Compound traditional Chinese medicine and application thereof in preparation of medicine for resisting eimeria tenella or treating coccidiosis

Abstract

The invention discloses a compound traditional Chinese medicine and application thereof in preparing a medicine for resisting eimeria tenella or treating coccidiosis, and relates to the technical field of livestock breeding. The compound traditional Chinese medicine comprises the following raw materials in parts by mass: 5-20 parts of hesperidin and 1-10 parts of citric acid. According to the invention, chicks are taken as experimental subjects, a chick coccidiosis model is established, and the effect of the compound traditional Chinese medicine on the chick coccidiosis is researched from the aspects of general conditions of the chicks, bloody stool conditions, coccidiocyst discharge conditions, cecum tissue lesion conditions, pathological detection, serum biochemistry, oxidation resistance detection and the like. The compound traditional Chinese medicine achieves a good anti-coccidiosis drug level, the required raw materials are low in cost, convenient to use and free of hormones and antibiotics, no toxic or side effect is caused to chickens, drug resistance cannot be caused, a new choice is provided for replacing chemical drugs for preventing and treating coccidiosis, and addition of anti-coccidiosis drugs in chicken feed is expected to be reduced or replaced.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry technology, specifically involving a compound traditional Chinese medicine and its application in the preparation of drugs for treating Eimeria tenella or coccidiosis. Background Technology

[0002] Coccidiosis is a common parasitic disease caused by a series of coccidia in the genus *Eimeria*. Its characteristic lesions are hemorrhagic changes in the chicken's intestines, leading to a range of clinical symptoms such as lethargy, ruffled and dull feathers, decreased or even complete loss of appetite, and bloody stools. This disease is most prevalent in chicks, especially those aged 8-20 days, where the morbidity rate is generally over 70%, and the mortality rate can reach 50% or higher. Besides chicks, chickens of other ages are also susceptible, but have a higher survival rate. Those that survive may experience stunted growth, malnutrition, and lethargy, directly reducing the economic benefits of poultry farming. In laying hens, infection with this disease will gradually reduce or even stop egg production, diminishing or eliminating their reproductive function, thus becoming a major threat to the poultry industry.

[0003] After sexual reproduction, coccidia oocysts are excreted in feces, contaminating their living environment and indirectly infecting other chickens. If not detected and controlled in time, this process will repeat, eventually leading to an epidemic in the region, posing a significant threat and loss to the poultry industry. Currently, the prevention and control of coccidiosis in chickens mainly relies on ionotropic drugs and chemically synthesized drugs. However, due to the long-term, large-scale, and even abused use of these drugs, the problem of drug resistance in coccidia is becoming increasingly prominent. Numerous reports indicate that almost all commercially available anticoccidial chemicals have developed drug-resistant strains, and multidrug resistance and cross-resistance are becoming increasingly serious. This widespread drug resistance often leads to poor or even failed control of coccidiosis in chickens. Once an outbreak occurs, farmers will face a predicament of having no drugs available, resulting in huge economic losses.

[0004] Due to the widespread existence of drug resistance, common chemical drugs are gradually losing their anticoccidial efficacy, making the control of coccidiosis in chickens increasingly difficult. It is conservatively estimated that my country spends over US$3 billion annually on the prevention and control of chicken coccidiosis, resulting in economic losses exceeding US$3 billion. To effectively control chicken coccidiosis, in addition to improving the skills of farmers and poultry farm employees, enhancing flock immunity, and improving flock health, it is even more important to respond to the national call for "banning antibiotics" and "antibiotic alternatives," actively seeking natural alternatives to anticoccidial drugs to more effectively prevent and control chicken coccidiosis while ensuring the health of chickens, the environment, humans, and society.

[0005] To maximize the health of animals, the environment, and humans, and to avoid excessive use of chemical drugs and vaccines, natural medicines have become an ideal approach. As the birthplace of Traditional Chinese Medicine (TCM), my country possesses abundant medicinal resources, a natural advantage. Effectively exploring anticoccidial TCM resources to improve anticoccidial efficacy and ensure the safety of chicken meat holds great promise.

