Application of paeonol in preparation of medicine or feed additive for treating or preventing chicken necrotic enteritis

Abstract

The invention discloses application of paeonol in preparation of a medicine or a feed additive for treating or preventing chicken necrotic enteritis. The medicine or feed additive is paeonol or is prepared into oral liquid, granules, tablets and powder by taking the paeonol as a main component and adding auxiliary materials, the use dosage is that the content of the paeonol is 10-100mg / kg of the weight of a chicken, or the paeonol is directly added into the feed, and the addition amount is 0.01-0.03% of the total weight of the chicken feed. The paeonol and the composition with the paeonol as the main component can effectively inhibit chicken enteritis pathogenic bacteria, and the paeonol and the composition with the paeonol as the main component have the advantages of being high in safety, stable in effect, free of toxic and side effects and the like and have the remarkable prevention and control effect on chicken necrotic enteritis.

Description

Technical Field

[0001] The present application relates to the technical field of prevention and treatment of chicken enteritis, and specifically to the application of paeonol in the preparation of drugs or feed additives for treating or preventing necrotic enteritis in chickens. Background Art

[0002] my country is a major producer and consumer of chicken. Ensuring the health and efficiency of broiler farming to improve chicken production and quality is related to the national economy and people's livelihood. However, necrotic enteritis (NE) seriously restricts the health and efficiency of broiler farming. NE is one of the main gastrointestinal diseases that endanger the healthy farming of broilers worldwide. It is mainly caused by infection with Clostridium perfringens (CP) type A and G. In the broiler production process, some abnormal environmental factors (such as heat stress), pathogens (such as coccidia) infection, and dietary types (such as wheat-based diets) provide conditions for CP infection. NE mainly occurs in the small intestine, mainly in the ileum, which leads to impaired physical and immune barrier functions of the broiler intestine, imbalance of intestinal flora structure, and then causes intestinal inflammatory damage.

[0003] Previously, the use of antibiotics was the main measure to prevent and control NE in broiler chickens. However, excessive use of antibiotics has brought many hazards: (1) Increased drug resistance: Long-term use of antibiotic additives can cause microorganisms to develop drug resistance, which can cause some pathogenic microorganisms to develop drug resistance through mutation, leading to a sharp increase in the number of antibiotic-resistant bacteria worldwide; (2) Excessive veterinary drug residues in animal-derived foods: Excessive use of veterinary drugs can cause excessive veterinary drug residues in animal-derived foods, posing a potential threat to human health; (3) Damage to the ecological environment: The discharge of metabolites from poultry that eat antibiotic-containing feed can damage the ecological environment and pollute drinking water or other agricultural products; (4) Impact on human health: If antibiotics remaining in livestock and poultry products are ingested by the human body, they will increase the burden on the liver and kidneys during metabolism, and long-term intake may cause lesions; (5) Reduced immunity: The abuse of antibiotics may cause an imbalance in the normal microbial system in the chicken, resulting in a decrease in the chicken's immune function and resistance. In view of the serious consequences of antibiotics, antibiotics have been banned on a large scale. However, since the ban on antibiotics in feed, pathogens in the gastrointestinal tract of broiler chickens have taken the opportunity to proliferate rapidly, which is bound to destroy their intestinal immune barrier and aggravate the negative impact of NE on broiler chickens.

[0004] It can be seen that NE is a key issue that needs to be urgently addressed in the healthy and efficient breeding of broilers in the post-antibiotic era. The prevention and control of NE through nutritional measures has gradually become one of the key topics of concern in broiler breeding. Summary of the invention

[0005] In view of this, the purpose of the present application is to use paeonol in the preparation of a drug or feed additive for treating or preventing necrotic enteritis in chickens. In this application, paeonol and a composition with paeonol as the main component can effectively inhibit the pathogenic bacteria of chicken enteritis. The paeonol and a composition with paeonol as the main component have the advantages of high safety, stable efficacy, and no toxic side effects, and have a significant preventive and control effect on necrotic enteritis in chickens.

[0006] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0007] Application of paeonol in preparing medicine or feed additive for treating or preventing necrotic enteritis in chickens.

[0008] Compared with the prior art, the technical solution provided by this application has at least the following advantages and effects:

[0009] 1. The paeonol provided in this application is derived from natural plants, has high safety, and has no toxic side effects. It can be used as a feed additive for a long time to prevent necrotic enteritis in chickens. Long-term use will not lead to the production of drug-resistant strains, which is an advantage that other anti-disease drugs do not have.

