ANTICOCCIDIAL COMPOSITION COMPRISING STILBENE-BASED COMPOUND, AND USE THEREOF

Abstract

The present application relates to an anticoccidial composition comprising a stilbene-based compound, and a use thereof. A composition comprising a compound or a salt thereof, according to one embodiment, has excellent effects of direct killing of protozoa that can induce coccidiosis, inhibiting the cell penetration of the protozoa and/or inhibiting the intracellular proliferation of the protozoa, and preventing, alleviating, and treating in vivo coccidiosis.

Claims

1-11. (canceled)

12. A method for preventing, alleviating or treating coccidiosis, comprising a step of administering a composition, comprising a compound of Chemical formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient, to an animal: ##STR00005## in which R.sub.1 and R.sub.2 may be each independently OH or OCH.sub.3.

13. The method according to claim 12, wherein the R.sub.1 is OCH.sub.3 and R.sub.2 is OCH.sub.3.

14. The method according to claim 12, wherein the R.sub.1 is OH and R.sub.2 is OH.

15. The method according to claim 12, wherein the R.sub.1 is OH and R.sub.2 is OCH.sub.3.

16. The method according to claim 12, wherein the coccidiosis is induced by an Eimeria sp. protozoan.

17. The method according to claim 12, 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.

18. The method according to claim 12, wherein the composition is a feed composition comprising 1% (w/w) or less of the active ingredient based on the total weight.

19. The method according to claim 12, wherein the composition is a feed additive.

20. A method for killing Eimeria sp. protozoan, or inhibiting cell invasion or propagation of Eimeria sp. protozoan, comprising a step of administering a compound of Chemical formula 1, or a salt thereof, to an animal: ##STR00006## in which R.sub.1 and R.sub.2 may be each independently OH or OCH.sub.3.

21. The method according to claim 20, wherein the R.sub.1 is OCH.sub.3 and R.sub.2 is OCH.sub.3.

22. The method according to claim 20, wherein the R.sub.1 is OH and R.sub.2 is OH.

23. The method according to claim 20, wherein the R.sub.1 is OH and R.sub.2 is OCH.sub.3.

Description

MODE FOR INVENTION

[0104] Hereinafter, the present invention will be described more specifically by the following examples. However, they are intended to illustrate the present invention only, but the scope of the present invention is not limited by these examples.

[0105] The present application 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 Stilbene Compound

Example 1-1. Experimental Facility and Research Design

[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 29 days Gender Male Number of broilers per cage 6 Number of repetitions per treatment group 3 repetitions Number of treatment groups 5 Total number of broilers 90 broilers Kind of challenge inoculation protozoa Eimeria tenella Number of challenge inoculation oocysts 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. General feed was fed according to the nutritional requirements of broilers, and the mixing ratio was described in Table 3.

TABLE-US-00003 TABLE 3 Content Raw material (%) Corn 69.01 Soybean meal 23.99 Soybean oil 2.75 Dicalcium phosphate 2 Calcium carbonate 1.4 Salt 0.35 Poultry Vit Mix 0.2 Poultry Mineral Mix 0.15 DL-Methionine 0.1 Choline-chloride, 60% 0.05 Total 100 Calculated values (DM basis, %) CP, % 16 Ca, % 1.19 Avail. P, % 0.54 Lys, % 1 Met, % 0.42 Cys + Met, % 0.65 TMEn, kcal/kg 3585

Example 1-2. Experimental Design

[0108] To the general feed (Table 3), each material (salinomycin (CAS No. 53003-10-4), resveratrol (sigma) or pterostilbene (sigma) was added at a concentration described in Table 4 below, respectively, and was self-mixed.

[0109] 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 6 1-day-age broilers in cages randomly placed for each of the control group or test group and breeding, general feed was fed for 14 days, and then the mixed feed prepared above was ingested by dividing by control group or test group.

[0110] Coccidiosis induction was performed by oral inoculation (challenge inoculation) in an oral tube type of 10,000 oocysts (eggs) with over 90% mature (sporulation) Eimeria tenella per one individual to 21-day-old broilers.

TABLE-US-00004 TABLE 4 Group Treatment Non-infected negative General feed control group Infected negative control group Eimeria tenella infection + general feed Positive control group Eimeria tenella infection + formulated feed comprising salinomycin 60 ppm Resveratrol treated group Eimeria tenella infection + formulated feed comprising resveratrol 125 ppm Pterostilbene treated group Eimeria tenella infection + formulated feed comprising pterostilbene 125 ppm

Example 1-3. Measurement of Anticoccidial Activity of Stilbene

[0111] The anticoccidial efficacy by test group designed in Example 1-2 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)

[0112] 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. The measured survival rate was described in Table 5 below.

