Application of gossypol in preparation of chicken feed or medicine for preventing and treating chicken coccidiosis

Abstract

The invention provides application of gossypol in preparation of chicken feed or medicine for preventing and treating chicken coccidiosis, and relates to the technical field of parasitic disease prevention and treatment. Experimental exploration proves that gossypol has a new application of preventing and treating chicken coccidiosis, a new scheme is provided for development of drugs and feeds for preventing and treating chicken coccidiosis, a new idea is provided for clinical control of chicken coccidiosis, and the application range of gossypol is expanded. Gossypol acts on chicks infected with chicken eimeria tenella disease, compared with chicks in a challenge control group, the reduction rate of oocyst discharge of chicks in a gossypol treatment group reaches 82.5%, the relative weight gain rate can reach 90.8%, the coccidiosis resistance index of the chicks can reach 167.4, and bleeding symptoms caused by chicken coccidiosis can be effectively reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of parasitic disease prevention and treatment, and particularly relates to application of gossypol in preparing chicken feed or medicine for preventing and treating chicken coccidiosis. Background Art

[0002] Coccidiosis, a parasitic protozoal disease primarily characterized by intestinal lesions, is one of the most common diseases in intensive chicken farms and is distributed worldwide. The morbidity rate is as high as 50-70%, with a mortality rate of 20-30%, and in severe cases, as high as 80%. Coccidiosis is characterized by hemorrhagic enteritis, bloody diarrhea, high morbidity in chicks, slowed growth, poor feed conversion, and high mortality. It significantly reduces chicken production performance and causes significant economic losses to the poultry industry. Coccidiosis is characterized by rapid onset, high morbidity, high mortality, and significant harm.

[0003] The main pathogens of coccidiosis include Eimeria tenella, E. necatrix, E. brunetti, E. acervulina, E. maxima, E. mitis, and E. praecox. Eimeria tenella is the most pathogenic and causes the highest mortality rate in chickens. It primarily parasitizes the intestinal epithelial cells of chickens. Clinically, it manifests as severe diarrhea with blood in the feces. Some affected chickens become comatose and unable to stand normally, and die 2-3 days after the onset of symptoms.

[0004] Currently, coccidiosis prevention and treatment methods include chemical treatment, antibiotics, and coccidiosis vaccination. However, the long-term use of chemical drugs can lead to drug resistance and drug residues in poultry products, effectively preventing coccidiosis in chickens and causing harm to humans. Coccidiosis vaccines, on the other hand, are not widely adopted due to their high cost, cumbersome administration, and the high risk of coccidiosis outbreaks in chicken farms. Therefore, developing an effective drug for coccidiosis prevention and treatment in chickens is an urgent need for those skilled in the art.

[0005] Gossypol is a yellow polyphenolic hydroxybisnaphthaldehyde compound found primarily in the roots, stems, leaves, and seeds of the mallow plant, cotton, with the highest concentration found in cottonseed kernels. Gossypol inhibits spermatogenesis and sperm motility and also has anti-tumor and antiviral activity. Currently, no studies have shown that this drug has a role in preventing and treating chicken coccidia. The molecular formula of gossypol is C 30 H 30 O8, CAS number: 303-45-7, chemical structure:

[0006] Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention aims to provide an application of gossypol in preparing chicken feed or medicine for preventing and treating chicken coccidiosis, wherein the preventing and treating chicken coccidiosis includes: preventing or treating chicken coccidiosis.

[0009] Preferably, the pathogen of chicken coccidiosis is Eimeria.

[0010] Preferably, the pathogen of chicken coccidiosis is Eimeria tenella.

[0011] Preferably, the prevention or treatment of chicken coccidiosis includes: inhibiting the growth of Eimeria tenella, reducing the excretion of Eimeria tenella oocysts, and increasing the survival rate and relative weight gain rate of chickens infected with Eimeria tenella.

[0012] Preferably, the chicken feed and pharmaceutical active ingredients for preventing and treating chicken coccidiosis include gossypol; gossypol can be used to prevent and treat chicken coccidiosis alone or in combination with one or more other active ingredients and / or one or more pharmaceutically acceptable carriers.

[0013] Preferably, the gossypol includes gossypol acetate, purified gossypol and gossypol formate.