[0006] The above background information is provided only to aid in understanding the inventive concept and technical solution of this invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above information was disclosed on the filing date of this patent application, the above background information should not be used to evaluate the novelty and inventiveness of this application. Summary of the Invention

[0007] This invention provides a compound traditional Chinese medicine and its application in the preparation of drugs for treating Eimeria tenella or coccidiosis, so as to effectively treat coccidiosis in chickens, provide a new approach to treating coccidiosis in chickens, and avoid drug resistance and drug residues caused by chemical drug treatment.

[0008] The technical solution provided by this invention is as follows:

[0009] A compound traditional Chinese medicine used in the preparation of drugs for treating coccidiosis or Eimeria tenella, comprising the following raw materials in parts by weight: 5-20 parts of hesperidin and 1-10 parts of citric acid.

[0010] Furthermore, an optimal solution was selected through experimentation, comprising the following parts by weight of raw materials: 10 parts hesperidin and 5 parts citric acid.

[0011] Furthermore, the purity of hesperidin is ≥97%, and the purity of citric acid is ≥99%.

[0012] Furthermore, the compound traditional Chinese medicine includes the active ingredient being the powder of the raw material or an aqueous or organic solvent extract of the raw material, and pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage form of the compound traditional Chinese medicine includes one or more of the following: powder, decoction, oral liquid, granules, pills, and tablets.

[0014] This invention also provides the application of a compound traditional Chinese medicine used in the preparation of drugs for treating or treating coccidiosis in the preparation of feed or feed additives.

[0015] Furthermore, the dosage of the compound traditional Chinese medicine added to the feed by mixing is 125-500g / ton of feed.

[0016] Furthermore, the compound traditional Chinese medicine is added to the feed at a dosage of 500g / ton of feed when administered by mixing.

[0017] Furthermore, the application of the compound traditional Chinese medicine in feed preparation, the feed preparation method includes the following steps: (1) weighing each raw material according to the formula; (2) mixing the powdered raw materials in a mixer according to the proportion to obtain the compound traditional Chinese medicine; (3) mixing the compound traditional Chinese medicine with a portion of the feed raw materials to obtain feed premixed with the compound traditional Chinese medicine; (4) mixing all the remaining feed raw materials with the feed premixed with the compound traditional Chinese medicine in multiple batches to obtain feed.

[0018] Hesperidin is a flavonoid compound with antioxidant and immunomodulatory functions. Studies have found that hesperidin has immunomodulatory properties. When chickens are infected with coccidia, their immune system is affected. Hesperidin can help enhance the activity of immune cells in chickens, such as increasing the proliferation of lymphocytes, allowing the body's immune defense system to function better and resist coccidia invasion and reproduction.

[0019] Hesperidin also possesses anti-inflammatory properties, inhibiting the release of inflammatory mediators (such as interleukin-1 and interleukin-6) and reducing intestinal inflammation. Coccidiosis infection can trigger intestinal inflammation. After the intestinal inflammation subsides with hesperidin, it helps maintain normal intestinal physiological functions, such as nutrient absorption, thereby improving the chicken's health and aiding in its recovery from coccidiosis infection.

[0020] Citric acid is an organic acid that can lower the pH level of the intestines. Coccidia require a suitable acid-base environment to survive and reproduce in the chicken's intestines. Lowering the intestinal pH can inhibit the growth and development of coccidia, reducing their infectivity and reproduction rate, thus achieving the effect of treating coccidiosis in chickens. This is because many coccidia are quite sensitive to pH in their survival and reproduction. In an acidic environment, the sporulation process of coccidia may be hindered, thereby reducing their infectivity.