[0010] 2. The in vitro test results of this application show that paeonol can inhibit the proliferation of common pathogenic bacteria in chickens, such as Clostridium perfringens, Escherichia coli, Salmonella, etc.

[0011] 3. The in vivo test results of this application show that paeonol can stimulate the proliferation of macrophages, inhibit the expression of pro-inflammatory cytokines, and promote the intestinal health of broiler chickens, thereby improving the feed conversion efficiency of broiler chickens with necrotic enteritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of the effect of paeonol on the ratio of monocytes to B lymphocytes in the blood of broilers infected with Clostridium perfringens provided in the examples of the present application, wherein A is the flow density diagram of monocytes and B lymphocytes in the blood of chickens in the control group (CTR), CP infection group (CC), and paeonol + CP infection group (PAE + CC), and B is the corresponding statistical analysis result.

[0013] Figure 2 The results of the effects of paeonol on the transcription levels of ileal immunity-related genes in broiler chickens infected with Clostridium perfringens are provided in the examples of the present application.

[0014] Figure 3 The results provided in the examples of the present application show the effect of paeonol on the transcription level of ileal barrier-related genes in broiler chickens infected with coccidia and Clostridium perfringens. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0016] Those skilled in the art will appreciate that, unless otherwise stated, the words "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the wording "including" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0017] Those skilled in the art will appreciate that, for any specific experimental steps or conditions not specified in the examples, the experiments may be carried out according to the conventional experimental steps or conditions described in the literature in the art; any raw materials or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0018] Those skilled in the art will understand that, unless otherwise specified in the present application, when a numerical range is given in an embodiment, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application are consistent with the prior art mastery of those skilled in the art and the records of this application, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of this application may also be used to implement this application.

[0019] Paeonol is a Chinese herbal medicine extracted from the root bark of peony flowers, and can also be extracted from the dried root bark of the ranunculaceae plant peony. Paeonol is a white or slightly yellow needle-shaped crystal with a melting point of about 49-51°C. It is easily soluble in ethanol and methanol and slightly soluble in water. Paeonol can be distilled with water vapor and has strong absorption in the ultraviolet region; the energy (molar absorption coefficient) of paeonol at a wavelength of 274nm is 862, so it can be measured using a spectrophotometer. Current studies have found that paeonol has analgesic, anti-inflammatory, antipyretic and allergic effects. Specifically, paeonol can relieve pain reactions caused by stimulation by substances such as tail pressure and acetic acid; it can relieve inflammatory reactions caused by formaldehyde, histamine, 5-hydroxytryptamine, bradykinin and endotoxin; it can relieve fever caused by vaccine immunization. In addition, paeonol also has a variety of biological effects such as antibacterial, anti-inflammatory, antioxidant, and regulation of blood pressure and blood lipids. Due to its green, safe and efficient characteristics, paeonol is used as a clinical drug in human medicine, such as paeonol ointment, but there are no reports on paeonol in livestock production.

[0020] The results of in vitro and in vivo tests in the examples of the present application show that paeonol can inhibit the proliferation of common chicken pathogens, such as Clostridium perfringens, Escherichia coli, Salmonella, etc.; paeonol can stimulate the proliferation of macrophages, inhibit the expression of pro-inflammatory cytokines, and promote the intestinal health of broiler chickens, thereby improving the feed conversion efficiency of broiler chickens with necrotic enteritis; the above results provide basic data for the development of paeonol in poultry health drugs and feed additives.

[0021] Based on this, the embodiments of the present application provide the use of paeonol in the preparation of a drug or feed additive for treating or preventing necrotic enteritis in chickens.

[0022] In certain embodiments, the intestinal inflammation caused by the necrotic enteritis of chickens is caused by Clostridium perfringens alone or mixed with coccidia.

[0023] In certain embodiments, the drug or feed additive is paeonol, that is, paeonol can be directly used as a drug or feed additive for treating or preventing necrotic enteritis in chickens.

[0024] In certain embodiments, the drug or feed additive is a paeonol composition comprising paeonol and excipients, wherein the excipients include at least one of anhydrous ethanol, propylene glycol, edible oil, water, lactose, maltose, soluble starch, montmorillonite, medical stone, zeolite powder, defatted rice bran, corn cob powder, bran, and white carbon black.

[0025] In certain embodiments, the aforementioned paeonol composition is a solution of paeonol mixed with anhydrous ethanol and water, wherein the concentration of paeonol is 10-120 mg / mL.