[0014] Preferably, the other active ingredients include Chinese medicinal materials and / or drugs that can prevent or treat chicken coccidiosis and alleviate the symptoms of chicken coccidiosis.

[0015] Preferably, the Chinese medicinal materials include areca nut, radix scutellariae, artemisia annua, herba groundgrass, herba agrimonii, pulsatillae, brucea javanica, sophora flavescens, milk thistle, pomegranate peel, turmeric, magnolia bark, etc.; the drugs include diclazuril, toltrazuril (toltrazuril), salinomycin, dinitolamide, sulfachloropyrazine sodium, monensin, arecoline, febrifugine, artemisinin and its derivatives, garlic essential oil, allicin, silymarin, total phenols of magnolia bark, etc.

[0016] Preferably, the carrier includes pharmaceutically acceptable diluents, wetting agents, adhesives, flash disintegrators, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, preservatives, pH regulators, surfactants, excipients, fillers and synergists.

[0017] Preferably, diluents include starch, sugars (such as sucrose, lactose, maltose), celluloses (such as microcrystalline cellulose, methylcellulose, hydroxypropyl methylcellulose), inorganic salts, etc.; wetting agents include water, ethanol, glycerol, etc.; binders include starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; disintegrants include starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium dicarboxymethyl cellulose, surfactants, etc.; lubricants include talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, polyethylene glycol, etc.; color, flavor and seasoning agents include pigments, sweeteners, spices, mucilage, etc.; solvents include water, Glycerol, ethanol, etc.; solubilizers include Tweens, sorbits, sulfates, sulfonates, etc.; cosolvents include organic acids (such as citric acid) and their salts, inorganic salts, polyethylene glycol, etc.; emulsifiers include spans, glycerol fatty acid esters, gum arabic, gelatin, agar, sodium alginate, etc.; antioxidants include sulfites, ascorbic acid, gallic acid and its salts, etc.; metal chelating agents include disodium edetate, polycarboxylic acid compounds, etc.; preservatives include parabens, quaternary ammonium compounds, chlorhexidine acetate, etc.; pH adjusters include hydrochloric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, citrates, etc.

[0018] Preferably, the dosage forms of the anti-coccidiosis drug include, but are not limited to, oral dosage forms, external preparations, suppositories, and sterile injectable solutions in the form of powders, dusts, granules, tablets, microcapsules, suspensions, emulsions, syrups, and sprays. It will be appreciated that the gossypol of the present invention can be combined with a suitable type of carrier to prepare drugs in various forms, such as liquid, gaseous, semisolid, and solid, to form corresponding dosage forms.

[0019] Preferably, the administration methods of the anti-coccidiosis drug include, but are not limited to, group administration, individual administration, and a combination of group and individual administration. It is understood that the gossypol described herein can be administered in a variety of ways, depending on the different excipients and dosage forms. For example, group administration methods include, but are not limited to: ① water-mixing administration: First, the drug's solubility in water must be understood; second, the drug dosage must be calculated based on water intake. If the drug's stability in water is poor, the "thirst medication method" can be considered; ② mixed-feed administration: The drug is mixed into feed for ad libitum consumption, but the drug and feed must be mixed evenly. An incremental dilution method can be used; and ③ aerosol administration. Individual administration methods include, but are not limited to: ① oral administration: This method offers the advantages of safety, convenience, and economy; ② injection administration: The advantages of rapid and complete drug absorption, accurate dosage, and effective efficacy. Injection administration includes subcutaneous injection, intramuscular injection, intravenous injection, intrathecal injection, crop injection, arterial injection, and joint capsule injection; and ③ topical administration: application, dusting, wet compress, instillation, enema, inhalation, and implantation.

[0020] Preferably, the chicken feed comprises gossypol and basal feed.

[0021] Preferably, the basic feed includes complete feed, concentrated feed, premix feed, concentrate mixture or mixed feed.

[0022] Beneficial effects of the present invention:

[0023] 1. Currently, gossypol has a relatively narrow application area, and there are no research reports on its application in the field of chicken coccidiosis. The present invention has experimentally demonstrated that gossypol has a new use in preventing and treating chicken coccidiosis, providing a new solution for the development of drugs and feeds for preventing and treating chicken coccidiosis, offering new ideas for clinical control of chicken coccidiosis, and expanding the scope of application of gossypol.