[0021] Citric acid can form chelates with some minerals (such as calcium and iron), making these nutrients easier for chickens to absorb. In cases of coccidiosis in chickens, intestinal function is impaired, and nutrient absorption capacity decreases. Citric acid, by promoting nutrient absorption, can enhance the chicken's constitution and improve its resistance to coccidiosis infection. Simultaneously, good nutritional status also helps repair the intestinal mucosa, as adequate nutrition is fundamental for the regeneration of intestinal mucosal cells and the maintenance of normal function. During coccidiosis, damage to the intestinal mucosa easily leads to secondary infections by pathogens. Citric acid has an inhibitory effect on some intestinal pathogens, reducing the risk of secondary infections. Its synergistic effect with hesperidin in combating coccidiosis helps chickens better cope with coccidiosis and potential concurrent bacterial infections.

[0022] This invention uses chicks as experimental subjects. By establishing a chick coccidiosis model, the effects of the compound traditional Chinese medicine described in this invention on chick coccidiosis were studied from aspects such as the general condition of chicks, bloody stools, coccidia oocyst excretion, cecal tissue lesions, pathological examination, serum biochemistry, and antioxidant detection.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) The compound traditional Chinese medicine of the present invention has significant efficacy. In the case of chickens infected with Eimeria tenella, it can reduce the amount of oocysts excreted, inhibit cecal tissue inflammation and oxidative stress, reduce cecal tissue structural damage, improve the survival rate and growth performance of infected chickens, and achieve a good level of anticoccidial drug.

[0025] (2) The raw materials required for this compound traditional Chinese medicine are inexpensive and easy to use. It does not contain hormones or antibiotics and does not cause any toxic side effects on chickens, nor does it lead to drug resistance. Therefore, this compound traditional Chinese medicine can provide a new approach to antibiotic-free, green and healthy farming models, and offers a new option for replacing chemical drugs for coccidiosis control. It is expected to reduce or replace the addition of anticoccidial drugs in chicken feed, alleviate chicken coccidiosis drug resistance, and contribute to the cause of "reducing and banning antibiotics" in livestock and poultry farming. Attached Figure Description

[0026] Figure 1 The image shows a magnified section of cecal tissue from chicks after pathological preparation and H&E staining, obtained using an optical microscope. Detailed Implementation

[0027] 1. Screening experiment on the mixing ratio of Chinese herbal monomers

[0028] 1.1 Material Preparation

[0029] 1.1.1 Drug: Hesperidin was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., with a hesperidin content of ≥97%.

[0030] Citric acid was purchased from Tianjin Aopusheng Chemical Co., Ltd., and its citric acid content is ≥99.5%.

[0031] Diclazuril is a conventional anticoccidial drug: Diclazuril solution was purchased from Hefei Zhonglong Shenli Animal Pharmaceutical Co., Ltd.

[0032] 1.1.2 Chicks: One-day-old chicks were provided by Beijing Agricultural College and raised in a sterilized, coccidioidom-free animal house; chicken cages, feed troughs, and water troughs were all strictly sterilized. They were fed sterilized feed and purified water, and had free access to food. One week before creating the animal coccidiosis model, the clinical symptoms of the flock were carefully observed and their feces were tested for the presence of coccidia oocysts. Weak chickens and chickens that might be infected with coccidiosis were culled, and the remaining chickens were kept for future use.

[0033] 1.1.3 Feed: Provided by Dabeinong Feed Mill (containing no anticoccidial ingredients).

[0034] 1.1.4 Mixing: Weigh each raw material according to the formula. First, put the powdered Chinese medicine monomers into the mixer according to the weight ratio of the raw materials and mix them evenly. Then, mix the monomer mixture with a small amount of feed evenly. Then, mix the remaining feed with the feed premixed with the Chinese medicine monomer mixture in four batches.

[0035] 1.1.5 Chicken coccidia oocysts: Eimeria tenella sporulated oocysts (gifted by China Agricultural University) were used after two weeks of passage and rejuvenation in chickens. The cecum was collected, and the oocysts were collected. When the sporulation rate reached more than 80% at 28℃, the oocysts were counted and stored in a 2.5% potassium dichromate solution at 4℃. They should be used within one month.

