Traditional Chinese medicine monomer composition for preventing and controlling necrotic enteritis in chickens

Abstract

The present invention discloses a Chinese medicine monomer composition, which is mainly composed of citric acid and magnolol. Studies have shown that the drug combination can effectively inhibit the growth of Clostridium perfringens and the formation of biofilm in vitro, wherein the weight ratio of citric acid to magnolol is 50:3, and a synergistic effect can be exerted. At the same time, in vivo experimental results show that the symptoms of necrotic enteritis in yellow-feathered broilers treated with the present invention are significantly alleviated, and improvements are made in terms of weight loss, feed conversion rate, lesion score, inflammatory factors, etc. Therefore, the Chinese medicine monomer composition of the present invention has a good effect on resisting Clostridium perfringens infection and enriches the theory of preventing and controlling Clostridium perfringens infection. The application of the present invention to replace antibiotics to treat necrotic enteritis in chickens can reduce breeding drug residues, create higher economic benefits and ensure human food safety.

Description

Technical Field

[0001] The invention belongs to the technical field of chicken enteritis prevention and control, and in particular relates to a traditional Chinese medicine monomer composition for preventing and controlling chicken necrotic enteritis. Background Art

[0002] The causative agent of necrotic enterocolitis is Clostridium perfringens, an anaerobic, Gram-positive bacillus found in soil, food, and the intestines of healthy animals. Clostridium perfringens is a component of the normal intestinal flora of animals and can multiply rapidly and cause disease under conditions such as heat and cold stress, moldy feed, and excessive stocking density. Current research has classified Clostridium perfringens into five serotypes, AE, based on the toxins it secretes. Types A and C are the primary pathogens of necrotic enterocolitis in poultry, with type A being the predominant. The clinical manifestations of C. perfringens infection are characterized by symptoms such as intestinal wall thinning, focal necrosis, and diarrhea. It is estimated that the poultry industry spends billions of dollars annually on the prevention, diagnosis, and treatment of necrotic enterocolitis.

[0003] In the past, adding antibiotics to feed or drinking water was the primary method for preventing and controlling necrotic enteritis. However, the widespread and even overuse of antibiotics in the livestock industry has inevitably led to environmental pollution, including soil and water sources, which in turn has led to increased drug residues in food, posing serious health risks and potential safety hazards to humans. Therefore, the development of green, safe, and healthy alternatives to antibiotics is becoming a hot topic. Natural compounds and traditional Chinese medicine monomers offer advantages such as low toxicity, high safety, and low residue levels, making them a key resource for screening effective drugs.

[0004] Necrotic enteritis is often clinically characterized by symptoms of internal fullness, heat, and pain, diarrhea, fever, loss of appetite, and foul stools. Traditional Chinese Medicine (TCM) diagnosis primarily involves spleen dysfunction, internal accumulation of dampness and turbidity, which then transforms into heat, or exposure to external pathogens, damaging the spleen and stomach and causing damp-heat. Plum blossoms have astringent and fluid-inducing properties, making them useful for chronic diarrhea and dysentery, as well as thirst caused by deficiency heat. Magnolia bark (Magnolia officinalis) can dry out dampness, eliminate phlegm, and relieve qi distension, making them useful for dampness-induced damage to the middle abdomen, as well as abdominal distension, vomiting, and diarrhea. The combination of these two herbs has the benefits of astringency, antidiarrheal properties, warming and clearing heat, drying dampness and stopping dysentery, and replenishing qi and blood. Plum blossoms are rich in citric acid, one of their primary antibacterial components. Magnolia bark phenol, a phenolic compound extracted from the traditional Chinese medicine Magnolia bark, is its primary medicinal component. Research has uncovered no studies domestically or internationally on the use of citric acid and magnolia bark phenol in the treatment of necrotic enteritis in chickens caused by Clostridium perfringens. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a traditional Chinese medicine monomer composition with reasonable formula and good effect for preventing and controlling necrotic enteritis in chickens.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The traditional Chinese medicine monomer composition is mainly composed of citric acid and magnolol.

