Compound Chinese herbal medicine for replacing antibiotics to treat chicken colibacillosis and application of compound Chinese herbal medicine

Abstract

The invention discloses a compound Chinese herbal medicine for replacing antibiotics to treat chicken colibacillosis, and belongs to the technical field of veterinary drugs. The raw material formula of the compound Chinese herbal medicine is selected from any one of the following components: (1) sappan wood, sanguisorba officinalis, pomegranate rind, coptis chinensis and fructus forsythiae; (2) preparing hematoxylon, sanguisorba officinalis, pomegranate rind, coptis chinensis, andrographis paniculata and liquorice; and (3) hematoxylon, sanguisorba officinalis, pomegranate rind, coptis chinensis, andrographis paniculata and honeysuckle. The compound Chinese herbal medicine components are carefully selected and reasonably matched, the synergistic effect of all the components is exerted, the treatment effect of the preferred compound Chinese herbal medicine is not remarkably different from that of antibiotics, and therefore the compound Chinese herbal medicine can replace the antibiotics to be applied to broiler chicken breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drugs, and in particular relates to a compound Chinese herbal medicine for treating chicken Escherichia coli disease instead of antibiotics and application thereof. Background Art

[0002] Colibacillosis in chickens is a general term for different types of infectious diseases in chickens caused by certain pathogenic serotypes of Escherichia coli. This disease has rich manifestations, such as pericarditis, perihepatitis, eye inflammation, air sac inflammation, peritonitis, synovitis, salpingitis, omphalitis, sepsis, Escherichia coli granuloma, etc. Chickens of different breeds and ages are affected, especially broilers. At the same time, the disease is a common secondary disease of chronic respiratory diseases, avian influenza, Newcastle disease and other diseases, leading to widespread prevalence and high morbidity and mortality. With the development of intensive breeding industry, the disease has become one of the diseases that cause huge economic losses in the poultry industry. At present, antibiotics are the conventional means of treating colibacillosis in chickens. However, the long-term irrational use of antibiotics in the breeding process has spawned a large number of drug-resistant strains and led to the proliferation of drug residues in livestock products. This dilemma has become a focus issue that needs to be overcome in the field of global breeding and food hygiene. Therefore, there is an urgent need for a treatment method that can replace antibiotics to effectively prevent and treat colibacillosis in chickens.

[0003] As a treasure in the treasure house of traditional Chinese medicine, Chinese herbal medicine has a wide range of materials, abundant reserves, and is safe, efficient, convenient, and has multiple functions such as antiviral, antibacterial, anti-inflammatory, and immunomodulatory. It has become a research hotspot in the field of antibiotic substitution. When Chinese herbal medicines and their compound preparations are used in the treatment of animal diseases, they show excellent advantages such as weak side effects, low drug residues, and significant prevention and treatment effects. In recent years, they have been widely popularized and applied in the practice of animal clinical disease prevention and treatment. In summary, compound Chinese herbal medicines have opened up new directions and paths for the treatment of chicken Escherichia coli disease, and have also laid a solid theoretical foundation for the research and development of safe and efficient antibiotic substitutes, which has far-reaching significance and great value in promoting the green and healthy development of the poultry industry.

[0004] At present, there is no literature report on the use of Sappan wood, Sanguisorba officinalis, Coptis chinensis, and pomegranate peel as the basic formula, and Forsythia suspensa, Scutellaria baicalensis, Andrographis paniculata, Licorice, and Honeysuckle as adjuvants to prepare a prescription for the treatment of Escherichia coli disease in chickens. Summary of the invention

[0005] The purpose of the present invention is to provide a compound Chinese herbal medicine for treating chicken Escherichia coli disease, which can improve the production performance and intestinal flora of sick chickens, thereby achieving the purpose of replacing antibiotics to treat chicken Escherichia coli disease.