[0024] 2. In the present invention, when gossypol was applied to chicks infected with Eimeria tenella, compared to the chickens in the challenge control group, the gossypol-treated group achieved a relative weight gain of 90.8%, a survival rate of 87.5-100%, and an 82.5% reduction in oocyst excretion. It also effectively reduced hemorrhagic symptoms caused by coccidiosis, indicating that gossypol can significantly reduce the development and reproduction of Eimeria tenella and oocyst formation in chickens, demonstrating its effectiveness in preventing and / or treating Eimeria tenella. Calculations show that the anticoccidial index of gossypol applied to chicks reached 167.4, making it a moderately effective anticoccidial drug. DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0026] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those skilled in the art in the field of the present invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0027] The various raw materials, reagents, instruments and equipment used in the examples can be purchased from the market or prepared by existing methods. Unless otherwise specified, all reagents used in the present invention are of analytical grade.

[0028] Example 1 Preparation of sporulated oocysts of Eimeria tenella

[0029] Ten 14-day-old SPF chicks were selected and each chicken was orally inoculated with 100 μL of oocyst suspension (i.e., 6,000 oocysts). After the inoculation, the chicks were fed with feed dried in a constant temperature drying oven at 80°C and boiled water in a coccidia-free environment.

[0030] Collect feces 6-7 days after infestation. Place the collected feces in a 5L measuring cup and add 2000mL of water. Stir thoroughly. Filter the feces through 80, 100, and 200 mesh sieves in sequence. Repeat the filtration 2-3 times and collect the filtrate. Centrifuge the collected filtrate for 5 minutes, discard the supernatant, and retain the fecal sediment. Add 20mL of water to the centrifuge cup and mix thoroughly using a vortex instrument. Centrifuge for 5 minutes and repeat the washing 3-4 times until the supernatant color shows no significant change. Add 3 times the volume of saturated salt water to the centrifuge cup containing the fecal sediment at the bottom. Use a vortex instrument to stir thoroughly and transfer the mixture to a 500mL glass beaker. Let it stand for 5 minutes. Then, divide the entire solution into 50mL centrifuge tubes, centrifuge for 5 minutes, and collect the supernatant. Dilute the supernatant with at least 7 times the volume of water, divide the solution into 50 mL centrifuge tubes, centrifuge for 5 min, and discard the supernatant. Rinse the oocysts at the bottom of each centrifuge tube with 20 mL of 2.5% potassium dichromate solution, collect the oocysts into culture bottles, and place them in a shaker at 28°C for aeration culture. After culture, store them at 4°C for later use.

[0031] Example 2 Evaluation of the effect of gossypol against chicken coccidia

[0032] 2.1 Test reagents and main instruments

[0033] 2.2 Experimental animals

[0034] Experimental animals: 150 one-day-old three-yellow chickens were purchased from Hunan Animal Husbandry and Veterinary Research Institute.

[0035] Feeding conditions: Strictly kept in a clean animal henhouse without coccidia, free access to food, sufficient light, and managed by a dedicated person.

[0036] 2.3 Reagents and materials

[0037] 2.3.1 Trial Drugs

[0038] Positive drug: 0.5% diclazuril solution, add 0.2 mL to every 1 L of water.

[0039] Test drug: 20 mg / mL gossypol acetate (2 g of gossypol acetate powder was weighed and dissolved in 100 mL of ultrapure water to prepare a 20 mg / mL aqueous solution).

[0040] Feed: It does not contain any anticoccidial drugs and is made by Hunan Animal Husbandry and Veterinary Research Institute.

[0041] 2.4 Test methods

[0042] 2.4.1 Trial Grouping

[0043] 200 one-day-old three-yellow chickens were fed to 13 days of age and weighed, and overweight and underweight chicks were eliminated. The remaining chicks were randomly divided into 6 groups, 16 chicks / group, and 4 replicates per group (n=4). There should be no statistical difference in body weight among the groups.

[0044] 2.4.2 Dosage and coccidia infection

[0045] The drugs were mixed into the feed according to the requirements in Table 1. At 12 days of age, all the groups except the non-infected and non-medicated group and the positive control group were given drugs until the end of the experiment. At 14 days of age, each chicken in the other groups except the non-infected and non-medicated group was orally infected with 1×10 5 The experimental group was gavaged once a day with 20 mg / mL gossypol diluted with ultrapure water according to the average body weight of the corresponding dose group.