[0008] The above-mentioned traditional Chinese medicine monomer composition mainly consists of citric acid and magnolol in a weight ratio of 50:4 to 300:2.

[0009] The above-mentioned traditional Chinese medicine monomer composition mainly consists of citric acid and magnolol in a weight ratio of 50:3.

[0010] The Chinese medicine monomer composition is used for preparing medicine for inhibiting Clostridium perfringens.

[0011] The above-mentioned Chinese medicine monomer composition is used to prepare medicine for preventing and controlling necrotic enteritis in chickens.

[0012] Necrotic enteritis in chickens is caused by infection with Clostridium perfringens type A.

[0013] The medicine is taken orally.

[0014] In response to the current problems in the treatment of necrotic enteritis in chickens, the inventors have developed a Chinese medicine monomer composition mainly composed of citric acid and magnolol. Studies have shown that this drug combination can effectively inhibit the growth of Clostridium perfringens and the formation of biofilms in vitro, and a synergistic effect can be exerted when the weight ratio of citric acid and magnolol is 50:3. At the same time, in vivo experimental results show that the symptoms of necrotic enteritis in yellow-feathered broilers treated with the present invention are significantly alleviated, and there are improvements in weight loss, feed conversion rate, lesion scores, inflammatory factors, etc. Therefore, the Chinese medicine monomer composition of the present invention has a good effect on resisting Clostridium perfringens infection, enriching the theory of preventing and controlling Clostridium perfringens infection. The application of the present invention to replace antibiotics in the treatment of necrotic enteritis in chickens can reduce the residue of breeding drugs, create higher economic benefits and ensure human food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the growth curve of Clostridium perfringens. In the figure: CP is the blank control group, the combination drug concentration is 0 μg / ml; 1×MIC is the 265 μg / ml Chinese medicine monomer combination drug group, 1 / 2×MIC is the 132.5 μg / ml Chinese medicine monomer combination drug group, and 1 / 4×MIC is the 66.25 μg / ml Chinese medicine monomer combination drug group.

[0016] Figure 2 This is a graph showing the test results of the biofilm-forming ability of Clostridium perfringens. In the graph: Control is the broth control group, CP is the blank control group, and the concentration of the combined drug is 0 μg / ml; 1×MIC is the 265 μg / ml Chinese medicine monomer combination drug group, 1 / 2×MIC is the 132.5 μg / ml Chinese medicine monomer combination drug group, and 1 / 4×MIC is the 66.25 μg / ml Chinese medicine monomer combination drug group.

[0017] Figure 3 This is a comparison chart of chick growth performance, in which: ADG is average daily gain; ADFI is average daily feed intake; FCR is feed conversion rate; among them, Control is the blank control group, CP is the infection group, C.P+1‰ is the low-dose treatment group, C.P+2‰ is the medium-dose treatment group, C.P+3‰ is the high-dose treatment group, and Lincomycin is the lincomycin treatment group.

[0018] Figure 4 This is a comparison chart of intestinal lesion scores in chicks. In the figure: Control is the blank control group, CP is the infection group, C.P+1‰ is the low-dose treatment group, C.P+2‰ is the medium-dose treatment group, C.P+3‰ is the high-dose treatment group, and Lincomycin is the lincomycin treatment group.

[0019] Figure 5 These are intestinal pathological sections. In the figure: A jejunal section of the blank control group, B jejunal section of the infected group, C jejunal section of the low-dose treatment group, D jejunal section of the medium-dose treatment group, E jejunal section of the high-dose treatment group, and F jejunal section of the lincomycin treatment group.

