ANTI-COCCIDIAL COMPOSITION COMPRISING MANGOSTEEN AND USES THEREOF

Abstract

The present application relates to an anti-coccidial composition comprising mangosteen, and the uses thereof. The composition comprising mangosteen according to an example has excellent effect of directly killing protozoa which can induce coccidiosis, effect of inhibiting cell penetration by protozoa and/or effect of inhibiting intracellular protozoan proliferation, and has excellent in vivo coccidiosis prevention, relief and therapeutic effects.

Claims

1-12. (canceled)

13. A method for preventing, alleviating or treating coccidiosis, comprising a step of administering a composition comprising a mangosteen, to an animal.

14. The method according to claim 13, wherein the mangosteen is mangosteen pericarp, mangosteen flesh, or a combination thereof.

15. The method according to claim 13, wherein the mangosteen is at least one selected from the group consisting of raw material, dried material, pulverized material and extract of the mangosteen.

16. The method according to claim 15, wherein the mangosteen extract is extracted by a solvent extraction method using an extraction solvent selected from the group consisting of water, linear or branched chain alcohols having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane and mixtures thereof.

17. The method according to claim 16, wherein the mangosteen extract is a mangosteen pericarp extract.

18. The method according to claim 13, wherein the coccidiosis is induced by an Eimeria sp. protozoan.

19. The method according to claim 13, wherein the preventing, alleviating, or treating coccidiosis is at least one selected from the group consisting of the following (1) to (4): (1) reduction of at least one selected from the group consisting of lesion score, fecal oocyst excretion amount and mortality; (2) inhibition of weight loss due to coccidiosis; (3) increase of an anticoccidial index (ACI); and (4) reduction of cell invasion of an Eimeria sp. protozoan, propagation of the protozoan in cells, or both of them.

20. The method according to claim 13, wherein the composition is a feed composition comprising the mangosteen at a concentration of 1% (w/w) or less based on the total weight.

21. The composition according to claim 13, wherein the composition is a feed additive.

22. A method for killing Eimeria sp. protozoan, or inhibiting cell invasion or propagation of Eimeria sp. protozoan, comprising a step of administering a mangosteen, to an animal.

23. The method according to claim 22, wherein the mangosteen is mangosteen pericarp, mangosteen flesh, or a combination thereof.

24. The method according to claim 22, wherein the mangosteen is at least one selected from the group consisting of raw material, dried material, pulverized material and extract of the mangosteen.

25. The method according to claim 24, wherein the mangosteen extract is extracted by a solvent extraction method using an extraction solvent selected from the group consisting of water, linear or branched chain alcohols having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane and mixtures thereof.

26. The method according to claim 25, wherein the mangosteen extract is a mangosteen pericarp extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0101] FIG. 1 shows the cell invasion rate inhibitory effect of the protozoa according to treatment of the mangosteen, xanthone, and anticoccidial agents (diclazuril, salinomycin).

[0102] FIG. 2 shows the propagation inhibitory effect of the protozoa in cells according to treatment of the mangosteen, xanthone, and anticoccidial agents (diclazuril, salinomycin).

[0103] FIG. 3 shows the result of measuring the acid resistance of the mangosteen extract.

[0104] FIG. 4 shows the result of measuring the heat resistance of the mangosteen extract.

MODE FOR INVENTION

[0105] The present invention will be described in more detail by the following examples, but it is not intended to limit the scope by the following examples.

EXAMPLE 1. IN VIVO ANTICOCCIDIAL ACTIVITY OF MANGOSTEEN

Example 1-1. Experimental Facility

[0106] An in vivo anticoccidial efficacy evaluation test was performed in an animal experiment facility in Gyeongsangnam-do, South Korea. One-day-old female Ross broilers were individually weighed and randomly divided into groups to use in an experiment. Matters and conditions for experimental design were described in Table 2.