[0036] 1.2 Experimental Methods

[0037] Two hundred and forty chickens from the same batch were randomly divided into ten treatments, with four replicates per treatment and six chickens per replicate, all fed the same way. The implementation methods are shown in the table below:

[0038] Table 1: Treatment methods for screening experimental groups of mixed proportions of Chinese herbal monomers

[0039]

[0040] 1.2.1 Feeding and Management

[0041] Prior to the experiment, the chicken coop was thoroughly cleaned and disinfected. Immunization program: The existing immunization program at the chicken farm was followed. Experimental period: From 1 day to 23 days of age. Weights were taken on days 7 and 14 to ensure uniform weight across all examples. Chickens were treated with parasites at day 14. Examples 1 to 10 were administered 2 × 10 g of parasite medication per chicken via gavage. 4 Individual sporozoites were administered to control groups 1 to 3 via gavage with an equal volume of physiological saline. Feed intake and diarrhea / bloody stools were recorded daily after 14 days of age. Feces were collected on days 4, 5, 6, 7, 8, and 9 after model establishment for subsequent egg counting. Sampling was performed at day 21 to observe the extent of cecal lesions. Finally, the relative weight gain, feed efficiency, and anticoccidial index (ACI) of each group of chicks were calculated.

[0042] The morbidity, cure rate, and mortality rate of chicks in each group were observed and recorded. The efficacy assessment method and standard anticoccidial index (ACI) were calculated according to the formula recommended by Merck & Co., Ltd.: ACI = (relative weight gain rate + survival rate) - (oocyst value + lesion value); relative weight gain rate = (weight gain of experimental group ÷ weight gain of blank control group) × 100%; survival rate = (number of surviving chicks in each group ÷ total number of chicks in each group) × 100%. Lesions were scored according to five levels, as shown in Table 2.

[0043] Table 2: Gravical Lesion Scoring Criteria

[0044]

[0045] The efficacy criteria are as follows: ACI ≥ 180 is considered excellent, 160 ≤ ACI ≤ 180 is good, 140 ≤ ACI ≤ 160 is moderate, 120 ≤ ACI ≤ 140 is poor, and ACI ≤ 120 is ineffective. The oocyst value is calculated from the occlusal contents gram oocyst count (OPG).

[0046] 1.3 Results of the screening experiment on the mixing ratio of traditional Chinese medicine monomers

[0047] 1.3.1 Clinical Manifestations

[0048] In control group 1, which was not inoculated with Eimeria tenella sporulated oocysts, the chicks were in good spirits, ate normally, and did not have bloody stools. In model group 1, control group 1, and examples 1-7, on the second day after inoculation with Eimeria tenella sporulated oocysts, the chicks showed symptoms such as lethargy and standing listlessly; obvious bloody stools began to appear on the fourth day, and the bloody stools were most severe on the fifth day, after which the bloody stools gradually decreased.

[0049] 1.3.2 Growth performance

[0050] Weigh the animals before 14 days of viral challenge, record their daily feed intake after viral challenge, weigh them before 21 days of dissection and sampling, and compile the data to calculate the average daily feed intake, average daily weight gain, and feed conversion ratio.

[0051] The results are shown in Table 3:

[0052] Table 3: ADFI, ADG, and FCR of different mixing ratios of Chinese herbal monomers

[0053]

[0054] * Values ​​of superscripts with different lowercase letters in the same line indicate significant differences (p < 0.05).

[0055] Table 3 shows that compared with the control group 1, the average daily weight gain of model group 1 was significantly lower (p < 0.05), and the feed conversion ratio was significantly higher (p < 0.05), proving that coccidiosis infection seriously affects the production performance of chicks. Except for the model group, the feed conversion ratio of all other groups decreased significantly (p < 0.05), proving that multiple mixing ratios can improve the production performance of chicks, with Example 4 being the most significant.

[0056] 1.3.3 Anticoccidial Index

[0057] In each embodiment, 12 chickens were dissected and their cecums were removed. The anticoccidial index of each group was calculated based on the lesion score, relative weight gain rate, survival rate and oocyst value. The results are shown in Table 4.

