Application of tree hemp in preparation of medicine for preventing and / or treating chicken necrotic enteritis disease

Abstract

The application discloses application of tree hemp in preparation of a medicine for preventing and / or treating chicken necrotic enteritis, and belongs to the technical field of medicines.The tree hemp can be in the form of medicinal material powder or tree hemp extract.The test result of the application shows that the tree hemp can reduce the mortality of broilers with necrotic enteritis and intestinal lesions of necrotic enteritis, and has a good application prospect in preventing and treating chicken necrotic enteritis.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the use of tree hemp in the preparation of drugs for the prevention and / or treatment of necrotizing enteritis in chickens. Background Technology

[0002] Clostridium perfringens ( Clostridium perfringens Clostridium perfringens, a Gram-positive anaerobic spore-forming bacillus, causes necrotic enteritis in chickens, a prominent type of intestinal disease in modern intensive poultry farming. Clostridium perfringens is widely distributed in nature, and its spores are highly resistant to the external environment. Autoclaving requires 20 minutes to completely kill it, while high-temperature treatment at 70℃ for 30-60 minutes can destroy the toxins it produces.

[0003] In addition to the classic alpha and beta toxins, studies have found that NetB toxin is a key virulence factor causing intestinal mucosal damage, disrupting barrier function by forming pores in the intestinal mucosal layer. The pathogenic mechanism of *Clostridium perfringens* also involves collagen adhesion and biofilm formation; clinical isolates generally exhibit strong collagen-binding capabilities, allowing them to colonize the intestinal surface more firmly. While this bacterium is a member of the normal intestinal flora and can exist in small amounts in the intestines of healthy chickens, it proliferates rapidly and produces toxins to cause disease under suitable environmental conditions. This opportunistic pathogenicity increases the difficulty of prevention and control.

[0004] Necrotic enteritis in chickens exhibits a clear seasonal pattern, with a significantly higher incidence rate during the warm and humid spring and summer seasons compared to the cold and dry autumn and winter seasons. This is closely related to the biological characteristics of *Clostridium perfringens*, which rapidly multiplies in environments ranging from 25-40°C. Chickens of all ages and breeds are susceptible, but there are significant age differences: broilers are most susceptible between 2 and 5 weeks of age, especially during the rapid growth period around 3 weeks; while laying hens primarily experience infection after 5 weeks of age, with infection during the laying period leading to a significant decrease in egg production.

[0005] Necrotic enteritis in chickens presents with diverse clinical phenotypes, which can be divided into three types: acute, subacute, and chronic. Acute cases are the most dangerous, often manifesting as sudden death in seemingly healthy chickens, with mortality rates rising to 10%-40% within a short period. Death is often preceded by no specific symptoms. Affected flocks exhibit lethargy, closed eyes, retracted necks, and ruffled feathers. The typical characteristic feces are dark brown, tarry, watery droppings, sometimes mixed with blood streaks and undigested feed particles. Subacute and chronic cases are primarily characterized by growth and developmental delays. Affected chickens show decreased feed intake, slow weight gain, and a significant reduction in overall flock uniformity. In laying hens, infection results in a 10%-30% decrease in egg production, poor eggshell quality, and the appearance of thin-shelled or sandy-shelled eggs. In cases with a longer disease course, anemia symptoms such as pale combs and wattles, and cyanosis of the leg skin are observed, closely related to impaired intestinal absorption and chronic blood loss.

[0006] Tree hemp is the bark of the tree hemp, a plant of the Urticaceae family. It grows in mixed limestone forests or bamboo forests at altitudes of 800-1300m and is distributed in Guangxi, Yunnan and other places. It has a bland taste and cool nature and has anthelmintic function. It is mainly used to treat ascariasis, but its efficacy in preventing and treating necrotizing enteritis in chickens has not been reported. Summary of the Invention

[0007] The purpose of this invention is to provide a new medicinal use for tree hemp.

[0008] The novel pharmaceutical use of tree hemp provided by this invention is its application in the preparation of drugs for the prevention and / or treatment of necrotizing enteritis in chickens.

[0009] Furthermore, the necrotizing enteritis in chickens is caused by Clostridium perfringens.

[0010] The Clostridium perfringens includes Clostridium perfringens types A, B, C, D, and E.

[0011] Specifically, the Clostridium perfringens is a chicken-derived Clostridium perfringens.

[0012] Furthermore, the medicinal form of the hemp tree can be a powder or an extract of the hemp tree.

[0013] Furthermore, the hemp seed extract may be an aqueous extract of hemp seed.

[0014] Specifically, the preparation method of the water extract of Hemp japonica includes the following steps: taking Hemp japonica and boiling it in water to extract the extract;

[0015] The decoction extraction is performed at least twice, specifically twice; during each extraction, the mass ratio of hemp to water can be 1:(8-12), specifically 1:10, and the extraction time can be 0.5-2 hours, specifically 1 hour.

[0016] When extracting by decoction multiple times, the decoctions are combined and concentrated after extraction.