[0006] In order to achieve the above technical objectives, the inventors used Sappan wood, Sanguisorba officinalis, Coptis chinensis, and Pomegranate peel as the basic formula, and added Fructus Forsythiae, Scutellaria baicalensis, Andrographis paniculata, Glycyrrhiza uralensis, and Honeysuckle as adjuvants. Orthogonal experiments were used to form 16 formulas at two levels of administration and no administration, and in vitro experiments were conducted. Finally, the following technical scheme was obtained:

[0007] A Chinese herbal medicine compound for treating chicken Escherichia coli disease instead of antibiotics. The raw material formula of the Chinese herbal medicine compound is selected from any one of the following: (1) sappan wood, sanguisorba officinalis, pomegranate peel, coptis chinensis and forsythia suspensa; (2) sappan wood, sanguisorba officinalis, pomegranate peel, coptis chinensis, andrographis paniculata and liquorice; (3) sappan wood, sanguisorba officinalis, pomegranate peel, coptis chinensis, andrographis paniculata and honeysuckle.

[0008] Further preferably, in the above-mentioned compound Chinese herbal medicine for replacing antibiotics to treat chicken Escherichia coli disease, the raw material ratio of formula (1) is 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2). In one embodiment of the present invention, the raw material ratio of formula (1) is 1: 1: 1: 1: 1: 1.

[0009] Further preferably, the composite Chinese herbal medicine for replacing antibiotics to treat chicken Escherichia coli disease as described above, wherein the raw material ratio of formula (2) is 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2). In one embodiment of the present invention, the raw material ratio of formula (2) is 1: 1: 1: 1: 1: 1: 1.

[0010] Further preferably, in the above-mentioned compound Chinese herbal medicine for replacing antibiotics to treat chicken Escherichia coli disease, the raw material ratio of formula (3) is 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2). In one embodiment of the present invention, the raw material ratio of formula (3) is 1: 1: 1: 1: 1: 1: 1.

[0011] At the same time, the present invention also provides a compound Chinese herbal medicine extract, which is obtained by mixing the Chinese herbal medicine raw materials in the formula, soaking in water, decocting and concentrating; or, the extract is obtained by soaking the Chinese herbal medicine raw materials in the formula in water, decocting and concentrating them separately, and then combining the Chinese herbal medicine concentrates;

[0012] The raw material ratio of the formula is selected from any one of the following by weight:

[0013] (1) Sappan wood: Sanguisorba officinalis: Punica granatum: Coptis chinensis: Forsythia suspensa = 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2);

[0014] (2) Sappan wood: Sanguisorba officinalis: Punica granatum: Coptis chinensis: Andrographis paniculata: Licorice root = 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2);

[0015] (3) Sappan wood: Sanguisorba officinalis: Punica granatum: Coptis chinensis: Andrographis paniculata: Honeysuckle = 1: (0.8~1.2): (0.8~1.2): (0.8~1.2): (0.8~1.2): (0.8~1.2).

[0016] In addition, the present invention also provides a preparation method of the above-mentioned compound Chinese herbal medicine extract, which comprises the following steps: taking weighed Chinese herbal raw materials, mixing them, placing them in a grinder and grinding them into coarse powder; soaking the coarse Chinese herbal raw material powder in 8 to 14 times water for 0.5 to 1.5 hours; heating to boiling over medium heat, simmering over low heat for 0.5 to 1 hour, filtering out the decoction, repeating the decoction of the filter residue in the same way for 1 to 2 times, and combining the decoctions; and concentrating the decoction under reduced pressure to 0.5 to 2 g of crude drug / mL.

[0017] Through animal experiments, it was found that the compound Chinese herbal extract of the present invention can improve the production performance of broilers infected with E. coli to a certain extent, promote the proliferation of beneficial bacteria, inhibit the colonization and growth of harmful bacteria, and is beneficial to regulating the balance of cecal microorganisms, verifying that there is no significant difference between the compound Chinese herbal extract and the antibiotic treatment effect; this shows that the compound Chinese herbal extract of the present invention can replace antibiotics to treat chicken E. coli disease, and has a good therapeutic effect on broiler enteritis caused by E. coli. Therefore, the present invention also provides a product use, that is, the use of the above-mentioned compound Chinese herbal extract in the preparation of veterinary drugs that replace antibiotics to treat chicken E. coli disease.