[0058] Table 4: Evaluation of the anticoccidial effects of different Chinese herbal monomers

[0059]

[0060] As shown in Table 4, Example 4 showed the best anticoccidial effect, that is, the anticoccidial effect was best when the mixing ratio of hesperidin and citric acid was 2:1.

[0061] 2. Screening experiment on dosage of compound traditional Chinese medicine

[0062] 2.1 Material preparation: Same as 1.1.

[0063] 2.2 Experimental Methods

[0064] A total of 168 chickens from the same batch were randomly divided into 7 treatments, with 4 replicates per treatment and 6 chickens per replicate, all fed the same way. The implementation methods are shown in the table below:

[0065] Table 5: Experimental Treatment Methods for Screening Dosage of Compound Traditional Chinese Medicine Additions

[0066]

[0067] 2.2.1 Feeding and Management

[0068] Same as 1.2.1.

[0069] 2.3 Results of the drug administration method screening experiment

[0070] 2.3.1 Clinical Manifestations

[0071] In control group 2 and Example 11, chicks were not inoculated with Eimeria tenella sporulated oocysts and showed good spirits, normal feed intake, and no bloody stools. In model group 2, control group 2, and Examples 8-10, chicks showed symptoms such as lethargy and standing listlessly on the second day after inoculation with Eimeria tenella sporulated oocysts; significant bloody stools began to appear on the fourth day, reaching their most severe stage on the fifth day, after which the bloody stools gradually decreased.

[0072] 2.3.2 Growth performance

[0073] Weigh the animals before 14 days of viral challenge, record their daily feed intake after viral challenge, weigh them before 21 days of dissection and sampling, and compile the data to calculate the average daily feed intake, average daily weight gain, and feed conversion ratio.

[0074] The results are shown in Table 6:

[0075] Table 6: ADFI, ADG, and FCR in the screening experiment of compound traditional Chinese medicine dosage addition

[0076]

[0077] * Values ​​of superscripts with different lowercase letters in the same line indicate significant differences (p < 0.05).

[0078] Table 6 shows that compared with the control group 2, the average daily weight gain of model group 2 was significantly lower (p < 0.05), while the feed conversion ratio was significantly higher (p < 0.05), proving that coccidiosis infection severely affects the production performance of chicks. Example 11, compared with the control group 2, showed a significantly higher average daily weight gain (p < 0.05) and a lower feed conversion ratio, proving that the additive has no biological toxicity and does not affect the production performance of chicks. Example 8, compared with model group 2, showed a significantly higher average daily weight gain (p < 0.05) and a lower feed conversion ratio (p < 0.05), proving that adding a high dose of hesperidin and citric acid combination can effectively help chicks infected with coccidiosis recover their production performance.

[0079] 2.3.3 Anticoccidial Index

[0080] In each embodiment, 12 chickens were dissected and their cecums were removed. The anticoccidial index of each group was calculated based on the lesion score, relative weight gain rate, survival rate and oocyst value. The results are shown in Table 7.

[0081] Table 7: Evaluation of the anticoccidial effect of compound traditional Chinese medicine additive dosage screening

[0082]

[0083] As shown in Table 7, Example 8 has the best anticoccidial effect. That is, adding the mixture of hesperidin and citric acid in the feed at a ratio of 2:1 and a dosage of 500 g / T can achieve the best anticoccidial effect. Therefore, 500 g / T is the optimal dosage.

[0084] 2.3.3 Pathological sections

[0085] Chicks were euthanized by cervical dislocation, and their intestinal tissue was removed. The cecum was carefully separated. Surgical forceps were used to fix the proximal cecum, and a small segment of approximately 2-3 cm was separated. The contents of the intestinal lumen were cleaned with PBS solution. The separated cecum segment was placed in a 4% formaldehyde solution, which was changed every 12 hours until the solution became clear. Pathological sections of the fixed cecum tissue were prepared and H&E stained. Each example was magnified to 100x (left) and 200x (right) using an optical microscope. Figure 1 .