[0046] Table 1 Grouping of the gossypol coccidia control test

[0047]

[0048]

[0049] 2.4.3 Index detection method

[0050] 2.4.4 Determination of the anticoccidial index (ACI)

[0051] The anticoccidial index (ACI) is calculated based on the statistical analysis of relative weight gain, survival rate, cecal lesions, and oocyst count. The formula is: ACI = (relative weight gain + survival rate) - (oocyst count + lesion count). According to the anticoccidial efficacy criteria in the "Coccidiology of Chickens": ACI < 120 indicates ineffective anticoccidial efficacy; 120 ≤ ACI < 160 indicates low anticoccidial efficacy; 160 ≤ ACI < 180 indicates moderate anticoccidial efficacy; and ACI ≥ 180 indicates high anticoccidial efficacy.

[0052] 2.4.5 Weight gain index

[0053] The chicks were weighed on an empty stomach at 14 and 21 days of age, with the weight at 14 days of age recorded as the initial weight and the weight at 21 days of age recorded as the final weight, and the relative weight gain rate was calculated.

[0054] Average weight gain = average final weight - average initial weight,

[0055] Relative weight gain rate = average weight gain of chicks in each group / average weight gain of negative control group × 100%.

[0056] 2.4.6 Mortality

[0057] During the whole process, the death of chickens was observed, and they were dissected immediately after death. The cecal lesions were observed to determine whether they died of coccidiosis and calculate the mortality rate.

[0058] 2.4.7 Cecal lesion scoring

[0059] On the 8th day after artificial infection with sporulated eggs of Eimeria tenella, the chicks were necropsied and the side with obvious cecal lesions was selected for scoring. Details are shown in Table 2. Cecal lesion value = average cecal score of each group of chicks × 10

[0060] Table 2 Lesion scoring rules

[0061]

[0062] 2.4.8 Oocyst value

[0063] Starting on day 6 after gavage with sporulated Eimeria tenella oocysts, feces were collected from each group until the end of the experiment. The feces from each group were thoroughly mixed. 2 g of feces to be tested were placed in a beaker. 10 mL of deionized water, followed by 50 mL of saturated saline, were added. After thorough mixing, 1 mL of the fecal solution was pipetted into a McMaster counting chamber and allowed to stand for 5 minutes. The number of eggs in two graduated chambers was counted under a microscope. The average of the two chambers' egg counts multiplied by 200 was the number of eggs per gram of feces. At day 21 of age, 2 g of cecal contents from each group of test chickens were collected and thoroughly mixed. Oocysts were counted under a microscope using a McMaster counting chamber. The number of oocysts per gram of feces (OPG) was calculated and averaged over three replicates.

[0064] The oocyst ratio = OPG of the test chickens in each treatment group / OPG of the test chickens in the positive control group × 100%, and the oocyst value is calculated based on the oocyst ratio.

[0065] Table 3 Calculation of oocyst value

[0066] Oocyst ratio / % 0～1％ 1～26％ 27～50％ 51～75％ 76～100％ Oocyst value 0 5 10 20 40

[0067] 2.4.9 Scoring bloody stools

[0068] On the 5th, 6th and 7th days after the oral administration of sporulated oocysts of Eimeria tenella, the bloody stools of each test group were observed and scored: 0 points means 100% of the stools were free of blood; 1 point means 25% of the stools were bloody; 2 points means 50% of the stools were bloody; 3 points means 75% of the stools were bloody; 4 points means 100% of the stools were bloody.

[0069] 2.5 Test results

[0070] 2.5.1 Relative weight gain rate

[0071] Table 4 Effect of gossypol acetate on the body weight of experimental chickens

[0072]

[0073] The results showed that the average weight gain and relative weight gain rate of the challenged control group were significantly lower than those of the blank group (P≤0.01); the average weight gain and relative weight gain rate of the medium and low dose groups of gossypol were not different from those of the blank group and diclazuril group (P≤0.05), indicating that 50-100 mg / kg of gossypol can effectively improve the weight loss of chickens caused by coccidiosis.

