Application of Erythrina bark in the preparation of drugs for the prevention and / or treatment of infectious coryza in chickens

Abstract

This invention provides the application of Erythrina bark in the preparation of drugs for the prevention and / or treatment of infectious coryza in chickens. The active ingredient of this drug can be composed solely of Erythrina bark, making the formulation easy and convenient to implement. It can be used as a substitute for antibiotics during the egg-laying period of chickens, with no drug residue requirements, reducing the economic loss of discarded eggs after the use of antibiotics in laying hens. It has a high cure rate for infectious coryza, is safe and has no toxic side effects, and is easy to use.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drugs, specifically relating to the use of Erythrina bark in the preparation of drugs for the prevention and / or treatment of infectious coryza in chickens. Background Technology

[0002] Infectious coryza is an acute or subacute respiratory infectious disease in chickens caused by *Avibacterium paragallinarum*. Its main characteristics are inflammation of the nasal mucosa, runny nose, and eyelid edema. The disease primarily affects growing and laying hens, causing a 10%-40% decrease in egg production. It has a very short incubation period and spreads rapidly, typically reaching the entire flock within 1-3 days. The disease has a high morbidity rate, especially after initial infection, often resulting in outbreaks with morbidity rates exceeding 70%, leading to significant economic losses.

[0003] Chickens of all ages can be infected, but those aged 4-12 months are most susceptible, especially first-laying hens. Adult chickens have a short incubation period, develop acute symptoms, and exhibit severe disease with a prolonged course. The disease can occur in any season, but is more common in autumn and winter. Infected and carrier chickens are the main sources of infection, primarily spread through dust and air, but also through contaminated feed, water, equipment, and the clothing of mobile workers.

[0004] Antibiotics that are effective in preventing and treating infectious coryza in chickens include sulfamethoxypyrimidine and doxycycline, but their use is restricted during the egg-laying period. Under the policy background of reducing and limiting antibiotics, egg farms basically have no effective antibiotics available when infectious coryza occurs, and traditional Chinese medicine has become an important alternative for the prevention and treatment of infectious coryza in chickens.

[0005] Erythrina bark is the dried bark of the Erythrina tree (also known as the coral tree or ebony tree), belonging to the legume family. It is bitter and neutral in nature, and enters the liver and kidney meridians. Its functions include dispelling wind and dampness, relaxing muscles and tendons, and promoting blood circulation; it is mainly used to treat rheumatic pain, lower back and knee pain, and traumatic injuries. However, its efficacy in preventing and treating infectious coryza in chickens has not been reported. Summary of the Invention

[0006] The purpose of this invention is to provide a new medicinal use for Erythrina bark.

[0007] The novel pharmaceutical use of Erythrina bark provided by this invention is its application in the preparation of drugs for the prevention and / or treatment of infectious coryza in chickens.

[0008] Furthermore, the infectious coryza in chickens is caused by Avianella paragallinarum.

[0009] The *Paragonimus* species include types A, B, and C.

[0010] According to a specific embodiment of the present invention, the *Paragonimus avianus* is *Paragonimus avianus* type B.

[0011] Furthermore, the medicinal form of the Erythrina bark can be Erythrina bark powder or Erythrina bark extract.

[0012] In some embodiments of the present invention, the preparation method of the Erythrina bark powder is as follows: the Erythrina bark is pulverized and then made into a powder.

[0013] In some embodiments of the present invention, the extract of Pittosporum tobira may be an aqueous extract of Pittosporum tobira.

[0014] Specifically, the preparation method of the aqueous extract of Erythrina bark includes the following steps: taking Erythrina bark and boiling it in water to extract the extract; The decoction extraction is performed at least twice, specifically twice; during each extraction, the mass ratio of Erythrina bark to water can be 1:(8-12), specifically 1:10, and the extraction time can be 0.5-2 hours, specifically 1.5 hours.

[0015] When extracting through multiple decoctions, the decoctions are combined and concentrated after extraction. The concentration may be to reduce the crude drug content to 1 g / mL.

[0016] In some embodiments of the present invention, the dosage form of the drug may be a powder, decoction, mixture or granules.

