in cages, mixed thoroughly, and randomly sampled 3 times, 1g each time. After flocculating the oocysts in 1g of feces with saline, the oocyst excretion was measured using a McMaster chamber. The results are shown in Table 6 below.

[0146] Table 6

[0147] Group Oocyte excretion volume / g Uninfected negative control group 0.00E+00 Negative control group of infection 1.90E+08 Salinomycin treatment group 1.30E+08 Oxanthrone treatment group 1.80E+08 Mangosteen extract treatment group 1.30E+08

[0148] The oocyst excretion rate (%) compared to the infected negative control group was calculated by dividing the oocyst excretion rate of each group by the oocyst excretion rate of the infected negative control group and multiplying by 100. The oocyst excretion rate index was calculated as follows: 0% to less than 1% compared to the infected negative control group; 5% to less than 26%; 10% to less than 26%; 20% to less than 51%; 20% to less than 76%; and 40% to less than 100%. These values ​​were then used to calculate the anticoccidial index.

[0149] As described above, the anticoccidial index of each experimental group measured by Equation 2 is shown in Table 7 below. As shown in Table 7, the anticoccidial index of the mangosteen extract treatment group was higher than that of the positive control group and the salinomycin treatment group, thus demonstrating excellent anticoccidial efficacy.

[0150] Table 7

[0151] Group Anticoccidial Index (ACI) Uninfected negative control group 197 Negative control group of infection 101 Salinomycin treatment group 141 Oxanthrone treatment group 122 Mangosteen extract treatment group 151

[0152] Example 2. Direct killing effect of mangosteen on Eimeria protozoa.

[0153] Example 2-1. Determination of the direct protozoan killing effect of mangosteen

[0154] In this embodiment, the direct killing ability of protozoa (spores) of three representative Eimeria species known to infect most farms (Eimeria tenella, Eimeria smotherum, and Eimeria giant) was assessed.

[0155] A certain amount of oocysts from various protozoa were placed in test tubes containing glass beads and crushed. The crushed oocyst cell walls and other debris were then removed. The internal sporangia were purified using a Percoll density gradient and washed with PBS solution. Sporangia from *Eimeria tenella*, *Eimeria squarrosa*, and *Eimeria giantiformis* were treated with reagents containing sodium taurocholate (Sigma-Aldrich, USA) and trypsin (Gibco, USA) respectively for decapacitation, incubated, and then washed once with PBS solution to obtain the protozoa.

[0156] Mangosteen pericarp extract, epicatechin, α-dextrin, xanthones, and the anticoccidial agents salinomycin and diclazuril (hereinafter, materials) were reacted with three species of Eimeria protozoa at concentrations ranging from 1 to 500 ppm. Viable protozoa (spores) were then counted under a microscope. The mortality rate (%) of protozoa treated with each material was then calculated compared to the PBS-treated negative control group, and is shown in Tables 8 to 10 and Table 12, respectively. The minimum concentration required to directly kill 100% of the protozoa is shown in Table 11 below.

[0157] Table 8

[0158]

[0159]

[0160] Table 9

[0161]

[0162] Table 10

[0163]

[0164] Table 11

[0165]

[0166] As shown in Table 11, in the xanthonone treatment group, xanthonone at concentrations up to 500 ppm did not have 100% direct killing effect on protozoa capable of inducing coccidia. Similarly, in the case of diclazuril, diclazuril at concentrations up to 500 ppm did not have 100% direct killing effect on protozoa (Eimeria tenella and Eimeria giant). In contrast, mangosteen extract, at significantly lower concentrations than other groups, could kill 100% of Eimeria tenella, Eimeria spp., and Eimeria giant, thus confirming its excellent protozoic killing effect.