[0026] In certain embodiments, the aforementioned paeonol composition is a solid mixture of paeonol and lactose or maltose, and at least one of soluble starch, bran, and white carbon black, wherein the mass fraction of paeonol in the solid mixture is 40% to 60%. The solid mixture can be used to prepare tablets or granules (for example, paeonol, lactose, and soluble starch are used to make tablets with a mass fraction of 50% of paeonol by conventional technology; or paeonol, maltose, and soluble starch are used to make tablets with a mass fraction of 50% of paeonol by conventional technology; or paeonol, maltose, and soluble starch are used to make granules with a mass fraction of 50% of paeonol by conventional technology; or paeonol, maltose, and soluble starch are used to make granules with a mass fraction of 50% of paeonol by conventional technology; the soluble starch component in the above-mentioned compositions can be used as any one or 2 to 3 of soluble starch, bran, and white carbon black as substitutes to form a new composition formula).

[0027] In certain embodiments, the aforementioned paeonol composition is a powdery mixture (i.e., powder) formed by paeonol and at least one of soluble starch, bran, and white carbon black, wherein the mass fraction of paeonol in the powdery mixture is 40% to 60% (for example, paeonol and white carbon black are conventionally prepared into a powder with a paeonol mass fraction of 50%; paeonol and bran are conventionally prepared into a powder with a paeonol mass fraction of 50%; paeonol and soluble starch are conventionally prepared into a powder with a paeonol mass fraction of 50%; or paeonol and soluble starch, bran, and white carbon black are conventionally prepared into a powder with a paeonol mass fraction of 50%).

[0028] In certain embodiments, when the drug or feed additive is a paeonol composition, its preparation type includes at least one of oral liquid, granules, tablets, and powders, and the preparation can be prepared by conventional pharmaceutical preparation methods.

[0029] In certain embodiments, the method of using the drug or feed additive comprises at least one of the following methods:

[0030] (1) Orally administering the drug to chickens at a dosage of 10 to 100 mg / kg of chicken body weight;

[0031] (2) The paeonol content is 0.01% to 0.03% by weight of the chicken feed; the paeonol granules, powder or tablets are added to the chicken feed as a feed additive;

[0032] (3) When used for daily prevention of chicken enteritis, the paeonol powder, granules or tablets are crushed and added to the feed or drinking water so that the paeonol content is 0.01% of the weight of the chicken feed or drinking water;

[0033] (4) When used for clinical treatment of chicken enteritis: orally administer the drug to chickens at a dosage of 100 mg / kg of chicken body weight; or grind the granules, powder or tablets into powder and feed directly to chickens at a dosage of 100 mg / kg of chicken body weight; or grind the granules, powder or tablets into powder and add it to the feed at a dosage of 0.03% of the weight of chicken feed; or grind the granules, powder or tablets into powder and add it to the drinking water at a dosage of 0.03% of the weight of drinking water, three times a day, morning, noon and evening, for one week.

[0034] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.

[0035] Example 1

[0036] This embodiment provides a method for preparing paeonol oral liquid, comprising the following steps:

[0037] (1) Weigh 10 g of paeonol and 30 mL of anhydrous ethanol;

[0038] (2) First, dissolve paeonol in anhydrous ethanol and mix well, then slowly add water for injection to make up to 100 mL, to obtain a paeonol oral solution with a concentration of 100 mg / mL, i.e., the paeonol oral solution D1.

[0039] Example 2

[0040] This embodiment provides a method for preparing paeonol powder, comprising the following steps:

[0041] (1) Weigh 50 g of paeonol, 20 g of wheat bran, and 30 g of soluble starch;

[0042] (2) Paeonol, wheat bran and soluble starch are mixed evenly and passed through a 60-mesh sieve to obtain a powder with a mass fraction of 50% of paeonol, i.e., paeonol powder D2.

[0043] Example 3

[0044] This embodiment provides a method for preparing a paeonol tablet, comprising the following steps:

[0045] (1) Weigh 50 g of paeonol, 20 g of lactose, and 30 g of soluble starch and set aside;

[0046] (2) Paeonol, lactose and soluble starch are mixed evenly, compressed and dried using a tablet press to obtain tablets with a mass fraction of 50% paeonol, namely paeonol tablets D3.

[0047] Example 4

[0048] This embodiment provides a method for preparing paeonol granules, comprising the following steps:

[0049] (1) Weigh 50 g of paeonol, 20 g of maltose, and 30 g of soluble starch and set aside;

[0050] (2) Paeonol, maltose and soluble starch are mixed and granulated, and then dried and granulated to obtain granules with a mass fraction of paeonol of 50%, namely, paeonol granules D4.