[0017] In some embodiments of the present invention, the active ingredient of the drug for preventing and / or treating infectious coryza in chickens may be only Erythrina bark.

[0018] In some embodiments of the present invention, the drug for the prevention and / or treatment of infectious coryza in chickens may also include pharmaceutically acceptable excipients.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipient refers to a drug carrier conventional in the pharmaceutical field, including at least one of fillers, binders, disintegrants, solubilizers, suspending agents, wetting agents, pigments, fragrances, solvents, surfactants, or flavoring agents.

[0020] When using Erythrina bark for the prevention and / or treatment of infectious coryza in chickens, the method of application includes the following 1) or 2): 1) Administer the extract of Erythrina bark orally; 2) Mix the Erythrina bark extract with drinking water and let the chickens drink freely. The volume ratio of the Erythrina bark extract to the drinking water can be 1:500.

[0021] The chickens mentioned in this invention include broiler chickens and laying hens.

[0022] This invention also protects the use of Erythrina bark in the preparation of products that inhibit and / or kill avian bacilli.

[0023] Furthermore, the inhibition and / or killing of *Paragonimus westermani* includes: inhibiting the growth and activity of *Paragonimus westermani*.

[0024] Furthermore, the product includes at least one of an inhibitor, a bactericide, and a disinfectant; Furthermore, the *Paragonimus paragallinarum* includes types A, B, and C.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects. When chickens develop infectious coryza, the external pathological manifestation is facial swelling. From the perspective of traditional Chinese veterinary medicine, this can be understood as wind, cold, and dampness invading the skin surface and stagnating in the meridians of the head and face, leading to poor blood circulation and water retention, manifesting as facial swelling, runny nose, and eyelid edema. Therefore, it is possible to explore solutions to clinical problems from the perspective of dispelling wind and dampness, while also taking into account antibacterial activity. Thus, this invention selects and studies medicinal materials with antibacterial effects, and combines them with directional screening based on the effects of dispelling wind and dampness. This has identified medicinal materials such as Erythrina bark, Coptis chinensis, Sophora flavescens, Stephania tetrandra, Phellodendron chinense, Dictamnus dasycarpus, and Scutellaria baicalensis. This invention records part of the process of screening a large number of drugs. The medicinal materials recorded in this invention have both anti-Haemophilus influenzae and anti-wind and dampness effects. Studies have found that Erythrina bark has a more significant effect in treating infectious coryza in chickens. It can not only effectively inhibit Avian bacillus paragallinarum, but also alleviate clinical symptoms, improve chicken growth and egg production performance in laying hens, and has a more prominent therapeutic effect among many screened medicinal materials with similar effects. Attached Figure Description

[0026] Figure 1 A scoring standard for clinical symptoms of infectious coryza in chickens; Figure 2 Images of some chickens in the experimental clinical blank group for infectious coryza in chickens; Figure 3 Images of some chickens from two experimental clinical trials for infectious coryza in chickens; Figure 4 Images of some chickens in the experimental clinical model group for infectious coryza in chickens; Figure 5 Images of some chickens in the experimental clinical control group for infectious coryza in chickens; Figure 6 Images of chickens with infectious coryza in a chicken farm. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Example Example 1: Take 1000g of Erythrina bark, crush it, and pass it through a 100-mesh sieve to obtain the product.

[0030] Example 2: Take 1000g of Erythrina bark, add 10 times the amount of water, decoct twice, 1.5 hours each time, combine the filtrates, and concentrate to a crude drug content of 1g / mL to obtain the product.

[0031] Comparative Example 1: Take 1000g of Coptis chinensis, add 10 times the amount of water, decoct twice, 1.5 hours each time, combine the filtrates, and concentrate to a crude drug content of 1g / mL.

[0032] Comparative Example 2: 1000g of Sophora flavescens, add 10 times the amount of water, decoct twice, 1.5 hours each time, combine the filtrates, and concentrate to a crude drug content of 1g / mL to obtain the final product.

[0033] Comparative Example 3: 1000g of Stephania tetrandra, add 10 times the amount of water, decoct twice, 1.5 hours each time, combine the filtrates, and concentrate to a crude drug content of 1g / mL to obtain the final product.