[0020] Figure 6 This is a comparison chart of various cytokines in chicken serum. In the figure: A is chicken immunoglobulin Y (IgY), B is chicken immunoglobulin A (IgA), C is chicken interleukin 1β (IL-1β), D is chicken interleukin 2 (IL-2), E is chicken tumor necrosis factor α (TNF-α), F is chicken superoxide dismutase (SOD), and G is chicken catalase (CAT); among them, Control is the blank control group, CP is the infection group, C.P+1‰ is the low-dose treatment group, C.P+2‰ is the medium-dose treatment group, C.P+3‰ is the high-dose treatment group, and Lincomycin is the lincomycin treatment group. DETAILED DESCRIPTION

[0021] Example 1. Determination of the Minimum Inhibitory Concentration (MIC) and Optimal Ratio of Citric Acid and Magnolia Noil

[0022] Cryopreserved tubes of the standard strain of Clostridium perfringens ATCC13124 (type A) were cultured overnight at 37°C in cooked meat medium. The culture was then streaked on TSC medium and anaerobically cultured for 24 hours. Black single colonies were picked and cultured in cooked meat medium until the logarithmic growth phase and stored for later use. Broth containing citric acid and magnolol was prepared in a test tube, and the MIC value against Clostridium perfringens was determined by the two-fold dilution method. A DMSO control group was also set up, and the MIC value was read after anaerobic culture at 37°C for 16 hours (Table 1). Based on the existing MIC values, the drugs were combined at different MIC multiples to determine the combined effect of citric acid and magnolol, and the optimal ratio was screened (Table 2).

[0023] Table 1

[0024]

[0025] Table 2

[0026]

[0027] Example 2: Effect of citric acid combined with magnolol on the growth curve of Clostridium perfringens

[0028] Dilute the prepared bacterial solution in a 1:1000 dilution into the cooked meat medium and culture at 37°C overnight. Pour the prepared bacterial solution into a conical flask containing 20 ml of cooked meat medium to make the bacterial solution concentration in the flask 1×10 5 CFU / ml. A medicinal solution was prepared with a weight ratio of citric acid to magnolol of 50:3 (the Chinese medicine monomer composition medicinal solution used in Example 3 below has the same ratio). The concentrations of the Chinese medicine monomer composition in the conical flask were adjusted to 265 μg / ml, 132.5 μg / ml, 66.25 μg / ml, and 0 μg / ml, respectively. The culture was anaerobically statically cultured at 37°C. The OD600 value of the bacterial culture was measured every 2 hours for 24 hours, and the corresponding growth curve was drawn. The results are shown in FIG. Figure 1 .

[0029] Example 3: Effect of citric acid combined with magnolol on biofilm formation of Clostridium perfringens

[0030] Take one of the prepared bacterial suspensions and dilute it to 1:1000 in cooked meat culture medium and culture it at 37°C overnight. Add the prepared bacterial suspension to a 24-well cell plate to make the bacterial concentration in the wells 1×10 5CFU / ml, and then adjust the concentration of the Chinese medicine monomer composition in the well to 265μg / ml, 132.5μg / ml, 66.25μg / ml and 0μg / ml, and set up 1ml of cooked meat culture medium as a control, and place it in an anaerobic environment at 37℃ for 72h. After the culture is completed, discard the culture medium, rinse with sterile PBS 3 times, and then stain with 1ml of 1% crystal violet for 30min after natural air drying. Discard the crystal violet, continue to rinse with sterile PBS 3 times, and air dry for 45min. Add 1ml of 95% ethanol solution to each well and dissolve for 30min. After blowing and mixing, aspirate 300μl from each well and add it to three independent wells of a 96-well plate, 100μl per well, and measure the absorbance at 595nm on an enzyme reader. The biofilm formation rate is judged according to the ultraviolet absorbance. The results are as follows Figure 2 .

[0031] Example 4: Therapeutic Effects of Necrotic Enteritis in Chickens

[0032] Fourteen-day-old yellow-feathered broilers were gavaged with 1 ml of a 1×10 9 CFU / ml of Clostridium perfringens ATCC13124 broth culture was used to establish a necrotic enteritis model caused by Clostridium perfringens in chickens.

[0033] Twelve hours after the first challenge, a Chinese medicine monomer composition consisting of citric acid and magnolol powder (98% purity) at a weight ratio of 50:3 was added to the feed. The mixture was mixed with the feed at a ratio of 1‰ for the low-dose group, 2‰ for the medium-dose group, and 3‰ for the high-dose group. Lincomycin-treated, infected, and blank control groups were also established. The blank control group received the same volume of cooked meat culture medium daily.