[0051] The formulas and parameters of various paeonol preparations prepared in the above examples are shown in Table 1 below:

[0052] Table 1

[0053] Example No. Composition No. Components Paeonol concentration Formulation type Example 1 D1 Paeonol, anhydrous ethanol, water for injection 100mg / mL Oral solution Example 2 D2 Paeonol, wheat bran, soluble starch 50% (mass fraction) powder Example 3 D3 Paeonol, lactose, soluble starch 50% (mass fraction) tablet Example 4 D4 Paeonol, maltose, soluble starch 50% (mass fraction) Granules

[0054] The above-mentioned paeonol preparation can be used as a medicine or feed additive to prevent or treat necrotic enteritis in chickens. When used for the prevention of chicken diseases in daily life, the paeonol preparation can be added to the feed at a dosage of 10 mg / kg of chicken body weight and used throughout the entire breeding cycle; when used for the clinical treatment of chicken diseases, the paeonol preparation can be orally administered at a dosage of 100 mg / kg of chicken body weight; or the paeonol preparation can be dissolved in water and administered or mixed with the daily diet at a dosage of 100 mg / kg of chicken body weight, and added to the feed three times a day in the morning, noon and evening for continuous use for one week; the dosage of the paeonol preparation can also be adjusted according to the severity of the chicken disease, and the dosage range is controlled within a paeonol content of 10 to 100 mg / kg of chicken body weight.

[0055] In vitro inhibitory effect of paeonol on pathogenic bacteria from chickens

[0056] The present application example verifies the in vitro inhibitory effect of paeonol on three chicken pathogens: Clostridium perfringens, Escherichia coli, and Salmonella. The verification method is as follows:

[0057] 1. Materials and Methods

[0058] 1.1 Bacteria: Clostridium perfringens, Escherichia coli, and Salmonella) were isolated, preserved, and donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0059] 1.2 Experimental drugs: Weigh paeonol (purchased from Sigma, purity > 99%), dissolve it with physiological saline to prepare a paeonol mother solution with a concentration of 0.1 g / mL, and then dilute it step by step with physiological saline to 0.05 g / mL, 0.025 g / mL, 0.0125 g / mL, and 0.0063 g / mL, respectively.

[0060] 1.3 Test process:

[0061] The Oxford cup method was used to evaluate the antibacterial effect of paeonol at different concentrations on Clostridium perfringens, Escherichia coli, and Salmonella. Physiological saline was used as a blank control group; enramycin (purchased from Sinopharm Group, purity>99%) was used as a positive control. In the inhibition zone test, enramycin was dissolved in physiological saline and its concentration was 0.1μg / mL; paeonol was set at 0.1g / mL, 0.05g / mL, 0.025g / mL, 0.0125g / mL, and 0.0063g / mL, with a total of 5 concentration gradients; each treatment group was repeated 3 times.

[0062] In the specific test process, Escherichia coli and Salmonella were cultured in LB medium and the concentration was 1×10 5CFU / mL of bacterial liquid; prepare LB solid culture plates and LB semi-solid culture medium, and when the LB semi-solid culture medium is cooled to about 37°C, take 100 μL of 1×10 6 Add CFU / mL of Escherichia coli and Salmonella liquid to LB semi-solid medium, shake well, and quickly pour onto LB solid culture plate, then evenly place Oxford cup on the culture plate, take out Oxford cup after semi-solid medium solidifies, add 50 μL of each treatment solution into the ring hole, and culture in a 37°C constant temperature incubator for 24 hours; then use a ruler (accuracy of 0.01 cm) to measure the diameter of the inhibition zone;

[0063] The Clostridium perfringens was cultured in cooked meat medium and the concentration was 1×10 5 Prepare cooked meat medium solid culture plates and cooked meat semi-solid culture medium. When the cooked meat semi-solid culture medium is cooled to about 37°C, take 100 μL of 1×10 5 CFU / mL of Clostridium perfringens solution was added to the cooked meat semi-solid culture medium and shaken well, and then quickly poured onto the cooked meat culture medium solid culture plate, after which an Oxford cup was evenly placed on the culture plate, and after the semi-solid culture medium solidified, the Oxford cup was taken out and 50 μL of each of the above treatment solutions was added into the ring hole, and then placed in a 37°C constant temperature incubator for 24 hours. The diameter of the inhibition zone was then measured using a ruler (accuracy of 0.01 cm).

[0064] 2. Test results:

[0065] The in vitro inhibitory results of paeonol on three chicken-derived pathogens, Clostridium perfringens, Escherichia coli and Salmonella, are shown in Table 2 below.