[0034] Comparative Example 4: 1000g of Phellodendron bark was decocted twice with 10 times the amount of water for 1.5 hours each time. The filtrates were combined and concentrated to a concentration of 1g / mL of raw herb.

[0035] Comparative Example 5: 1000g of Dictamnus dasycarpus root bark, add 10 times the amount of water, decoct twice, 1.5 hours each time, combine the filtrates, and concentrate to a raw drug content of 1g / mL to obtain the product.

[0036] Comparative Example 6: 1000g of Scutellaria baicalensis was decocted twice with 10 times the amount of water for 1.5 hours each time. The filtrates were combined and concentrated to a concentration of 1g / mL of raw herb.

[0037] Experimental Example 1 The in vitro antibacterial effects of Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6 on *Avianella paragallinarum* were observed.

[0038] In this experiment, different concentrations of drugs were mixed and dissolved in liquid culture medium, bacteria were inoculated, and the minimum inhibitory concentration (MIC) of the embodiment was determined by the growth of the bacteria.

[0039] Experimental materials: Example 2, drugs prepared by comparative examples 1, 2, 3, 4, 5 and 6; Avian bacillus paragallinarum strain B; TSB medium; diluent (sterile physiological saline); 37°C incubator.

[0040] The strain of *Paragonimus paragallinarum* type B was obtained by collecting typical pathogenic materials from chicken farms where infectious coryza occurred. After processing, purifying, and culturing the pathogenic materials, the Shengtai Testing Center conducted morphological and cultural characteristic identification, physiological and biochemical characteristic identification, molecular biological identification, and serotyping identification to determine that the isolated strain was *Paragonimus paragallinarum* type B.

[0041] Experimental methods: Preparation of Haemophilus paragallinarum bacterial culture. Culture medium preparation: Weigh 30g of trypsin-soybean broth (TSB) medium and 10g of yeast extract, add 945mL of purified water, shake thoroughly to dissolve, autoclave at 121℃ for 15min, cool, add 50mL of inactivated newborn calf serum and 5mL of filtered sterilized 1% NAD solution, shake thoroughly to obtain the culture medium. Pick a single Haemophilus paragallinarum colony and add it to the culture medium, incubate at 37℃ for 24h.

[0042] Preparation of culture medium containing the drug in the example: The drug was serially diluted with physiological saline to prepare test solutions of different concentrations. 2.5 mL of each dilution of test solution was added to a test tube containing 2.5 mL of double-concentration TSB culture medium and diluted to 1:256.

[0043] Take 20 μL of bacterial suspension and inoculate it into a test tube containing the culture medium of the example to serve as the experimental group sample.

[0044] The same method was used to inoculate test tubes containing the drug from the example, which served as positive control samples.

[0045] Take two test tubes containing culture medium and treat them with physiological saline instead of the drug to serve as negative control samples.

[0046] The samples from the example group, the positive control group, and the negative control group were placed in an incubator at 37°C and cultured for 24 hours. The results were observed. The positive control group showed obvious turbidity, so the culture was stopped and the results were observed.

[0047] Evaluation method: When the positive control tube shows bacterial growth (turbidity) and the negative control tube shows no bacterial growth (clearness), the lowest concentration at which no bacterial growth occurs in the example group is the minimum inhibitory concentration (MIC) of the sample against the test bacteria.

[0048] Experimental results: Table 1. Minimum inhibitory concentration (MIC) of different samples against Haemophilus paragallinarum

[0049] The results showed that the negative and positive controls were valid. Examples 2 and Comparative Examples 1-6 all showed in vitro anti-Haemophilus paragallinarum activity. Coptis chinensis showed the best in vitro anti-Haemophilus activity, while Stephania tetrandra and Dictamnus dasycarpus showed the worst activity.

[0050] Experiment Example 2 Clinical trials for the prevention and treatment of infectious coryza in chickens Cleaning and disinfection of the experimental animal room: Formaldehyde fumigation was used, with 250g of potassium permanganate and 500mL of formaldehyde mixed and fumigated for 24 hours; ventilation was carried out for 24 hours after disinfection.