[0018] Compared with the prior art, the present invention has the following beneficial effects: by selecting the components of the compound Chinese herbal medicine and reasonably combining them, the synergistic effect of each component is brought into play, and there is no significant difference in the therapeutic effect between the preferred compound Chinese herbal medicine and antibiotics, so it can replace antibiotics in broiler farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the effect of different concentrations of formula 4 on cell survival rate.

[0020] Figure 2 The figure shows the effect of different concentrations of formula 7 on cell survival rate.

[0021] Figure 3 The figure shows the effect of different concentrations of formula 8 on cell survival rate.

[0022] Figure 4 The figure shows the effect of different concentrations of hematoxylin on cell survival rate.

[0023] Figure 5 The figure shows the effect of different concentrations of Sanguisorba officinalis on cell survival rate.

[0024] Figure 6 The effect of different concentrations of Coptis chinensis on cell survival rate.

[0025] Figure 7 The effect of different doses of Escherichia coli infection on cytotoxicity.

[0026] Figure 8 The effects of different Chinese herbal medicines on cell survival rate.

[0027] Fig. 9 To investigate the effects of different Chinese herbal medicines on the inflammatory factor IL-10 in chicken intestinal epithelial cells infected with Escherichia coli.

[0028] Fig.10 To investigate the effects of different Chinese herbal medicines on the inflammatory factor INF-γ in chicken intestinal epithelial cells infected with Escherichia coli. DETAILED DESCRIPTION

[0029] The present invention is further described below by test examples. It should be understood that the following test examples are only used to illustrate the technical scheme and technical effects of the present invention, and are not intended to limit the scope of protection of the present invention. Simple improvements to the method under the overall conception are within the scope of protection claimed by the present invention.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0031] Example 1: Preparation and antibacterial effect of single Chinese herbal medicine

[0032] Weigh 500 g each of Scutellaria baicalensis, Coptis chinensis, Honeysuckle, Andrographis paniculata, Licorice, Forsythia suspensa, Sappan wood, Pomegranate peel, and Sanguisorba officinalis, grind them into powder, add appropriate amount of cold water (preferably to cover the medicinal materials), soak for 1 hour, then boil over medium heat, boil for 30 minutes over low heat, filter out the decoction, add appropriate amount of warm water, and continue to boil once according to the above method, combine the two decoctions, and concentrate them to 500 mL under reduced pressure using a rotary evaporator. At this time, the concentration of the medicinal solution is equivalent to 1 g of the crude drug in 1 mL of the medicinal solution.

[0033] The minimum inhibitory concentration of each drug solution was determined by 96-well plate microtiter method, and the in vitro antibacterial activity of each drug solution against the test bacteria was compared. Using a 96-well polystyrene microtiter plate, 100 μL of ordinary nutrient broth was added to each row of wells 1 to 10 in turn; 100 μL of the test drug solution was added to the first well, mixed thoroughly, and 100 μL was added to the second well, and then two-fold serial dilution was performed to the tenth well. Finally, 100 μL was drawn from the tenth well and discarded to form 10 different concentrations (500.00 mg / mL, 250.00 mg / mL, 125.00 mg / mL, 62.50 mg / mL, 31.25 mg / mL, 15.63 mg / mL, 7.81 mg / mL, 3.91 mg / mL, 1.95 mg / mL, 0.98 mg / mL), and then 10 μL of Escherichia coli solution (concentration of 10 8 CFU / mL), the 11th well is the negative control (no pathogen), and the 12th well is the positive control (no test solution). The 96-well plate is placed in a 37°C constant temperature incubator for 18-24 hours, and the lowest drug concentration in the clear transparent well is the minimum inhibitory concentration (MIC).

[0034] Pipette 0.1 mL of the culture from each well above the minimum inhibitory concentration, pour into the agar medium, mix evenly, and then culture in a 37°C constant temperature incubator for 24 hours. The minimum concentration at which no bacteria grows is the minimum bactericidal concentration (MBC) of the drug for this type of bacteria.