[0066] Table 2

[0067]

[0068] As shown in Table 2, different concentrations of paeonol showed antibacterial effects on chicken-derived Clostridium perfringens, Escherichia coli, and Salmonella, and the diameter of the inhibition zone increased with the increase of paeonol dosage, indicating that with the increase of paeonol dosage, its antibacterial effect is better. Among the three chicken-derived pathogens, paeonol has the most obvious antibacterial effect on Clostridium perfringens.

[0069] Effects of paeonol on growth performance and intestinal health of broiler chickens infected with Clostridium perfringens

[0070] The present application example verifies the effect of paeonol on the growth performance and intestinal health of broilers infected with Clostridium perfringens, and the verification method is as follows:

[0071] 1. Materials and Methods

[0072] The experiment involved 288 healthy AA+ Broiler male chicks were evenly divided into 3 treatment groups, each treatment group was divided into 8 replicate groups, each replicate group included 12 chickens; the 3 treatment groups were control group, CP infection group, paeonol + CP infection group; broilers in the control group and CP infection group were fed with a "corn-soybean meal" type basal diet, and the diet formula was formulated according to the nutritional requirements standard of Chinese broilers (NY / T33-2004); 0.01% paeonol (purchased from Sigma, purity > 90%) was added to the basal diet of broilers in the paeonol + CP infection group; on the 14th to 21st day of the experiment, 1 mL CP (CVCC2030 strain, dose of 1.0×10 9 CFU / mL), and the control group broilers were gavaged with an equal volume of CP medium; during the experiment, the broilers were free to eat and drink, and a 24-hour lighting system was used; on the 22nd day of the experiment, all broilers were fasted for 8 hours, and then weighed and the feed consumption was counted, and growth performance-related indicators such as average daily gain (ADG), average daily feed intake (ADFI), and feed-to-weight ratio (FCR) were calculated. After that, one broiler with uniform weight was selected from each repeated group to collect subwing venous blood and separate serum for testing; further, sodium pentobarbital (at a dose of 50 mg / kg live weight) was injected into the subwing vein for anesthesia, slaughtered, and ileum tissue was dissected to make slices for observation, and ileum samples were taken for subsequent analysis.

[0073] 2. Data Analysis

[0074] The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups, with P < 0.05 as the standard of significant difference and P < 0.01 as the standard of extremely significant difference. The results are expressed as mean ± standard deviation.

[0075] 3. Test results

[0076] 3.1 Effect of paeonol on growth performance of broilers infected with Clostridium perfringens

[0077] Table 3 shows the effects of paeonol on the growth performance of broilers infected with Clostridium perfringens, where a, b, and c are significant differences. Data in the table with different letters on their shoulders indicate significant differences P<0.05.

[0078] Table 3

[0079] Test items Control group CP infection group Paeonol + CP infection group P-value d21 average body weight (g) <![CDATA[695.12±11.43 a ]]> <![CDATA[535.92±43.21 c ]]> <![CDATA[595.31±32.19 b ]]> <0.001 ADFI(g) <![CDATA[44.36±2.43 a ]]> <![CDATA[40.32±1.09 b ]]> <![CDATA[40.80±2.21 b ]]> 0.036 ADG(g) <![CDATA[33.11±1.21 a ]]> <![CDATA[25.52±1.92 c ]]> <![CDATA[28.34±0.88 b ]]> <0.001 FCR <![CDATA[1.34±0.08 c ]]> <![CDATA[1.58±0.12 a ]]> <![CDATA[1.44±0.09 b ]]> 0.012

[0080] As shown in Table 3, Clostridium perfringens infection caused a decrease in the average body weight, average daily feed intake, and average daily weight gain of broilers, and an increase in the feed-to-weight ratio; adding paeonol to the diet can alleviate the decrease in the average body weight and average daily weight gain of broilers, and the increase in the feed-to-weight ratio caused by Clostridium perfringens infection.

[0081] 3.2 Effects of paeonol on blood biochemical parameters of broiler chickens infected with Clostridium perfringens

[0082] Table 4 shows the effects of paeonol on blood biochemical indices of broiler chickens infected with Clostridium perfringens, where a, b, and c are markers of significant differences.

[0083] Table 4

[0084]

[0085]

[0086] As shown in Table 4, the addition of paeonol to the diet can alleviate the increase in the levels of alanine aminotransferase (ALT), γ-aminotranspeptidase (GGT), uric acid (UA), and lactate dehydrogenase (LDH) in broiler plasma caused by Clostridium perfringens infection. When the liver is damaged, the levels of ALT and GGT in plasma increase; in addition, the increase in LDH content in plasma can reveal intestinal damage. The results show that the addition of paeonol to the diet can alleviate the negative effects of Clostridium perfringens infection on the liver and intestines of broiler chickens.