[0051] SPF chickens were weighed and grouped: 300 SPF chickens over 30 days old were removed, and the remaining 270 SPF chickens were divided into 9 groups of 30 chickens each. The weights of the groups were weighed and adjusted so that the weight difference between the groups was less than 3%, and the data were recorded.

[0052] Preparation of bacterial culture medium TSA plates: Weigh 40g of tryptic soybean agar (TSA) medium, add 845ml of purified water, shake well and heat until fully dissolved, autoclave at 121℃ for 15min, cool to about 60℃, add 50mL of healthy sterile newborn calf serum, 100mL of yeast extract and 5mL of filtered sterile 1% NAD solution, shake well and pour into sterile petri dishes to make plates.

[0053] TSB medium: Weigh 30g of tryptic soybean broth (TSB) medium and 10g of yeast extract, add 945mL of purified water, shake well to dissolve, autoclave at 121℃ for 15min, cool, add 50ml of inactivated newborn calf serum and 5mL of filtered sterilized 1% NAD solution, shake well to obtain the medium.

[0054] Standards for the Preparation and Control of Highly Toxic Substances Take freeze-dried Avian bacillus paragallinarum type B (same strain as in Example 1) as the basic seed, streak it onto a TSA plate, and incubate it at 37°C for 16-24 hours. Pick several typical colonies and inoculate them into 100-200 mL of TSB medium. Incubate at 37°C for 12-24 hours to harvest. Dilute the medium 50 times with sterile physiological saline for challenge and use.

[0055] Storage and usage period: Store below -70℃, and use within 7 days.

[0056] Model establishment and grouping: Example 2; Comparative Examples 1-6; Model control group; Blank control group. Except for the blank control group, the challenge dose for each group was 1×10⁻⁶. 7 CFU / 0.2 mL / animal, the infection site was the infraorbital sinus, and the blank control group was injected with physiological saline.

[0057] Disease diagnosis criteria: Symptoms are defined as meeting one of the following criteria: ① Nasal discharge ranging from clear to serous-viscous secretions, and tearing; ② Swelling of the infraorbital sinus and surrounding areas on one or both sides of the face. If asymptomatic, a score of 0 is given. If symptoms are present, the severity of clinical symptoms is observed by comparing the affected chickens with the control group. Eye swelling and mucous nasal discharge are scored from mild to severe as 1, 2, and 3 (see [link to relevant documentation]). Figure 1 ).

[0058] After the chickens were challenged with the virus, they were given the corresponding medication via gavage for 7 days. Each chicken was given 1 mL of the extracted drug solution, ensuring that the amount of drug solution administered to each group was consistent.

[0059] After the drug was administered to the group, each group was given sufficient food and water.

[0060] Experimental results: Table 2. Efficacy of each group in preventing and controlling infectious coryza in chickens

[0061] Note: Same letters indicate no significant difference (P≥0.05), different letters indicate significant difference (P<0.05). Table 2 shows that after 7 days of drug use, the drug prepared in Example 2 had the best effect on reducing swelling in chickens caused by Haemophilus paragallinarum infection, and showed a significant difference compared with the model group. Compared with the scheme of the present invention, Comparative Examples 1-6 did not show better effects. It can be seen that the drugs with better or worse in vitro antibacterial effects than those in Example 2 may not necessarily have the same effect as those that can be effectively treated by oral administration for infectious coryza in chickens. Similarly, drugs with antirheumatic effects may not necessarily have the same effect as those that can be effectively treated by oral administration for infectious coryza in chickens.

[0062] Experimental Example 3 The following case studies verify the effectiveness of Example 2 in preventing and treating infectious coryza in chickens.

[0063] To verify the clinical efficacy of this invention against infectious coryza in chickens over a large area, an efficacy verification experiment of Example 2 was conducted in a poultry farm infected with infectious coryza. Because antibiotics that are sensitive to laying hens could not be used, the experiment was divided into Example 2 and a negative control group. The efficacy of Example 2 was compared after 7 days of administration.

[0064] Materials and methods Experimental animals: A chicken farm in Qingyuan City, Guangdong Province, where infectious coryza was occurring.