TABLE-US-00002 TABLE 2 Category Experiment variable Breeding type Cage Broiler stocking age 1-day-old Total experiment period 22 days Gender Female Number of broilers per cage 20 Number of repetitions per treatment group 2 repetitions Number of treatment groups 5 Total number of broilers 200 broilers Kind of challenge inoculation protozoa Eimeria tenella Number of challenge inoculation protozoa 10,000 oocysts oral inoculation/broiler

[0107] A breeding farm was managed according to the Korean poultry breeding management guidelines. Cages and the breeding farm were cleaned and disinfected before starting the test. The temperature of 40 to 41° C. and the humidity of 40 to 50% of the breeding farm were maintained, and it was continuously monitored.

Example 1-2. Preparation of Mangosteen Extract

[0108] Raw powder 120 kg of drying and pulverizing a mangosteen (Garcinia mangostana L.

[0109] source: Heung-ill herb. Co.,Ltd; country of origin: China, Changsha, Changsha herbway biotech of Wangcheng district. Co.,Ltd; purchased (acquired) on June, 2020) pericarp was extracted twice by immersing in a constant-temperature water bath using 75% ethanol (grain alcohol, purified ethyl alcohol) as a solvent. After extraction, it was filtered using filter paper, and then concentrated under reduced pressure using a rotary decompression concentrator, and powdered to finally obtain about lkg of extracted powder, and it was used in the subsequent experiment.

Example 1-3. Experimental Design

[0110] For the feed, Korea Feed Al-choi product was used, and each material (salinomycin (Cheil Bio Cheilsalino-60 product), xanthone (Sigma Aldrich X600 product), and the mangosteen extract prepared in Example 1-2 above) was added to the feed, respectively, at a concentration described in Table 3 below to be self-mixed. Antibiotics and supplements were not used in general feed and mixed feed, and no anticoccidial agent other than each material was added. Broilers were fed ad libitum throughout the experiment period. After putting 20 1-day-age broilers in cages randomly placed for each of the control group or test group and breeding, general feed was fed for 7 days, and then the mixed feed prepared above was ingested by dividing by control group or test group.

[0111] The feed formulation administered to the control group (negative control group or positive control group) and test group and whether coccidiosis was induced by Eimeria tenella were described in Table 3 below. 20 1-day-old broilers were put in a cage randomly arranged for each test group, and after 7 days of feeding the general feed, the formulated feed as described in Table 3 was divided into each test group and ingested.

[0112] 14-day-old broilers were orally inoculated with 10,000 oocysts (90% sporulated Eimeria tenella oocysts) per individual to induce coccidiosis.

TABLE-US-00003 TABLE 3 Group Treatment Non-infected negative control General feed group Infected negative control group Eimeria tenella infection + general feed Positive control group 1 Eimeria tenella infection + formulated (salinomycin treated group) feed comprising salinomycin 60 ppm Xanthone treated group Eimeria tenella infection + formulated feed comprising xanthone 62.5 ppm Mangosteen extract treated Eimeria tenella infection + formulated group feed comprising mangosteen extract 62.5 ppm

Example 1-4. Measurement of Anticoccidial Activity of Mangosteen

[0113] The anticoccidial efficacy by test group designed in Example 1-3 above was shown as an anticoccidial index (ACI), and the anticoccidial index was calculated by Equation 2 below. The ACI score is out of 200 points, and the higher the ACI score, the more excellent the anticoccidial ability, and when it is 120 points or more to less than 140 points, it is determined that it is effective as an anticoccidial material, and when it is 140 points or more to less than 160 points, it is determined that it is excellent as an anticoccidial material, and when it is 160 points or more, it is determined that the anticoccidial effect is very excellent. (Luis Miguel De Pablos et al., Anticoccidial activity of maslinic acid against infection with Eimeria tenella in chickens, Parasitol Res, 2010)

Anticoccidial index (ACI)=(survival rate after challenge inoculation (%))+(daily weight gain compared to negative control group (RWG, %))−(lesion score×10)−(fecal oocyst excretion amount index)   (Equation 2)

[0114] 1) Survival rate: the number of dead individuals was recorded daily, and post mortem autopsy was performed to determine a cause of mortality, and the number of individuals who died due to causes other than coccidiosis was excluded. The survival rate (%) up to the 8th day after challenge inoculation was used for anticoccidial index calculation.