[0035] The results are shown in Table 1:

[0036] Table 1: Antibacterial effects of single Chinese herbal medicines

[0037]

[0038] According to the test statistical results in Table 1, Sappan wood, Sanguisorba officinalis, and Coptis chinensis have the best antibacterial effect on Escherichia coli, with MIC of 31.25 mg / mL and MBC of 62.5 mg / mL; followed by pomegranate peel with MIC of 62.5 mg / mL and MBC of 125 mg / mL. Therefore, the basic antibacterial formula was determined to be composed of Sappan wood, Sanguisorba officinalis, Coptis chinensis, and Pomegranate peel, and Forsythia suspensa, Scutellaria baicalensis, Andrographis paniculata, Licorice, and Honeysuckle were added as adjuvants.

[0039] Example 2: Preparation and antibacterial effect of compound Chinese herbal medicine

[0040] (1) Preparation of compound Chinese herbal medicines: Based on the results of the minimum inhibitory concentration and minimum bactericidal concentration of single Chinese herbal medicines, Chinese herbs such as Sappan wood, Sanguisorba officinalis, Pomegranate peel, and Coptis chinensis were selected as the basic formula A; Chinese herbs such as Forsythia suspensa, Scutellaria baicalensis, Andrographis paniculata, Licorice, and Honeysuckle were added as adjuvants, namely B, C, D, E, and F. Orthogonal experiments were used to form 16 formulas at two levels: drug administration and no drug administration. Drug administration was level 1, and no drug administration was level 2. The content of each single Chinese herbal medicine raw material in each formula is the same, and the corresponding Compound Chinese herbal medicine liquid The preparation method of the compound Chinese herbal medicine liquid is as follows: weigh the Chinese herbal medicine raw materials, mix them, and grind them into coarse powder in a grinder; soak the coarse Chinese herbal medicine raw material powder in 10 times water for 1 hour; heat it to boiling over medium heat, simmer it over low heat for 0.5 hours, filter out the decoction, repeat the decoction twice in the same way, and combine the decoctions; and concentrate the decoction under reduced pressure to 1 g of crude drug / mL.

[0041] Table 2: Orthogonal test table

[0042]

[0043] (2) Antibacterial effect of compound Chinese herbal medicines: The minimum inhibitory concentration of each drug solution was determined by 96-well plate microtiter method, and the in vitro antibacterial activity of each drug solution against the test bacteria was compared. Use a 96-well polystyrene microplate, add 100 μL of common nutrient broth to wells 1 to 10 in each row; add 100 μL of test solution to well 1, mix thoroughly, then pipette 100 μL into well 2, and then dilute to well 10 by two times, finally pipette 100 μL from well 10 and discard, forming 10 different concentrations (500.00 mg / mL, 250.00 mg / mL, 125.00 mg / mL, 62.50 mg / mL, 31.25 mg / mL, 15.63 mg / mL, 7.81 mg / mL, 3.91 mg / mL, 1.95 mg / mL, 0.98 mg / mL), then add 10 μL of Escherichia coli solution (concentration 10 8 CFU / mL), the 11th well is the negative control (no pathogen), and the 12th well is the positive control (no test solution). The 96-well plate is placed in a 37°C constant temperature incubator for 18-24 hours, and the lowest drug concentration in the clear well is the MIC.

[0044] Pipette 0.1 mL of the culture from each well above the minimum inhibitory concentration, pour into the agar medium, mix evenly, and then culture in a 37°C constant temperature incubator for 24 hours. The minimum drug concentration without bacterial growth is calculated as MBC.

[0045] (3) Screening of compound Chinese herbal medicines: The compound Chinese herbal medicines with the best antibacterial effect are screened out based on the minimum inhibitory concentration and minimum bactericidal concentration of the compound Chinese herbal medicines.