[0065] Test drug: Example 2.

[0066] Experimental method: The diagnosis of infectious coryza in chickens was made by clinical symptoms, necropsy changes and bacterial isolation and identification. The experimenters randomly divided the sick chickens into two groups inside the building. After modifying the water line, one group used the drug prepared in Example 2 (i.e., 1 liter of drug solution in 500 liters of water) and drank it all in 4-6 hours. The other group used clean water as a negative control.

[0067] Diagnostic criteria for infectious coryza in chickens Clinical symptoms: Sick chickens exhibit symptoms such as nasal mucosal inflammation, runny nose, and eyelid edema. Figure 6 The chickens exhibit difficulty breathing, sometimes shaking their heads, and have green, watery droppings. The diseased chickens also show the typical unilateral facial edema.

[0068] Post-mortem changes: Caseous exudate was observed in the sinuses, and mucus or pus accumulated in the trachea. White pus drained from the swollen eyelids upon pricking. No obvious lesions were found in the liver, heart, kidneys, bursa of Fabricius, or digestive tract.

[0069] Bacterial isolation and identification: Nasal cavity and infraorbital sinus secretions from diseased chickens were collected, smeared, Gram-stained, and examined under a microscope. Gram-negative cocci were found, which were pleomorphic and occasionally filamentous, with capsules around the bacteria.

[0070] Pathogen detection: RT-PCR / PCR method was used to detect pathogens.

[0071] The test results are shown in Table 3: Table 3. Pathogen detection results

[0072] Note: "+" indicates a positive result; "-" indicates a negative result. The results showed that the pathogen detection was positive for HPG-A type.

[0073] Methods for determining treatment effectiveness: feed intake, egg production rate of laying hens, and morbidity rate (morbidity rate is determined by random sampling).

[0074] The criteria for determining the disease are: obvious swelling of the chicken's face or a viscous fluid discharge from the nostrils. The determination of whether a chicken is sick is performed by the same person to avoid the influence of subjective factors.

[0075] Experimental results: Table 4. Regional clinical efficacy of Example 2 against infectious coryza in chickens

[0076] Note: Same letters indicate no significant difference in the same column (P≥0.05), different letters indicate significant difference in the same column (P<0.05). The results in Table 4 show that the effect of Example 2 is significantly better than that of the negative control group, indicating that after treatment with Example 2, the chickens' appetite increased, clinical symptoms were significantly alleviated, and the incidence rate was significantly reduced, all of which were significantly improved compared with the control group.

[0077] The current clinical approach to preventing and treating infectious coryza in chickens commonly involves the use of antibiotics, but sensitive antibiotics are contraindicated during the egg-laying period. This invention uses a pure traditional Chinese medicine preparation instead of antibiotics, which can be used during the egg-laying period and shows a significant improvement compared to the control group. It fills the gap of having no available medication for infectious coryza in laying hens during the egg-laying period, providing a more effective solution for the prevention and treatment of infectious coryza in chickens.

[0078] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of Erythrina bark in the preparation of drugs for the prevention and / or treatment of infectious coryza in chickens.

2. The application according to claim 1, characterized in that: The infectious coryza in chickens is caused by Avian bacillus paragallinarum.

3. The application according to claim 1, characterized in that: The medicinal form of the Erythrina bark is Erythrina bark powder or Erythrina bark extract.

4. The application according to claim 3, characterized in that: The extract of Erythrina bark is an aqueous extract of Erythrina bark.

5. The application according to any one of claims 1-4, characterized in that: The erythrina bark is the sole active ingredient of the drug.

6. The application according to any one of claims 1-5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The application according to any one of claims 1-6, characterized in that: The dosage form of the drug is powder, decoction, mixture or granules.

8. Application of Erythrina bark in the preparation of products that inhibit and / or kill avian bacilli.

9. The application according to claim 8, characterized in that: The inhibition and / or killing of *Paragonimus westermani* includes: inhibiting the growth and activity of *Paragonimus westermani*.

10. The application according to claim 8 or 9, characterized in that: The product includes at least one of inhibitors, bactericides, and disinfectants.

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