[0115] 2) Daily weight gain: body weight was measured for each cage before challenge inoculation with a protozoan into an individual and at the 7th day after challenge inoculation, and the difference was divided by the number of days to calculate the daily weight gain (ADG, g/d). ‘Daily weight gain compared to the negative control group (RWG, %)’which was calculated by dividing the daily weight gain (ADG, average daily gain; g/d) of each experimental group by the weight gain (ADG, g/d) of the non-infected negative control group and multiplying by 100 was used for anticoccidial index calculation.

[0116] The daily weight gain (ADG, g/d) measured in each control group and test group and the daily weight gain compared to the negative control group (RWG, %) were shown in Table 4 below.

TABLE-US-00004 TABLE 4 Intermediate End body weight body weight (21-day-old, on Initial (14-day-old, the 7th day of body weight before challenge challenge Day 14~Day Treatment group (7-day-old) inoculation) inoculation) 21 ADG RWG Non-infected negative control group 197.1 473.7 956.8 65.99 100 Infected negative control group 200.1 493.2 886.9 53.98 82 Salinomycin treated group 192.9 455.9 917.6 62.46 94 Xanthone treated group 189.8 470.9 887.0 56.42 85 Mangosteen extract treated group 200.8 506.5 973.9 65.51 99

[0117] 3) Lesion scoring: On the 8th day after challenge inoculation, an autopsy was conducted for 4 broilers per cage, and the intestines were incised and opened. Scoring was performed for each coccidial lesion in the appendix region of the broilers. The lesion scoring method was performed by referring to Johnson JK & Reid WM (1970) document (Joyce Johnson, W.Malcolm Reid, Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens, Experimental parasitology, 1970). The lesion score is on a scale of 0-4, and 0 point corresponds to normal appendix, and 1 point is a mild infection symptom, and 2 points is a moderate infection symptom, and 3 points is a severe infection symptom and 4 points is a case of showing a very severe infection symptom or causing death. The appendix lesion score measured in each control group and test group was described in Table 5 below. The measured appendix lesion score was used for calculation of the anticoccidial index by multiplying by 10.

TABLE-US-00005 TABLE 5 Group Appendix lesion score Non-infected negative control group 0 Infected negative control group 3.1 Salinomycin treated group 2.7 Xanthone treated group 2.4 Mangosteen extract treated group 1.9

[0118] 4) Fecal oocyst excretion amount: the entire feces on the 6˜8th day of challenge inoculation were collected by cage, mixed evenly, and then randomly sampled 3 times in total by lg each. After floating the oocysts in lg feces using salt water, the oocyst excretion amount was measured using McMaster chamber, and the result was described in Table 6 below.

TABLE-US-00006 TABLE 6 Group Oocyst excretion amount/gram Non-infected negative control group 0.00E+00 Infected negative control group 1.90E+08 Salinomycin treated group 1.30E+08 Xanthone treated group 1.80E+08 Mangosteen extract treated group 1.30E+08

[0119] The oocyst excretion amount (%) compared to the infected negative control group was calculated by dividing the oocyst excretion amount by each group into the oocyst excretion amount of the infected negative control group and multiplying by 100. The oocyst excretion amount index was calculated as 0 when the calculated oocyst excretion amount compared to the infected negative control group was at a level of 0% to less than 1%, 5 when it was 1% or more to less than 26%, 10 when it was 26% or more to less than 51%, 20 when it was 51% or more to less than 76%, and 40 when it was 76% or more to 100% or less, and this was used for anticoccidial index calculation.

[0120] As aforementioned, the anticoccidial index by each test group measured by Equation 2 above was shown in Table 7 below. As shown in Table 7, it could be seen that the anticoccidial index in the mangosteen extract treated group was higher than the positive control group, salinomycin treated groups, so it showed the excellent anticoccidial efficacy.