[0087] 3.3 Effects of paeonol on blood D-xylose content and ileum morphology in broilers infected with Clostridium perfringens

[0088] Table 5 shows the effects of paeonol on the blood D-xylose content and ileum morphology of broilers infected with Clostridium perfringens, where a, b, and c are significant differences.

[0089] Table 5

[0090]

[0091] As shown in Table 5, the addition of paeonol to the diet can alleviate the increase in ileal crypt depth and the decrease in the ratio of villus height to crypt depth caused by Clostridium perfringens infection in broilers, and alleviate the decrease in plasma D-xylose content in broilers caused by Clostridium perfringens infection. The higher the intestinal villi height, the larger the contact area with nutrients in the intestinal cavity and the better the absorption efficiency. Therefore, the higher the intestinal villi height and the ratio of villus height to crypt depth indicate better intestinal absorption function. In addition, D-xylose can be absorbed by the intestine but cannot be used by the body. Therefore, the intestinal absorption function can be evaluated by evaluating the D-xylose content in the blood. The results of the study showed that the addition of paeonol to the diet can alleviate the negative impact of Clostridium perfringens infection on the intestinal absorption function of broilers.

[0092] 3.4 Effects of Paeonol on Proportion of Intestinal Immune Cells and Content of Pro-inflammatory Cytokines in Broilers Infected with Clostridium perfringens

[0093] Figure 1 The results showing the effects of paeonol on the proportions of monocytes and B lymphocytes in the blood of broilers infected with Clostridium perfringens are as follows. Among them Figure 1 A is the flow cytometry density map of monocytes and B lymphocytes in the blood of chickens in the control group (CTR), Clostridium perfringens infection group (CC), and paeonol + Clostridium perfringens infection group (PAE+CC). Figure 1 B is the corresponding statistical analysis results (**0.001 < P < 0.01, ***P < 0.001). Among them, monocytes / macrophages are represented by MON + and B lymphocytes are represented by BU1 + . The monocytes and B lymphocytes in whole blood were analyzed using a Navios EX flow cytometer (Beckman Coulter Corp., Fullerton, CA, USA). The test antibodies CD45, Bu-1, and Mon were all purchased from Southern Biotechnology Associates Inc., Birmingham, AL. Specifically, gating was performed under CD45 + to exclude the interference of red blood cells in chicken blood.

[0094] It can be seen from Figure 1 that Clostridium perfringens infection upregulates the proportion of monocytes in the blood of broilers and downregulates the proportion of B lymphocytes in the blood of broilers. Adding paeonol to the diet can upregulate the proportion of monocytes in the blood of broilers and relieve the downregulation of the proportion of B lymphocytes in the serum of broilers caused by Clostridium perfringens infection.

[0095] Figure 2 The results showing the effects of paeonol on the transcriptional levels of immune-related genes in the ileum of broilers infected with Clostridium perfringens are as follows. Among them, the black columns represent the control group, the gray columns represent the infection + paeonol group, and the colored columns represent the infection group. NF-κB represents nuclear transcription factor κB, MyD88 represents myeloid differentiation factor 88, NOD1 represents nucleotide-binding oligomerization domain-containing protein 1, IL-1β represents interleukin 1β, TNF-α represents tumor necrosis factor α, IFN-γ represents interferon γ, FOXP3 represents forkhead box protein P3, IL-10 represents interleukin 10, TGF-β represents transforming growth factor β, MHC-II represents major histocompatibility complex-II, LYZ represents lysozyme, and Leap2 represents liver-expressed antimicrobial peptide 2, where *0.01 < P < 0.05, **0.001 < P < 0.01, ***P < 0.001.

[0096] It can be seen from Figure 2It can be seen that Clostridium perfringens infection up-regulates MyD88, NOD1, IL-1β, TNF-α, IFN-γ, and IL-10 in the ileum of broilers, and down-regulates the transcription levels of TGF-β, LYZ, and Leap-2. Adding paeonol to the diet can alleviate the up-regulation of MyD88, NOD1, IL-1β, TNF-α, IFN-γ, and IL-10 in the ileum of broilers caused by Clostridium perfringens infection. The results of the study showed that adding paeonol to the diet can alleviate the intestinal inflammatory response of broilers caused by Clostridium perfringens infection.