[0046] The results are shown in Table 3:

[0047] Table 3: Antibacterial effects of compound Chinese herbal medicines

[0048]

[0049] According to the test statistical results in Table 3, the best antibacterial effect of formula 4 on Escherichia coli is obtained, with a MIC of 15.625 mg / mL and an MBC of 31.25 mg / mL; secondly, the MIC of formula 7 and formula 8 on Escherichia coli is 62.5 mg / mL and the MBC is 125 mg / mL. Therefore, formula 4, formula 7 and formula 8 are selected for the next test. The compound Chinese herbal medicine formula of formula 4 is Su Mu, Sanguisorba officinalis, pomegranate peel, Coptis chinensis, and Forsythia suspensa, and the mass ratio of each raw material is 1:1:1:1:1; the compound Chinese herbal medicine formula of formula 7 is Su Mu, Sanguisorba officinalis, pomegranate peel, Coptis chinensis, Andrographis paniculata, and Licorice, and the mass ratio of each raw material is 1:1:1:1:1:1; the compound Chinese herbal medicine formula of formula 8 is Su Mu, Sanguisorba officinalis, pomegranate peel, Coptis chinensis, Andrographis paniculata, and Honeysuckle, and the mass ratio of each raw material is 1:1:1:1:1:1:1.

[0050] Example 3: Dose screening of compound Chinese herbal medicine

[0051] Purchase 18-year-old chicken embryos to isolate, culture and identify chicken embryo intestinal epithelial cells. Inoculate the isolated chicken embryo intestinal epithelial cells into a 96-well cell culture plate, and add 100 μL of 5×10 4 After the cells were cultured at 37°C and 5% CO2 for 24 hours, the culture medium was discarded and 100 μL of basal culture medium (DMEM / F12 culture medium) containing E. coli was added to each well. The multiplicity of infection (the ratio of bacteria to cells during infection, MOI) was 0, 0.1, 1, 10, and 100. After further culture at 37°C and 5% CO2 for 3 hours, 6 hours, and 9 hours, the cell activity was determined using the MTT kit.

[0052] The isolated chicken embryo intestinal epithelial cells were inoculated into 96-well cell culture plates using the MTT method, and 100 μL of 1×10 5 / mL cell suspension. After the cells were cultured at 37°C and 5% CO2 for 48 hours, the culture medium was discarded, and 100 μL of DMEM / F12 culture medium with double antibodies, no serum, and no compound Chinese herbal medicine solution was added to each well, and starved for 8 hours. Then the cells were gently washed twice with 100 μL PBS (pH = 7.4). Then DMEM / F12 culture medium containing 7 concentrations of compound Chinese herbal medicine solution (preparation method is the same as Example 2) was added, and the culture was continued for 6 hours. Then the cell viability was determined using the MTT kit, and the cell survival rate was calculated by measuring the absorbance at 570nm to reflect the toxicity of the compound Chinese herbal medicine to primary intestinal epithelial cells, providing a reference for the optimal additive dose of each compound Chinese herbal medicine in the Escherichia coli infection cell model. Each treatment was repeated 6 times.

[0053] The isolated primary chicken embryo intestinal epithelial cells were inoculated into 6-well cell culture plates, and 2 mL of 1×10 6 / mL cell suspension. After the cells were cultured at 37°C and 5% CO2 for 48 hours, the culture medium was discarded and replaced with DMEM / F12 culture medium containing double antibodies, serum-free, and non-compound Chinese herbal medicine solution, and starved for 8 hours. Then the cells were gently washed twice with 2mL PBS (pH=7.4). 2mL of DMEM / F12 cell culture medium containing Escherichia coli and non-compound Chinese herbal medicine solution was added to each well, and the MOI was 0, 0.1, 1, and 10, respectively. Each treatment was repeated 4 times. The multiplicity of infection was measured. Escherichia coli was inoculated into LB culture medium and cultured at 37°C overnight. The bacterial solution was then centrifuged at 1000g for 10 minutes. The obtained bacterial precipitate was washed twice with sterile PBS and finally resuspended in DMEM / F12 cell culture medium for infecting cells to create an E. coli infected cell model. The isolated primary chicken embryo intestinal epithelial cells were inoculated into a 96-well cell culture plate, and 200μL of a concentration of 2.5×10 6After 48 hours of culture, the culture medium was replaced with DMEM / F12 medium without compound Chinese herbal medicine, serum, and double antibody. After starvation for 8 hours, the cells were washed twice with PBS. They were divided into 8 treatment groups: control group, infection group, and 6 different compound Chinese herbal medicine treatment groups. The control group was added with 200 μL of DMEM / F12 cell culture medium without any treatment, the first two groups were added with 200 μL of DMEM / F12 cell culture medium without double antibody, and each compound Chinese herbal medicine treatment group was added with an equal amount of DMEM / F12 cell culture medium containing the corresponding compound Chinese herbal medicine liquid (preparation method is the same as Example 2), and the drug concentration was selected with reference to the results of the effect of the compound Chinese herbal medicine on the cell viability of primary intestinal epithelial cells. After 6 hours of treatment, the supernatant was discarded and the cells were washed twice with PBS, and 200 μL of DMEM / F12 cell culture medium without compound Chinese herbal medicine containing Escherichia coli (MOI=1) was added. The control group was added with an equal amount of DMEM / F12 cell culture medium without extract, and the cell supernatant was collected after continued cultivation for 4 hours. The supernatant was centrifuged at 4000g for 10 minutes to determine the content of lactate dehydrogenase (LDH), interleukin-10 (IL-10), and interferon-γ (INF-γ), and the kit was used for determination to reflect the therapeutic effect of the compound Chinese herbal medicine on Escherichia coli infected cells. Each treatment had 6 replicates.