TABLE-US-00007 TABLE 7 Group Anticoccidial index (ACI) Non-infected negative control group 197 Infected negative control group 101 Salinomycin treated group 141 Xanthone treated group 122 Mangosteen extract treated group 151

EXAMPLE 2. DIRECT KILLING EFFECT AGAINST Eimeria sp. PROTOZOAN OF MANGOSTEEN

Example 2-1. Measurement of Protozoal Direct Killing Effect of Mangosteen

[0121] In the present example, protozoan (sporozoite) direct killing ability evaluation was conducted against 3 representative Eimeria kinds known to be infected in most farms (E. tenella, E. acervulina, E. maxima).

[0122] A certain amount of oocysts of each protozoan was put in a tube containing glass beads and pulverized, and then to remove the crushed oocyst cell wall and other debris, internal sporocysts were purified using the percoll density gradient, and washed with PBS solution. A reagent comprising sodium taurocholic acid (Sigma aldrich, USA) and trypsin (Gibco, USA), respectively, was treated to sporocysts of Eimeria tenella, Eimeria acervulina and Eimeria maxima for excystation and they were incubated, and then they were washed with PBS solution once and protozoa were obtained.

[0123] After reacting the mangosteen pericarp extract, epicatechin, alpha-mangostin, xanthone and anticoccidial agents, salinomycin and diclazuril (hereinafter, material) with 3 kinds of Eimeria protozoa at various concentrations of 1 to 500 ppm, respectively, only alive protozoa (sporozoites) were counted through microscopic observation. Then, the death rate (%) of the protozoa when each material was treated compared to the PBS-treated negative control group was calculated and shown in Table 8 to Table 10 and Table 12, respectively, and the minimum concentration which directly killed the protozoa by 100% was shown in Table 11 below.

TABLE-US-00008 TABLE 8 Eimeria acervulina protozoal death rate (%) Mangosteen pericarp ppm Diclazuril Salinomycin Xanthone extract 500 44 100 25 100 250 12 100 11 100 125 23 73 0 100 100 0 60 0 100 50 0 0 0 100 10 0 0 0 91 5 0 0 0 65 1 0 0 0 16

TABLE-US-00009 TABLE 9 Eimeria tenella protozoal death rate (%) Mangosteen pericarp ppm Diclazuril Salinomycin Xanthone extract 500 44 100 15 100 250 35 100 0 100 125 0 74 10 100 100 0 46 0 100 50 0 0 0 100 10 0 0 0 100 5 0 0 0 83 1 0 0 0 59

TABLE-US-00010 TABLE 10 Eimeria maxima protozoal death rate (%) Mangosteen pericarp ppm Diclazuril Salinomycin Xanthone extract 500 30 100 12 100 250 16 97 5 100 125 0 65 0 100 100 0 59 0 100 50 0 0 0 100 10 0 0 0 84 5 0 0 0 48 1 0 0 0 17

TABLE-US-00011 TABLE 11 Minimum concentration for killing 100% protozoa (ppm) Eimeria Eimeria Eimeria Treatment group tenella acervulina maxima Negative control group — — — Salinomycin 500 250 500 Diclazuril >500 500 >500 Xanthone >500 >500 >500 Mangosteen pericarp 10 50 50 extract

[0124] As shown in Table 11, in the xanthone treatment group, there was no direct killing effect of 100% against the protozoa capable of inducing coccidium up to 500 ppm, and in case of diclazuril, against Eimeria tenella and Eimeria maxima, there was no direct killing effect of 100% against the protozoa up to 500 ppm. In the mangosteen extract, at a significantly lower concentration than other groups, 100% of Eimeria tenella, Eimeria acervulina and Eimeria maxima could be killed, so it could be confirmed that the protozoal killing effect was significantly excellent.