[0097] Effects of paeonol on growth performance and intestinal health of broiler chickens infected with coccidia and Clostridium perfringens

[0098] The present application example verifies the effect of paeonol on the growth performance and intestinal health of broilers infected with coccidia and Clostridium perfringens, and the verification method is as follows:

[0099] 1. Materials and Methods

[0100] The experiment involved 288 healthy AA + Broiler male chicks were divided into 3 treatment groups, each treatment group was divided into 8 replicate groups, each replicate group included 12 chickens; the 3 treatment groups were: control group, coccidia and Clostridium perfringens combined infection group (CCP), paeonol + CCP group; broilers in the control group and CCP infection group were fed a "corn-soybean meal" type basal diet, and the diet formula was formulated in accordance with the Chinese broiler nutrition requirement standard (NY / T33-2004). Paeonol (purchased from Sigma, purity > 90%) was added to the basal diet of broilers in the paeonol + CCP group at 0.01% of feed weight. On the 8th and 10th days of the experiment, the broilers in the CCP group and the paeonol + CCP group were vaccinated with an ultra-high dose of coccidia vaccine (purchased from Foshan Zhengdian Company, the dose was 30 times the recommended dose) to establish intestinal coccidia infection. On the 14th to 21st days of the experiment, the broilers in the CCP group and the paeonol + CCP group were gavaged with 1 mL of CP (CVCC2030 strain, the dose was 1.0×10 9 CFU / mL), and the control group broilers were gavage-fed with an equal volume of CP medium; during the experiment, broilers were allowed to eat and drink freely, and a 24-h lighting system was adopted; on the 22nd day of the experiment, all broilers were fasted for 8 h, and then weighed and the feed consumption was counted, and growth performance-related indicators such as average daily gain (ADG), average daily feed intake (ADFI), and feed-to-weight ratio (FCR) were calculated; thereafter, one broiler with uniform weight was selected from each replicate group to collect subwing venous blood and separate serum for testing; further, sodium pentobarbital (at a dose of 50 mg / kg live weight) was injected into the subwing vein for anesthesia, and the broilers were slaughtered, and the ileum tissue was dissected to make slices for observation, and ileum samples were taken for subsequent analysis.

[0101] 2. Data Analysis

[0102] The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups, with P < 0.05 as the standard of significant difference and P < 0.01 as the standard of extremely significant difference. The results are expressed as mean ± standard deviation.

[0103] 3. Test results

[0104] 3.1 Effect of paeonol on growth performance of broilers infected with coccidia and Clostridium perfringens

[0105] Table 6 shows the effects of paeonol on the growth performance of broilers co-infected with coccidia and Clostridium perfringens, where a, b, and c are significant differences. Data in the table with different letters on their shoulders indicate significant differences, P<0.05.

[0106] Table 6

[0107] Test items Control group CCP Group Paeonol + CCP group P-value d21 average body weight (g) <![CDATA[683.06±10.39 a ]]> <![CDATA[577.51±31.77 b ]]> <![CDATA[561.96±51.34 b ]]> <0.001 ADFI(g) 47.43±1.46 46.73±2.54 46.94±3.58 0.535 ADG(g) <![CDATA[33.86±0.54 a ]]> <![CDATA[28.31±1.67 b ]]> <![CDATA[27.49±2.71 b ]]> <0.001 FCR <![CDATA[1.40±0.05 c ]]> <![CDATA[1.65±0.07 a ]]> <![CDATA[1.53±0.08 b ]]> <0.001

[0108] As shown in Table 6, the combined infection of coccidia and Clostridium perfringens caused the average body weight and average daily weight gain of broilers to decrease, and the feed-to-weight ratio to increase. The addition of paeonol to the diet can alleviate the increase in the feed-to-weight ratio of broilers caused by the combined infection of coccidia and Clostridium perfringens.

[0109] 3.2 Effects of paeonol on serum D-xylose content and ileal morphology in broilers infected with coccidia and Clostridium perfringens

[0110] Table 7 shows the effects of paeonol on serum D-xylose content and ileum morphology in broilers co-infected with coccidia and Clostridium perfringens, where a, b, and c are significant differences.

[0111] Table 7

[0112]

[0113] As shown in Table 7, the addition of paeonol to the diet can alleviate the increase in the depth of the ileal crypts caused by the combined infection of coccidia and Clostridium perfringens, and increase the ratio of the ileal villus height to the crypt depth. In addition, the addition of paeonol to the diet can alleviate the decrease in the plasma D-xylose content of broilers caused by the combined infection of coccidia and Clostridium perfringens. The experimental results further confirmed that paeonol can improve the intestinal absorption function of broilers with enteritis.