[0054] Depend on Figure 1-Figure 10 The experimental statistical results show that MOI = 1 and MOI = 10 significantly increased the release of LDH, indicating that the toxicity of E. coli to cells increased (P < 0.05). The optimal additive dose of formula 4, formula 7 and Su Mu in the E. coli infected cell model was 3.91 mg / mL, while the optimal additive dose of formula 8 and Coptis chinensis in the E. coli infected cell model was 7.81 mg / mL, and the optimal additive dose of Sanguisorba officinalis in the E. coli infected cell model was 1.95 mg / mL. Therefore, the optimal additive doses of formula 4, formula 7, formula 8, Su Mu, Sanguisorba officinalis and Coptis chinensis in the E. coli infected cell model were determined.

[0055] The results of LDH showed that the cell death rates of formula 4 and formula 7 were not significantly different from those of the control group (P>0.05), while compared with the three single herbs of Su Mu, Sanguisorba officinalis, and Coptis chinensis, the cell death rates of formula 4, formula 7, and formula 8 were significantly reduced (P<0.05); the results of IL-10 and INF-γ showed that the therapeutic effect of formula 4 was not significantly different from that of the control group (P>0.05), while compared with the three single herbs, the IL-10 content of formula 4 was significantly increased (P>0.05), and the INF-γ content was significantly reduced (P>0.05). It can be seen that the therapeutic effect of formula 4 in the Escherichia coli infected cell model was significantly better than that of the three single herbs. Therefore, formula 4 was selected for animal experiments. The compound Chinese herbal medicine formula of formula 4 is Su Mu, Sanguisorba officinalis, pomegranate peel, Coptis chinensis, and Forsythia suspensa, and the mass ratio of each raw material is 1:1:1:1:1.

[0056] Example 4: Effect of Compound Chinese Herbal Medicine Formula 4 on Escherichia coli Disease in Broiler Chickens

[0057] 1 Test method

[0058] Three hundred one-day-old healthy broiler chickens were randomly divided into five treatment groups, each with six replicates and ten chickens in each replicate, and the chickens were reared for 28 days.