[0074] 2.5.2 Mortality

[0075] Table 5 Effect of gossypol acetate on mortality of experimental chickens

[0076]

[0077] The results showed that compared with the 75% mortality rate of the control group treated with poison, the diclazuril treatment group and the medium-dose gossypol treatment group had a 100% survival rate, and the high and low-dose gossypol groups had an 87.5% survival rate, indicating that gossypol can effectively alleviate the death of chickens caused by coccidia infection.

[0078] 2.5.3 Cecal lesion scoring

[0079] Table 6 Effects of gossypol acetate on cecal lesions in experimental chickens

[0080]

[0081] The results showed that the lesion value of the coccidia infection group was 34.2, which was significantly higher than that of the blank group, diclazuril group and gossypol group (P≤0.05); the lesion value of the medium-dose gossypol group was 15.8, which was significantly lower than that of the high- and low-dose gossypol groups (P≤0.05), indicating that gossypol can effectively reduce the cecal mucosal damage caused by coccidiosis in chickens.

[0082] 2.5.4 Oocyst Value

[0083] Table 7 Effect of gossypol acetate on the oocyst value of experimental chickens

[0084]

[0085] The results showed that the oocyst reduction rate in the diclazuril group was 99.9%, while the reduction rates in the high-, medium-, and low-dose gossypol groups were 67.2%, 82.5%, and 78.7%, respectively, significantly lower than those in the challenge control group (P < 0.01). The results indicate that gossypol can effectively reduce oocyst excretion in infected chickens, but its effectiveness is less than that of diclazuril.

[0086] 2.5.5 Scoring bloody stools

[0087] Table 9 Effect of gossypol acetate on bloody stool scores of experimental chickens

[0088]

[0089] The results showed that the coccidia infection group had obvious bloody stools, which was significantly higher than that of the gossypol treatment group (P < 0.05), indicating that gossypol can effectively alleviate the bleeding symptoms caused by chicken coccidia.

[0090] 2.5.6 ACI Calculation

[0091] Table 8 Effect of gossypol acetate on ACI of experimental chickens

[0092]

[0093] The results showed that the ACI in the diclazuril-treated group was above 180, while the ACI in the infected group was below 120, indicating that the positive drug control group and model group were established. The ACI in the medium-dose gossypol group was between 160 ≤ ACI and < 180, indicating a moderate anticoccidial effect. The ACI in the high- and low-dose gossypol groups was between 120 ≤ ACI and < 160, indicating a low anticoccidial effect. This indicates that gossypol, at a dose of 100 mg / kg, can achieve the effect of a moderate anticoccidial drug.

[0094] 2.6 Test Conclusion

[0095] Gossypol can treat chicken coccidiosis by reducing chicken mortality, oocyst excretion, cecal mucosal damage, bloody stool excretion and restoring weight gain. When the dosage is 100 mg / kg (BW), the ACI is 167.4, which shows that it is a medium-effective anticoccidial drug.

[0096] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. Application of gossypol in preparing chicken feed or medicine for preventing and treating chicken coccidiosis.

2. According to claim 1, it is characterized in that The control of chicken coccidiosis includes: preventing or treating chicken coccidiosis.

3. According to claim 2, it is characterized in that The pathogen of chicken coccidiosis is Eimeria.

4. According to claim 3, it is characterized in that The pathogen of chicken coccidiosis is Eimeria tenella.

5. According to claim 4, it is characterized in that The drug for preventing and treating chicken coccidiosis includes gossypol, which can be used for preventing and treating chicken coccidiosis alone or in combination with one or more other active ingredients and / or one or more pharmaceutically acceptable carriers.

6. According to claim 5, it is characterized in that The carrier includes pharmaceutically acceptable diluents, wetting agents, adhesives, flash disintegrating agents, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, preservatives, pH regulators, surfactants, excipients, fillers and synergists.

7. According to claim 5, it is characterized in that The other active ingredients include traditional Chinese medicines and / or drugs that can prevent or treat chicken coccidiosis or alleviate the symptoms of chicken coccidiosis.

8. According to claims 1-7, it is characterized in that The gossypol includes gossypol acetate, refined gossypol and gossypol formate.

9. An anti-chicken coccidiosis drug, characterized in that: The active ingredients include gossypol.

10. A chicken feed resistant to coccidiosis, characterized in that: The active ingredients include gossypol.

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