[0114] 3.3 Effect of paeonol on the transcriptional level of ileal barrier-related genes in broiler chickens infected with coccidia and Clostridium perfringens

[0115] Figure 3The results show the effect of paeonol on the transcriptional level of ileal barrier-related genes in broiler chickens infected with coccidia and Clostridium perfringens. The black column represents the control group (CTR), the gray column represents the infection + paeonol group (PAE + CCP), and the colored column represents the infection group (CCP). ZO-1 represents zonula occludens 1, Mucin-2 represents mucin 2, and Claudin-1 represents tight junction protein 1. **0.001 <P<0.01,***P<0.001。

[0116] As can be seen from the figure, the higher the expression levels of ZO-1, Mucin-2 and Claudin-1 in the intestine, the stronger the intestinal barrier function and the stronger the ability of the intestine to resist pathogen infection. Figure 3 The results showed that the combined infection of coccidia and Clostridium perfringens downregulated the transcription levels of ileal ZO-1, Mucin-2 and Claudin-1, indicating that the combined infection of coccidia and Clostridium perfringens caused the damage of intestinal barrier function. The addition of paeonol to the diet could alleviate the downregulation of ileal Mucin-2 and Claudin-1 transcription levels caused by the combined infection of coccidia and Clostridium perfringens, indicating that paeonol could alleviate the negative impact of the combined infection of coccidia and Clostridium perfringens on the intestinal barrier function of broilers.

[0117] In summary, paeonol can be used to prepare drugs or feed additives for treating or preventing necrotic enteritis in chickens, and the prepared drugs or feed additives have the ability to resist various pathogens that cause necrotic enteritis in chickens, such as Clostridium perfringens. The dosage forms of the drugs and feed additives include oral liquid, granules, powders, tablets and the like.

[0118] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. Application of paeonol in the preparation of medicines or feed additives for treating or preventing necrotic enteritis in chickens.

2. The use according to claim 1, characterized in that: The chicken necrotic enteritis is an intestinal inflammation caused by Clostridium perfringens alone or in combination with coccidia.

3. The use according to claim 1, characterized in that: The medicine or feed additive is paeonol.

4. The use according to claim 1, characterized in that: The medicine or feed additive is a paeonol composition comprising paeonol and auxiliary materials, wherein the auxiliary materials include at least one of anhydrous ethanol, propylene glycol, edible oil, water, lactose, maltose, soluble starch, montmorillonite, medical stone, zeolite powder, defatted rice bran, corn cob powder, bran, and white carbon black.

5. The use according to claim 4, characterized in that: The paeonol composition is a solution formed by mixing paeonol with anhydrous ethanol and water, wherein the concentration of paeonol is 10-120 mg / mL.

6. The use according to claim 4, characterized in that: The paeonol composition is a solid mixture of paeonol and lactose or maltose, and at least one of soluble starch, bran and white carbon black, wherein the mass fraction of paeonol in the solid mixture is 40% to 60%.

7. The use according to claim 4, characterized in that: The paeonol composition is a powdery mixture of paeonol and at least one of soluble starch, bran and white carbon black, wherein the mass fraction of paeonol in the powdery mixture is 40% to 60%.

8. The use according to claim 4, characterized in that: When the medicine or feed additive is a paeonol composition, its preparation type includes at least one of oral liquid, granules, tablets, and powders, and the preparation can be prepared by conventional pharmaceutical preparation methods.

9. The use according to claim 1, characterized in that: The method for using the drug or feed additive includes at least one of (1) to (4): (1) Orally administering the drug to chickens at a dosage of 10 to 100 mg / kg of chicken body weight; (2) The paeonol content is 0.01% to 0.03% by weight of the chicken feed; the paeonol granules, powder or tablets are added to the chicken feed as a feed additive; (3) When used for daily prevention of chicken enteritis, the paeonol powder, granules or tablets are crushed and added to the feed or drinking water so that the paeonol content is 0.01% of the weight of the chicken feed or drinking water; (4) When used for clinical treatment of chicken enteritis: orally administer the drug to chickens at a dosage of 100 mg / kg of chicken body weight; or grind the granules, powder or tablets into powder and feed directly to chickens at a dosage of 100 mg / kg of chicken body weight; or grind the granules, powder or tablets into powder and add it to the feed at a dosage of 0.03% of the weight of chicken feed; or grind the granules, powder or tablets into powder and add it to the drinking water at a dosage of 0.03% of the weight of drinking water, three times a day, morning, noon and evening, for one week.

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