[0059] Grouping: A blank group (basic diet), B challenge group (basic diet + 1×10 8 CFU / mL Escherichia coli), C antibiotic group (basic diet + drinking water supplemented with enrofloxacin + 1×10 8 CFU / mL Escherichia coli), D challenge + low-dose compound Chinese herbal medicine group (basic diet + drinking water supplemented with compound Chinese herbal medicine + 1×10 8 CFU / mL Escherichia coli), E challenge + high-dose compound Chinese herbal medicine group (basic diet + drinking water supplemented with compound Chinese herbal medicine + 1×10 8 CFU / mL Escherichia coli). On days 11-13 of age, each chicken in the challenge group, antibiotic group, challenge + low-dose compound Chinese herbal medicine group, and challenge + high-dose compound Chinese herbal medicine group was orally administered 1 mL 1×10 8CFU / mL Escherichia coli suspension was used to induce the establishment of an Escherichia coli infection model, and the blank group was gavaged with an equal amount of sterile broth medium. After E. coli inoculation, the antibiotic group was fed with enrofloxacin, and 1L of water contained 1g of enrofloxacin. The low-dose group of the challenge + compound Chinese herbal medicine and the high-dose group of the challenge + compound Chinese herbal medicine were fed with compound Chinese herbal medicine. The low-dose group contained 8mL1g / mL of compound Chinese herbal medicine liquid (preparation method is the same as Example 2) in 1L of water, and the high-dose group contained 16mL 1g / mL of compound Chinese herbal medicine liquid (preparation method is the same as Example 2) in 1L of water, and fed continuously for 7 days. The broilers in the blank group and the challenge group continued to be fed with normal basal diets and drinking water. All experimental broilers were free to eat and drink water, and other feeding and management were handled according to routine procedures. At 21 days and 28 days of age, one chicken was randomly selected from each replicate to collect blood from the subwing vein, and the serum was separated by centrifugation and stored at -20°C for index determination. After the animals were killed by bleeding from the jugular vein, the abdominal cavity was immediately opened, the small intestine was removed, and the contents of the cecum were collected and stored in a -80°C refrigerator for intestinal flora determination.

[0060] 2. Determination of indicators

[0061] 2.1 Production performance

[0062] The initial body weight of chicks in each replicate was weighed before the start of the experiment. The empty stomach weight of chicks was weighed in replicates every week during the experiment. The feed and residual feed amount of each replicate were accurately recorded during the experiment. The average daily gain (ADG), average daily feed intake (ADFI) and feed-to-weight ratio (F / G) were calculated based on the data.

[0063] 2.2 Cecal intestinal microorganisms

[0064] Take 1g of cecal contents and place it in 9mL of normal saline for 10min of shaking, i.e. 10-fold dilution, centrifuge and take the supernatant, then repeat 10 times. 2 ~10 8 The content of microorganisms in the cecal contents was determined by plate colony counting method: lactic acid bacteria (MRS medium), Escherichia coli (VRBA medium) and total colony count (PCA medium). The unit is lg CFU / g.

[0065] 3 Experimental results

[0066] 3.1 Production performance

[0067] Table 4: Production performance of broiler chickens in each group

[0068]

[0069]

[0070] From the experimental statistical results in Table 4, it can be seen that before E. coli infection (1-14 days), there were no significant differences in the production performance indicators of broiler chickens in each group (P>0.05). One week after E. coli infection (15-21 days), E. coli infection significantly reduced ADFI and ADG compared with the control group (P<0.05). Two weeks after E. coli infection (22-28 days), there were no significant differences in ADG and ADFI in the antibiotic group, the low-dose compound Chinese herbal medicine group, and the high-dose compound Chinese herbal medicine group compared with the control group (P>0.05). During the entire experimental period (1-28 days), Escherichia coli infection significantly reduced ADG (P<0.05), while the antibiotic group, low-dose compound Chinese herbal medicine group and high-dose compound Chinese herbal medicine group had significantly increased ADG on the basis of Escherichia coli infection (P<0.05); Escherichia coli infection significantly increased ADFI and F / G (P<0.05), and the antibiotic group, low-dose compound Chinese herbal medicine group and high-dose compound Chinese herbal medicine group had significantly reduced F / G on the basis of Escherichia coli infection (P<0.05), while ADFI was significantly increased (P<0.05).

[0071] 3.2 Cecal intestinal microorganisms

[0072] Table 5: Comparison of cecal intestinal microbial counts in broiler chickens of various groups

[0073]

[0074]

[0075] It can be seen from the experimental statistical results in Table 5 that at 21 days of age, E. coli infection significantly reduced the number of lactic acid bacteria in the cecum of broiler chickens (P<0.05) and increased the number of E. coli (P<0.05). Compared with the challenge group, the antibiotic group, the low-dose compound Chinese herbal medicine group and the high-dose compound Chinese herbal medicine group can significantly reduce the number of E. coli (P<0.05) and increase the number of lactic acid bacteria (P<0.05). At 28 days of age, the challenge group significantly increased the content of E. coli (P<0.05) and significantly reduced the number of lactic acid bacteria (P<0.05). Compared with the challenge group, the antibiotic group, the low-dose compound Chinese herbal medicine group and the high-dose compound Chinese herbal medicine group can significantly increase the number of lactic acid bacteria (P<0.05).