[0034] At 14 and 21 days of age, the chicks were weighed, their feed intake was recorded, and their average daily gain and feed conversion rate were calculated. Figure 3 At 21 days of age, the animals were sacrificed by cervical dislocation after blood was collected from the jugular vein. The duodenum to cecum were collected, and the pathological changes were observed and scored with the naked eye. The pathological changes were scored using a 6-point system: 0 (no obvious damage), 1 (thinning and brittle intestinal wall), 2 (1-5 necrotic foci), 3 (6-15 necrotic foci), 4 (16 or more necrotic foci), 5 (2-3 cm long flake necrosis), 6 (large patches of diffuse necrosis). The results are as follows: Figure 4 The jejunum was fixed in 4% formaldehyde and stained with HE to prepare pathological sections. The pathological histological changes were observed under a microscope. The results were as follows: Figure 5 .

[0035] The blood was centrifuged to separate the serum, and the chicken immunoglobulin Y (IgY), chicken immunoglobulin A (IgA), chicken interleukin 1β (IL-1β), chicken interleukin 2 (IL-2), chicken tumor necrosis factor α (TNF-α), chicken superoxide dismutase (SOD), and chicken catalase (CAT) were detected using Elisa kits. The above kits were purchased from Nanjing Boyan Biotechnology Co., Ltd. Figure 6 .

[0036] The results of the experimental study showed that DMSO had no effect on the growth of Clostridium perfringens at a concentration of 5%, the minimum inhibitory concentration of citric acid was 2 mg / ml, and the minimum inhibitory concentration of magnolol was 40 μg / ml, and there was a good synergistic effect when the mass ratio was 50:3 (250 μg / ml citric acid + 15 μg / ml magnolol); under the action of 1 times the MIC value of the monomer combination, it had a significant inhibitory effect on the growth of Clostridium perfringens and the formation of biofilm.

[0037] In the in vivo challenge experiment, when infected with Clostridium perfringens, the growth performance of chicks decreased significantly, intestinal lesions were severe, intestinal morphology was significantly changed, the levels of antibodies and antioxidant enzymes in the serum decreased significantly, while the levels of inflammatory factors in the serum increased significantly.

[0038] All treatment groups showed improvement in the damage caused by C. perfringens infection. The high-dose group showed the greatest effect in terms of average daily weight gain, while the medium-dose group showed the most significant effect in terms of feed conversion rate. This result may be due to the high-dose group inhibiting digestion and gastrointestinal motility, which had a certain impact on absorption. The medium-dose group and the lincomycin group significantly reduced the changes in jejunal morphology caused by C. perfringens infection, but the difference in lesion scores between the low-, medium-, and high-dose groups and the lincomycin-treated group was not significant. In serum testing, each treatment group increased antibody levels, reversed the changes in inflammatory factors caused by C. perfringens infection, and enhanced the body's antioxidant capacity, with the medium-dose treatment showing the greatest effect.

[0039] In summary, the Chinese medicine monomer composition (citric acid + magnolol) for preventing and controlling necrotic enteritis in chickens of the present invention has a certain inhibitory effect on Clostridium perfringens, has a good preventive and control effect on infection caused by it, and can be used for on-site treatment of necrotic enteritis in chickens.

Claims

1. A Chinese medicinal monomer composition for inhibiting Clostridium perfringens, characterized in that: The traditional Chinese medicine monomer composition consists of citric acid and magnolol in a weight ratio of 50:

3.

2. Use of a Chinese medicine monomer composition in the preparation of a medicament for inhibiting Clostridium perfringens, characterized in that: The traditional Chinese medicine monomer composition consists of citric acid and magnolol in a weight ratio of 50:

3.

3. Use of a Chinese medicine monomer composition in the preparation of a medicament for treating necrotic enteritis in chickens caused by infection with Clostridium perfringens type A, characterized in that: The traditional Chinese medicine monomer composition consists of citric acid and magnolol in a weight ratio of 50:

3.

4. The use according to claim 2 or 3, characterized in that: The medicine is an oral medicine.

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