TABLE-US-00012 TABLE 12 Eimeria tenella protozoal death rate Mangosteen ppm pericarp extract Epicatechin Alpha-mangostin 15.6 100 0 93.3 7.8 93.3 0 50 3.9 40 0 23.3

[0125] As shown in Table 12, the mangosteen extract could kill 100% of the Eimeria tenella protozoan at the significantly lower concentration, 15.6 ppm than the individual components comprised in the mangosteen, epicatechin and alpha-mangosteen, so it could be confirmed that the protozoal killing effect was significantly excellent. By LC-MS analysis (LC-MS analysis conditions are as follows: LC was waters acquity UPLC i-class, and for MS, waters xevo G2-XS(Q-TOF) product was used. For the used column information, waters acquity BEH C18 column was used.), the components and contents in the mangosteen extract were analyzed and this result was shown in Table 13. As shown in Table 13, the content of the alpha-mangostin in the mangosteen extract was at a level of about 12%, and considering this, the anticoccidial effect of the mangosteen extract itseft was superior to that of the alpha-mangostin.

TABLE-US-00013 TABLE 13 Active ingredient in mangosteen extract (%) Alpha mangostin 12.3% Gamma mangostin 1.06% Quercetin 0.008% Epicatechin 0.03%

EXAMPLE 3. EXAMPLE 3. CELL INVASION AND INTRACELLULAR PROPAGATION INHIBITORY EFFECT AGAINST EIMERIA PROTOZOA OF MANGOSTEEN

[0126] In the present example, using MDBK cell line which is a representative animal cell known to cause Eimeria infection and propagation, the inhibition ability of intracellular protozoan invasion and intracellular protozoal propagation of the mangosteen was investigated.

Example 3-1. Cell Invasion Inhibitory Effect of Protozoan According to Mangosteen Treatment

[0127] 100,000 MDBK cells (purchased from ATCC) were aliquoted in a 24-well plate, and then incubated at a temperature of 37° C. for 12 hours. The protozoan of Eimeria acervulina was obtained similar to the method of Example 2 above. 200,000 protozoa per one well were added to wells in which cells were aliquoted, and each material (mangosteen, xanthone and anticoccidial agents, salinomycin and diclazuril) was treated to the cells by concentration (1 or 10 Oppm), and cultured at a temperature of 41° C. for 24 hours. A negative control group is a MDBK cell infected by protozoa, and a positive control group means a group in which salinomycin or diclazuril solution was incubated with the protozoan. Thereafter, in order to remove the protozoa that did not invade cells, the cells were washed twice using PBS solution. After removing the cells and protozoa inside the cells through pipetting, DNA was extracted from the cells, and PCR was performed using E.acervulina ACE gene-specific primers. The sequences of the used primers were described in Table 14 below.

TABLE-US-00014 TABLE 14 Nucleotide sequence Primer (5′.fwdarw.3′) SEQ ID NO E.tenella ITS-1 Forward TGGAGGGGATTATGAGAG SEQ ID NO: 1 GA Reverse CAAGCAGCATGTAACGGA SEQ ID NO: 2 GA E.acervulina ACE Forward GCAGTCCGATGAAAGGTAT SEQ ID NO: 3 TTG Reverse GAAGCGAAATGTTAGGCC SEQ ID NO: 4 ATCT

[0128] Ct values before/after washing for each material were compared and corrected with the ΔCt value of the negative control group to calculate the cell invasion inhibition rate (%) of the protozoa through treatment of each material, and the result was shown in FIG. 1. As shown in FIG. 1, it could be confirmed that the cell invasion inhibition efficacy against Eimeria acervulina in the mangosteen extract treatment group was excellent.

Example 3-2. Intracellular Propagation Inhibition Effect of Protozoan According to Mangosteen Treatment

[0129] 100,000 MDBK cells (purchased from ATCC) were aliquoted in a 24-well plate, and then incubated at a temperature of 37° C. for 12 hours. The protozoan of Eimeria tenella was obtained similar to the method of Example 2 above. 200,000 protozoa per one well were added to wells in which cells were aliquoted, and cultured at a temperature of 41° C. for 24 hours, and then the cells were washed using PBS solution twice to remove the protozoa which were not attached to the cells. Each material (mangosteen, xanthone and anticoccidial agents, salinomycin and diclazuril) was treated to the cells by concentration, and further cultured at a temperature of 41° C. for 24 hours. A negative control group is a MDBK cell infected by protozoa, and a positive control group means a group in which salinomycin and diclazuril solution was incubated with the protozoa. After removing the cells and protozoa propagated inside the cells through pipetting, DNA was extracted from the cells, and PCR was performed using E. tenella ITS-1 (Internal transcribed spacer-1) gene-specific primers. The sequences of the used primers were described in Table 14 above.