[0076] 4 Conclusion

[0077] (1) Adding compound Chinese herbal medicine to drinking water can improve the production performance of broilers infected with E. coli to a certain extent. The low-dose compound Chinese herbal medicine group had the best effect. There was no significant difference between the normal control group and the antibiotic group.

[0078] (2) Adding compound Chinese herbal medicine to drinking water can significantly reduce the number of Escherichia coli in the cecum of broiler chickens infected with Escherichia coli, increase the number of lactic acid bacteria in the cecum, and help regulate the balance of cecal microorganisms.

[0079] Based on the above test results, adding 8mL of 1g / mL compound Chinese herbal medicine to 1L of drinking water has the best effect.

Claims

1. A compound Chinese herbal medicine for treating chicken Escherichia coli disease instead of antibiotics, characterized in that: The raw material formula of the compound Chinese herbal medicine is selected from any one of the following: (1) sappan wood, sanguisorba officinalis, pomegranate peel, coptis chinensis and forsythia suspensa; (2) sappan wood, sanguisorba officinalis, pomegranate peel, coptis chinensis, andrographis paniculata and liquorice; (3) sappan wood, sanguisorba officinalis, pomegranate peel, coptis chinensis, andrographis paniculata and honeysuckle.

2. The Chinese herbal medicine for treating chicken Escherichia coli disease as an alternative to antibiotics according to claim 1, characterized in that: The raw material ratio of formula (1) by weight is: Sappan wood: Sanguisorba officinalis: Pomegranate peel: Coptis chinensis: Forsythia suspensa = 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2).

3. The Chinese herbal medicine for treating chicken Escherichia coli disease instead of antibiotics according to claim 1, characterized in that: The raw material ratio of formula (2) by weight is: Sappan wood: Sanguisorba officinalis: Pomegranate peel: Coptis chinensis: Andrographis paniculata: Licorice = 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2).

4. The Chinese herbal medicine for treating chicken Escherichia coli disease instead of antibiotics according to claim 1, characterized in that: The raw material ratio of formula (3) by weight is: Sappan wood: Sanguisorba officinalis: Pomegranate peel: Coptis chinensis: Andrographis paniculata: Honeysuckle = 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2).

5. A compound Chinese herbal medicine extract, characterized in that: The extract is obtained by mixing the Chinese herbal raw materials in the formula, soaking them in water, boiling them and concentrating them; or, the extract is obtained by soaking the Chinese herbal raw materials in the formula in water, boiling them and concentrating them separately, and then combining the concentrated Chinese herbal liquids; The raw material ratio of the formula is selected from any one of the following by weight: (1) Sappan wood: Sanguisorba officinalis: Punica granatum: Coptis chinensis: Forsythia suspensa = 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2); (2) Sappan wood: Sanguisorba officinalis: Punica granatum: Coptis chinensis: Andrographis paniculata: Licorice root = 1: (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2); (3) Sappan wood: Sanguisorba officinalis: Punica granatum: Coptis chinensis: Andrographis paniculata: Honeysuckle = 1: (0.8~1.2): (0.8~1.2): (0.8~1.2): (0.8~1.2): (0.8~1.2).

6. A method for preparing the compound Chinese herbal medicine extract according to claim 5, characterized in that: The method comprises the following steps: taking weighed Chinese herbal medicine raw materials, mixing them, placing them in a grinder and grinding them into coarse powder; soaking the coarse Chinese herbal medicine raw material powder in 8 to 14 times water for 0.5 to 1.5 hours; heating to boiling over medium heat, decocting over low heat for 0.5 to 1 hour, filtering out the decoction, repeatedly decocting the filter residue in the same way for 1 to 2 times, and combining the decoctions; and concentrating the decoction under reduced pressure to 0.5 to 2 g of crude drug / mL.

7. Use of the compound Chinese herbal medicine extract as claimed in claim 5 in the preparation of veterinary drugs for treating chicken Escherichia coli disease instead of antibiotics.

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