[0113] 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. 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 5 below.

[0114] 3) Lesion scoring: On the 8th day after challenge inoculation, an autopsy was conducted for 6 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 J K & Reid W M (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. A lesion index was calculated by multiplying the measured appendix lesion score by 10, and this was used for anticoccidial index calculation. The measured lesion score and lesion index in each control group and test group were described in Table 5 below.

[0115] 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 1 g each. After floating the oocysts in 1 g feces using salt water, the oocyst excretion amount was measured using McMaster chamber, and the result was described in Table 5 below. The result data were statistically treated using SAS Mixed Model (PROC MIXED), and data having a significant difference of p<0.05 were indicated by superscripts. 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 the result was described in Table 5 below, and this was used for anticoccidial index calculation.

TABLE-US-00005 TABLE 5 Group Control group Test group Compound Non-infected Infected negative negative control group control group Salinomycin Resveratrol Pterostilbene Survival rate 100 100 100 100 100 after challenge inoculation (%) BWG after 439.sup.a 298.sup.e 360.sup.cd 381.sup.bcd 389.sup.bc challenge inoculation (g/d) Daily weight 100  67  82  87  89 gain compared to negative control group (RWG, %) Lesion score  0.sup.d  3.4.sup.a  2.3.sup.c  2.95 2.8.sup.b Lesion index  0  34  23  29  28 Oocyst excretion  0.sup.c 1.3.E+08.sup.a 6.3.E+07.sup.b 5.2.E+07.sup.b 6.3.E+07.sup.b amount/g Oocyst index  0  40  10  10  10 ACI 200  93 149 149 151

[0116] As aforementioned, the anticoccidial index by each test group measured by Equation 2 above was shown in Table 5 above.

[0117] As shown in Table 5, in the Eimeria tenella-infected negative control group, the weight gain compared to the non-infected negative control group was reduced and the lesion score and oocyst excretion amount were increased. In the salinomycin treatment group used as a positive control group, compared to the infected negative control group, an effect of increasing the weight gain (p<0.05) and reducing the lesion score and oocyst excretion amount was shown (p<0.05). The resveratrol or pterostilbene treatment group showed an efficacy equivalent or superior to the salinomycin treatment group, and therefore, it showed a significant improvement in weight gain and a significant decrease in lesion index and oocyst excretion amount. As the result of calculating and comparing the anticoccidial index, resveratrol (149 points) and pterostilbene (151 points) showed an anticoccidial efficacy equivalent or superior to an anticoccidial agent, salinomycin (149 points).

Example 2. Direct Killing Effect Against Eimeria sp. Protozoan of Stilbene Compound

[0118] In the present example, protozoan (sporozoite) direct killing ability evaluation was conducted against 1 representative Eimeria kind known to be infected in most farms (E. tenella).

[0119] A certain amount of oocysts of each coccidial 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 for excystation of the internal sporozoites, and they were incubated, and then they were washed with PBS solution once and the protozoa were obtained.

[0120] After reacting the pterostilbene, pinostilbene and anticoccidial agents, diclazuril and gallic acid (hereinafter, material) with the protozoan of Eimeria tenella at various concentrations of 16 to 500 ppm, respectively, only alive protozoa (sporozoites) were counted through microscopic observation. Then, the direct death rate (%) of the protozoan by material concentration was shown in Table 6 below.

TABLE-US-00006 TABLE 6 Protozoal death rate by material Treatment treatment concentration (%) group 500 250 125 62 32 16 Pterostilbene 100 100 100 74.2 63.4 33.3 Pinostilbene 61.3 50.5 0 0 0 0 Diclazuril 44 35 0 0 0 0 Gallic acid 32 26 0 0 0 0

[0121] As shown in Table 6 above, in the gallic acid and diclazuril treatment groups, the death rate of 50% or more against the protozoan (sporozoite) capable of inducing coccidium was not shown at 500 ppm or less. The pterostilbene treatment group showed the death rate of 50% or more against an Eimeria tenella protozoan at 32 ppm or more, and thereby, it could be confirmed that the protozoal killing effect was significantly excellent. In addition, pinostilbene showed the protozoal death rate of 50% or more at 250 ppm or more, and thereby, it could be confirmed that the protozoal killing effect was significantly excellent in the same manner.

[0122] From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics. In this regard, it should be understood that the examples described above are illustrative and not restrictive in all respects. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described below and equivalent concepts rather than the detailed description above, in the scope of the present application.