[0167] Table 12

[0168]

[0169] As shown in Table 12, the mangosteen extract, at a concentration of 15.6 ppm, significantly lower than that of the individual components contained in mangosteen (epicatechin and α-dextrin), could kill 100% of the *Eimeria tenella* protozoa, thus confirming the excellent protozoic killing effect of the mangosteen extract. The components and contents of the mangosteen extract were analyzed by LC-MS (LC-MS analysis conditions: LC was Waters Acquity UPLC i-class, MS was Waters Xevo G2-XS (Q-TOF) product. For column information, a Waters Acquity BEH C18 column was used). The results are shown in Table 13. As shown in Table 13, the α-dextrin content in the mangosteen extract was approximately 12%. Considering this, the anticoccidial activity of the mangosteen extract itself is superior to that of α-dextrin.

[0170] Table 13

[0171]

[0172] Example 3. Inhibitory effect of mangosteen on cell invasion and intracellular proliferation of Eimeria protozoa.

[0173] In this embodiment, the inhibitory effect of intracellular protozoan invasion and intracellular protozoan proliferation on mangosteen was studied using the MDBK cell line, a representative animal cell line known to cause infection and proliferation of Eimeria spp.

[0174] Example 3-1. Inhibitory effect of mangosteen treatment on cell invasion of protozoa.

[0175] 100,000 MDBK cells (purchased from ATCC) were aliquoted into 24-well plates and incubated at 37°C for 12 hours. Protozoa of *Eimeria tenella* were obtained similarly to the method described in Example 2 above. 200,000 protozoa were added to each well of the cell-allotted plates, and the cells were treated with each material (mangosteen, xanthones, and the anticoccidial agents salinomycin and diclazuril) at a concentration (1 ppm or 10 ppm) and cultured at 41°C for 24 hours. The negative control group consisted of MDBK cells infected with protozoa, and the positive control group consisted of cells incubated with either salinomycin or diclazuril solution. Subsequently, the cells were washed twice with PBS to remove protozoa from uninvaded cells. After removing the cells and protozoa within them using a pipette, DNA was extracted from the cells and PCR was performed using *Eimeria tenella* ACE gene-specific primers. The sequences of the primers used are described in Table 14 below.

[0176] Table 14

[0177]

[0178] The Ct values ​​of each material before and after washing were compared and corrected with the ΔCt value of the negative control group to calculate the cell invasion inhibition rate (%) of each material on protozoa. The results were presented in... Figure 1 As shown in the image. Figure 1 As shown, it can be confirmed that the mangosteen extract treatment group has an excellent inhibitory effect on cell invasion of Eimeria tenella.

[0179] Example 3-2. Inhibitory effect of mangosteen treatment on intracellular proliferation of protozoa.

[0180] 100,000 MDBK cells (purchased from ATCC) were aliquoted into 24-well plates and incubated at 37°C for 12 hours. Eimeria tenella protozoa were obtained similarly to the method described in Example 2 above. 200,000 protozoa were added to each well of the cell aliquots and cultured at 41°C for 24 hours. The cells were then washed twice with PBS to remove protozoa not attached to the cells. Cells were treated with each material (mangosteen, xanthones and anticoccidial agents, salinomycin and diclazuril) at the specified concentrations and cultured for an additional 24 hours at 41°C. The negative control group consisted of MDBK cells infected with protozoa, and the positive control group consisted of cells incubated with either salinomycin or diclazuril solution. After removing cells and proliferating protozoa within the cells using a pipette, DNA was extracted from the cells and PCR was performed using primers specific to the Eimeria tenella ITS-1 (Internal Transcriptional Spacer-1) gene. The sequences of the primers used are described in Table 14 above.

[0181] The Ct values ​​in the material treatment group and the negative control group were compared to calculate the intracellular protozoan (spore) proliferation inhibition rate (%). The results showed that... Figure 2 It is displayed in the middle.

[0182] like Figure 2 As shown, the mangosteen extract treatment group exhibited a significantly superior inhibitory effect on the intracellular protozoan proliferation of Eimeria tenella compared to other groups.

[0183] Example 4. Cytotoxicity evaluation of mangosteen

[0184] In this embodiment, the effect of mangosteen extract on cell survival was studied using MTS analysis (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole, Promega, USA).

[0185] The cell lines used for cytotoxicity evaluation were (1) MDBK (Madin-Darby bovine kidney epithelial cells) and (2) RAW264.7 (mouse macrophages), both of which were purchased from ATCC and used.