[0130] The Ct values in the material treatment groups compared to the negative control group were compared to calculate the intracellular protozoan (sporozoite) propagation inhibition rate (%), and the result was shown in FIG. 2.

[0131] As shown in FIG. 2, the mangosteen extract treatment group showed a significantly excellent intracellular protozoal propagation inhibition effect of E. tenella than other groups.

EXAMPLE 4. EVALUATION OF CYTOTOXICITY OF MANGOSTEEN

[0132] In the present example, using MTS analysis method (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, promega, USA), an effect on survival of cells of the mangosteen extract was investigated.

[0133] The cell lines utilized in cytotoxicity evaluation were (1) MDBK(Madin-Darby Bovine Kidney Epithelial Cells) and (2) RAW264.7(Mouse macrophage cell), and all of them were purchased from ATCC and used.

[0134] Each cell was aliquoted at a concentration of 1×10.sup.5 cell/well in a 96-well cell culture plate, and cultured at 37° C. for 24 hours, and then each material (mangosteen extract, xanthone, alpha-mangostin and epicatechin) was treated by concentration (3.1˜500 ppm/well) and cultured at a temperature of 37° C. After 14 hours, MTS solution was added to the cell culture solution, and incubated for 2 hours, and the absorbance was measured at a wavelength of 490nm by a microplate reader, and the cell survival rate (%) compared to the 5% DMSO treatment group, which was a negative control group, was calculated, and the cytotoxic minimum concentration where cells of 50% or more were survived was measured, and the result was shown in Table 15 below. In addition similar to the method of Example 2-1 above, the concentration of killing 100% of the Eimeria tenella protozoa of each material was measured, and the result was shown in Table 15.

[0135] As shown in Table 15, it could be confirmed that the cell safety was excellent as the mangosteen extract did not show cytotoxicity up to about 62.5 ppm, and also, it could be confirmed that the mangosteen extract showed an excellent protozoal killing effect at a significantly lower concentration than the concentration at which cytotoxicity was shown.

TABLE-US-00015 TABLE 15 Alpha Mangosteen Cell mangosteen Xanthone Epicatechin extract Minimum concentrataion of cytotoxicity at which cells of 50% or more survive (ppm) MDBK ≥7.8 ≥62.5 ≥500 ≥62.5 RAW264.7 ≥15.6 ≥62.5 ≥500 ≥62.5 Minimum concentrataion of killing protozoa 100% (ppm) Eimeria tenella ≥31.3 ≥500 ≥500 ≥10

EXAMPLE 5. EVALUATION OF ACID RESISTANCE OF MANGOSTEEN

[0136] In the presen example, the acid resistance of the mangosteen itselt to be evaluated. Hydrochloric acid (HC1) solution was added to the mangosteen extract prepared in Example 1-2 above, to adjust to be pH 2, 3 and 5.5, and then it was left at a temperature of 40° C. for 1 hour. Then, it was neutralized (pH 7.0) by adding sodium hydroxide (NaOH) solution, and 200,000 Eimeria acervuilna (E. acervuilna) protozoa (sporozoites) were exposed to the ginkgo leaf powder solution at various concentrations of 1 to 100 ppm diluted using PBS solution, and they were reacted at a temperature of 41° C. for 4 hours. Thereafter, the protozoal death rate (%) was measured by the similar method to Example 2 above, and this was shown in FIG. 3. As shown in FIG. 3, it could be confirmed that the mangosteen extract did not lose the killing efficacy against the protozoa inducing coccidiosis even under a strong acidic condition.