[0086] Depend on Figure 1It can be seen that Eimeria tenella severely damages the intestinal mucosa. Using H&E staining, the structural changes of the intestinal epithelial tissue can be observed directly. In control group 2, the intact striated border of the intestine, neatly arranged intestinal glands, and tightly connected cells in the upper mucosa were visible. In model group 2, the intestinal mucosa was severely damaged, with some intestinal mucosa sloughing off into the intestinal lumen. Under a 200x microscope, Eimeria tenella at different developmental stages could be clearly observed. The intestinal glands were densely occupied by Eimeria tenella, losing their original structure, and the cells in the mucosa were loosely distributed. Example 9 was similar to model group 2, with the intestinal mucosa severely damaged, with some intestinal mucosa sloughing off into the intestinal lumen. Under a 200x microscope, Eimeria tenella at different developmental stages could be clearly observed. The intestinal glands were densely occupied by Eimeria tenella, losing their original structure, and the cells in the mucosa were loosely distributed. In example 10, compared to example 9, the intestinal mucosa was more intact, the egg density was also reduced, and a small number of inflammatory cells were present. In example 8, the intact striated border of the intestine, neatly arranged intestinal glands, and tightly connected cells in the upper mucosa were visible, with a very small number of coccidia oocysts parasitizing between the intestinal cells.

[0087] Those skilled in the art will recognize that numerous variations are possible with respect to the above description, and therefore the embodiments are merely illustrative of one or more specific implementations.

[0088] Although exemplary embodiments of the invention have been described and illustrated, those skilled in the art will understand that various changes and substitutions can be made thereto without departing from the spirit of the invention. Furthermore, many modifications can be made to adapt specific situations to the doctrine of the invention without departing from the central concepts of the invention described herein. Therefore, the invention is not limited to the specific embodiments disclosed herein, but may include all embodiments and equivalents that fall within the scope of the invention.

Claims

1. A compound traditional Chinese medicine comprising the following raw materials in parts by weight: 5-20 parts of hesperidin and 1-10 parts of citric acid.

2. The compound traditional Chinese medicine according to claim 1, characterized in that, The ingredients include the following parts by weight: 10 parts hesperidin and 5 parts citric acid.

3. The compound traditional Chinese medicine according to claim 1 or 2, characterized in that, The purity of hesperidin is ≥97%, and the purity of citric acid is ≥99%.

4. A compound traditional Chinese medicine, characterized in that, The active ingredient includes the powder of the compound traditional Chinese medicine as described in any one of claims 1-3, or the water or organic solvent extract of the raw material, and pharmaceutically acceptable excipients.

5. The compound traditional Chinese medicine according to any one of claims 1-4, characterized in that, The dosage forms of the compound traditional Chinese medicine include one or more of the following: powder, decoction, oral liquid, granules, pills, and tablets.

6. The use of the compound traditional Chinese medicine according to any one of claims 1-5 in the preparation of drugs for treating coccidiosis or Eimeria tenella.

7. The use of a compound traditional Chinese medicine according to any one of claims 1-5 in the preparation of feed or feed additives.

8. The application of the compound traditional Chinese medicine according to claim 7 in the preparation of feed, characterized in that, The compound traditional Chinese medicine is added to feed at a dosage of 125-500g / ton of feed when administered by mixing.

9. The application of the compound traditional Chinese medicine according to claim 8 in the preparation of feed, characterized in that, The compound traditional Chinese medicine is added to the feed at a dosage of 500g / ton of feed when administered by mixing.

10. The application of the compound traditional Chinese medicine according to claim 7 in the preparation of feed, characterized in that, The method for preparing the feed includes the following steps: (1) weighing each raw material according to the formula; (2) mixing the powdered raw materials in a mixer according to the proportion to obtain the compound Chinese medicine; (3) mixing the compound Chinese medicine with a portion of the feed raw materials to obtain the feed premixed with the compound Chinese medicine; (4) mixing all the remaining feed raw materials with the feed premixed with the compound Chinese medicine in multiple batches to obtain the feed.

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