[0186] Each cell was divided into 1×10 5 Cells per well were aliquoted into 96-well cell culture plates and cultured at 37°C for 24 hours. Then, each material (mangosteen extract, xanthones, α-dextrin, and epicatechin) was treated at concentrations (3.1 ppm to 500 ppm / well) and cultured at 37°C. After 14 hours, MTS solution was added to the cell culture medium, and the cells were incubated for 2 hours. The absorbance was measured at 490 nm using a microplate reader. Cell viability (%) was calculated compared to the 5% DMSO treatment group (negative control), and the lowest cytotoxic concentration for 50% or more cell survival was measured. The results are shown in Table 15 below. Furthermore, using a method similar to that in Example 2-1 above, the concentrations of each material that resulted in 100% killing of *Eimeria tenella* protozoa were determined, and the results are shown in Table 15.

[0187] As shown in Table 15, the cell safety can be confirmed as excellent, as the mangosteen extract did not show cytotoxicity at concentrations up to approximately 62.5 ppm, and the mangosteen extract also showed excellent protozoan killing activity at concentrations significantly lower than those exhibiting cytotoxicity.

[0188] Table 15

[0189]

[0190] Example 5. Evaluation of the acid resistance of mangosteen

[0191] In this embodiment, the acid tolerance of mangosteen itself was evaluated. Hydrochloric acid (HCl) solution was added to the mangosteen extract prepared in Examples 1-2 above, adjusting the pH to 2, 3, and 5.5, and then allowed to stand at 40°C for 1 hour. Then, by adding sodium hydroxide (NaOH) solution to neutralize (pH 7.0), 200,000 giant Eimeria coccidia protozoa (spores) were exposed to mangosteen extract solutions of different concentrations (1 ppm to 100 ppm) diluted with PBS solution and reacted at 41°C for 4 hours. The protozoan mortality rate (%) was then measured using a method similar to that in Example 2 above. Figure 3 As shown in the image. Figure 3 As shown, it can be confirmed that mangosteen extract does not lose its killing effect on protozoa that induce coccidiosis even under strongly acidic conditions.

[0192] Example 6. Evaluation of the heat resistance of mangosteen

[0193] In this embodiment, the heat resistance of mangosteen was evaluated. The mangosteen extract prepared in Examples 1-2 above was exposed to 85°C–95°C for 10 minutes, then cooled and diluted with PBS solution to concentrations of 1 ppm, 10 ppm, 50 ppm, and 100 ppm. 200,000 *Eimeria tenella* protozoa were reacted with different concentrations of mangosteen extract (1 ppm–100 ppm) at 41°C for 4 hours. The protozoan mortality rate (%) was then measured using a method similar to that in Example 2 above. Figure 4 It is displayed in the middle.

[0194] like Figure 4 As shown, it can be confirmed that mangosteen extract does not lose its killing effect on protozoa that induce coccidiosis, even under adverse conditions of high temperature.

[0195] Example 7. Evaluation of the effect of mangosteen extract intake on broiler growth.

[0196] In this embodiment, the effect of mangosteen extract on broiler growth (improvement or attenuation) was evaluated.

[0197] Example 7-1. Experimental Facilities

[0198] Efficacy evaluation trials were conducted at an animal testing facility in Gyeongsangnam-do, South Korea. One-day-old female Ross broilers were individually weighed and randomly assigned to groups for the experiment. Experimental design considerations and conditions are described in Table 16.

[0199] Table 16

[0200] category Experimental variables Aquaculture type cage Broiler chicken free-range age 1 day old Total experimental period 14 days gender female Number of broilers / cage 20 Number of repetitions / treatment group 2 repetitions Treatment group number 6 groups Total number of broiler chickens 240 broiler chickens

[0201] The farm was managed according to the Korean Poultry Farming Management Guidelines. The cages and the farm were cleaned and disinfected before the trial began. The farm was maintained at a temperature of 40°C–41°C and a humidity of 40%–50%, and this was continuously monitored.