EXAMPLE 6. EVALUATION OF HEAT RESISTANCE OF MANGOSTEEN

[0137] In the present example, the heat resistance of the mangosteen was to be evaluated. The mangosteen extract prepared in Example 1-2 above was exposed at a temperature of 85 to 95° C. for 10 minutes, and then heat was cooled and it was diluted with PBS solution at a concentration of 1, 10, 50, 100 ppm. 200,000 Eimeria acervuline (E. acervulina) protozoa were reacted to the mangosteen extract at various concentrations of 1 to 100 ppm at a temperature of 41° C. for 4 hours, and the protozoal death rate (%) was measured by the similar method to Example 2 above, and this was shown in FIG. 4.

[0138] As shown in FIG. 4, it could be confirmed that the mangosteen extract did not lose the killing efficacy against the protozoa inducing coccidiosis even under an adverse condition of high temperature.

EXAMPLE 7. EVALUATION OF BROILER GROWTH EFFECT DUE TO MANGOSTEEN EXTRACT INTAKE

[0139] In the present example, an effect (improvement or degradation) on broiler growth of the mangosteen extract to be evaluated.

Example 7-1. Experimental Favility

[0140] An efficacy evaluation test was performed in an animal experiment facility in Gyeongsangnam-do, South Korea. One-day-old female Ross broilers were individually weighed and randomly divided into groups to use in an experiment. Matters and conditions for experimental design were described in Table 16.

TABLE-US-00016 TABLE 16 Category Experiment variable Breeding type Cage Broiler stocking age 1-day-old Total experiment period 14 days Gender Female Number of broilers per cage 20 Number of repetitions per treatment group 2 repetitions Number of treatment groups  6 Total number of broilers 240 broilers

[0141] A breeding farm was managed according to the Korean poultry breeding management guidelines. Cages and the breeding farm were cleaned and disinfected before starting the test. The temperature of 40 to 41° C. and the humidity of 40 to 50% of the breeding farm were maintained, and it was continuously monitored.

Example 7-2. Experimental Design

[0142] For the feed, Korea Feed A1-choi product was used, and each material (salinomycin (Cheil Bio Cheilsalino-60 product), xanthone (Sigma Aldrich X600 product), and the mangosteen extract prepared in Example 1-2 above) was added to the feed, respectively, at a concentration described in Table 17 below to be self-mixed. Antibiotics and supplements were not used in general feed and mixed feed, and no anticoccidial agent other than each material was added. Broilers were fed ad libitum throughout the experiment period. After putting 20 1-day-age broilers in cages randomly placed for each of the control group or test group and breeding, general feed was fed for 7 days, and then the mixed feed prepared above was ingested by dividing by control group or test group. The feed formulation administered to the control group (negative control group or positive control group) and test group was described in Table 17 below.

TABLE-US-00017 TABLE 17 Group Treatment group Negative control group General feed Salinomycin treated group Formulated feed comprising salinomycin (positive control group) 60 ppm Xanthone treated group 1 Formulated feed comprising xanthone 250 ppm Xanthone treated group 2 Formulated feed comprising xanthone 125 ppm Mangosteen extract treated Formulated feed comprising mangosteen group extract 125 ppm

Example 7-3. Calculation of Daily Weight Gain of Broilers

[0143] The body weight before feeding and on the 7th day after feeding the feed of Table 16 above into individuals was measured, and the difference was divided by the number of days to calculate the daily weight gain (ADG, g/d), and the result was shown in Table 18. For comparison between 6 treatment groups, Duncan post-test multiple comparison method was used, and statistical significance was verified using a SAS statistical program, and the result was shown in Table 18.

[0144] As shown in Table 18, in the mangosteen extract treated group, compared to the negative control group, positive control group and xanthone treated group, the daily weight gain was significantly increased. On the other hand, in case of the xanthone treated group, the daily weight gain was rather reduced compare to the negative control group.

TABLE-US-00018 TABLE 18 Initial End End-initial Daily body weight body weight body weight weight gain Duncan Treatment group (g) (g) (7 days, g) (ADG) p < 0.05 Negative control group 193 512 319 45.57 bc Salinomycin treated group 198 526 328 46.86 ab (positive control group) Xanthone treated group 1 201 485 284 40.57 d Xanthone treated group 2 202 509 307 43.86 bcd Mangosteen extract treated 208 539 331 47.29 a group