[0202] Example 7-2. Experimental Design

[0203] Feed A1-choi products from South Korea were used. Various ingredients (salinomycin (Cheil Bio Cheilsalino-60 product), xanthone (Sigma Aldrich X600 product), and mangosteen extract prepared in Examples 1-2 above) were added to the feed separately and self-mixed at the concentrations described in Table 17. No antibiotics or supplements were used in the general feed or mixed feed, and no anticoccidial agents were added except for the individual ingredients. Broilers were fed freely throughout the experiment. Twenty one-day-old broilers were randomly placed in each control or experimental group cage and fed a general feed for 7 days. Then, the prepared mixed feed was divided into control or experimental groups for consumption. The feed formulations administered to the control group (negative control group or positive control group) and experimental group are described in Table 17 below.

[0204] Table 17

[0205] Group Processing Group negative control group General feed Salinomycin treatment group (positive control group) Compound feed containing 60 ppm salinomycin Oxanthraquinone treatment group 1 Compound feed containing 250 ppm of xanthones Oxanthraquinone treatment group 2 The feed contains 125 ppm of xanthones. Mangosteen extract treatment group Compound feed containing 125 ppm mangosteen extract

[0206] Example 7-3. Calculation of daily weight gain in broilers

[0207] The body weight before feeding and the body weight on day 7 after feeding the individuals with the feed listed in Table 16 above were measured. The difference was divided by the number of days to calculate the daily weight gain (ADG, g / day), and the results are shown in Table 18. For comparisons among the six treatment groups, the Duncan post-test multiple comparison method was used, and statistical significance was verified using the SAS statistical program, and the results are shown in Table 18.

[0208] As shown in Table 18, in the mangosteen extract treatment group, the daily weight gain was significantly increased compared to the negative control group, positive control group, and xanthonone treatment group. On the other hand, in the xanthonone treatment group, the daily weight gain was actually decreased compared to the negative control group.

[0209] Table 18

[0210] sequence list <110> CJ CheilJedang Corporation <120> Anticoccidial Compositions Containing Mangosteen and Their Uses <130> OPP20211037KR <150> KR 10-2020-0125245 <151> 2020-09-25 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic E. tenella ITS-1 positive <400> 1 tggaggggat tatgagagga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic E. tenella ITS-1 reverse <400> 2 caagcagcat gtaacggaga 20 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic E. acervulina ACE-positive <400> 3 gcagtccgat gaaaggtatt tg 22 <210> 4 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic *Eimeria acervulina* ACE reverse <400> 4 gaagcgaaat gttaggccat ct 22

Claims

1. Use of mangosteen in the preparation of feed compositions for the prevention or mitigation of coccidiosis. in, The mangosteen is an ethanol extract of mangosteen pericarp, which is obtained by extracting mangosteen pericarp with a 70 to 80 v / v ethanol solution, wherein the coccidiosis is induced by protozoa of the genus Eimeria sp.

2. The use according to claim 1, wherein, The prevention or mitigation of coccidiosis is selected from at least one of the following groups (1) to (4): (1) Reduce at least one of the groups selected from those consisting of lesion score, fecal oocyst excretion, and mortality; (2) Inhibit weight loss caused by coccidiosis; (3) Increase the anticoccidial index (ACI); and (4) Reduce cell invasion of Eimeria protozoa, proliferation of protozoa in cells, or both.

3. The use according to claim 1, wherein, The feed composition is a feed containing mangosteen at a concentration of less than 1 w / w% based on total weight.

4. The use according to any one of claims 1 to 3, wherein, The feed composition is a feed additive.

5. Use of mangosteen in the preparation of pharmaceutical compositions for the prevention or relief of coccidiosis. in, The mangosteen is an ethanol extract of mangosteen pericarp, which is obtained by extracting mangosteen pericarp with a 70 to 80 v / v ethanol solution, wherein the coccidiosis is induced by protozoa of the genus Eimeria sp.

6. Use of mangosteen in the preparation of antiprotozoan compositions against Eimeria protozoa. in, The mangosteen is an ethanol extract of mangosteen pericarp, which is obtained by extracting mangosteen pericarp with a 70 to 80 v / v ethanol solution.

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