[0017] Furthermore, the active ingredient in the drug for preventing and / or treating necrotizing enteritis in chickens described in this invention may be only hemp seed.

[0018] Furthermore, the drug for preventing and / or treating necrotizing enteritis in chickens according to the present invention may also include other active ingredients for preventing and / or treating necrotizing enteritis in chickens.

[0019] Furthermore, the medicament for the prevention and / or treatment of necrotizing enteritis in chickens according to the present invention may also include pharmaceutically acceptable excipients.

[0020] Furthermore, the pharmaceutically acceptable excipients refer to conventional drug carriers in the pharmaceutical field, including at least one of fillers, binders, disintegrants, solubilizers, suspending agents, wetting agents, pigments, fragrances, solvents, surfactants, or flavoring agents.

[0021] When using tree hemp for the prevention and / or treatment of necrotic enteritis in chickens, the method of use includes the following 1) or 2) or 3):

[0022] 1) Administer the extract of Hemp seed tree orally, 0.5 mL / animal;

[0023] 2) Mix the hemp tree extract with drinking water and let the chickens drink freely. The volume ratio of the hemp tree extract to the drinking water can be 1:500.

[0024] 3) After mixing the hemp seed powder with the feed evenly, feed it directly. The mass ratio of the hemp seed powder to the feed can be 4:1000.

[0025] The chickens mentioned in this invention include broiler chickens and laying hens.

[0026] This invention also protects the use of tree hemp in the preparation of products that inhibit and / or kill Clostridium perfringens.

[0027] Furthermore, the inhibition and / or killing of Clostridium perfringens includes: inhibiting the growth and activity of Clostridium perfringens.

[0028] Furthermore, the product includes at least one of an inhibitor, a bactericide, and a disinfectant;

[0029] Furthermore, the Clostridium perfringens includes Clostridium perfringens types A, B, C, D, and E.

[0030] Specifically, the Clostridium perfringens is a chicken-derived Clostridium perfringens.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Through extensive preliminary screening, the inventors of this invention discovered that tree hemp can prevent and treat necrotizing enteritis in chickens, improve intestinal lesions caused by necrotizing enteritis, improve fecal condition, and improve feed absorption, showing promising prospects for widespread application in the prevention and treatment of necrotizing enteritis in chickens. Attached Figure Description

[0033] Figure 1 Chicken intestines (for the blank group);

[0034] Figure 2 Chicken intestines for the model group;

[0035] Figure 3 The chicken intestines of the positive drug group;

[0036] Figure 4 The chicken intestines of Example 1 group;

[0037] Figure 5 The chicken intestines of Example 2 group;

[0038] Figure 6 The chicken intestines of Example 3 group;

[0039] Figure 7 The chicken intestines of Example 4 group;

[0040] Figure 8 The chicken intestines of Example 5 group;

[0041] Figure 9 Images of lesions from clinical autopsy;

[0042] Figure 10 The feces of the chickens before the administration of the medication;

[0043] Figure 11 The images show chicken droppings five days after the medication was administered. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] Example 1:

[0047] Take 1000g of hemp seed, add 10 times the amount of water, decoct twice, 1 hour each time, combine the decoctions, concentrate to 1000mL, centrifuge to remove impurities, and make up to 1000mL.

[0048] Example 2

[0049] Take 1000g of chestnut shells, add 10 times the amount of water, decoct twice, 1 hour each time, combine the decoctions, concentrate to 1000mL, centrifuge to remove impurities, and make up to 1000mL.

[0050] Example 3

[0051] Take 1000g of dried plums, add 10 times the amount of water, decoct twice, 1 hour each time, combine the decoctions, concentrate to 1000mL, centrifuge to remove impurities, and make up to 1000mL.

[0052] Example 4

[0053] Take 1000g of small juniper, add 10 times the amount of water, decoct twice, 1 hour each time, combine the decoctions, concentrate to 1000mL, centrifuge to remove impurities, and make up to 1000mL.

[0054] Example 5

[0055] Take 1000g of costus root, add 10 times the amount of water, decoct twice, 1 hour each time, combine the decoctions, concentrate to 1000mL, centrifuge to remove impurities, and make up to 1000mL.

[0056] Example 6

[0057] Take 50,000g of hemp seed material, crush it, and pass it through a 40-mesh sieve.

[0058] The drugs prepared in the above embodiments were provided by Beijing Shengtai Technology Co., Ltd.

[0059] Pharmacodynamic studies:

[0060] I. In vitro antibacterial tests of Examples 1-5

[0061] 1. Purpose of the experiment

[0062] The in vitro anti-closing effects of Examples 1-5 were tested.

[0063] 2. Test strains

[0064] Clostridium perfringens toxin type A, CVCC2030, was purchased from the China Institute of Veterinary Drug Control and the China Veterinary Microbial Culture Collection Center (CVCC).

[0065] 3. Test Samples

[0066] Examples 1-5 were prepared by Beijing Shengtai Technology Co., Ltd., and the medicinal materials were purchased from Anguo Jufu Chinese Herbal Medicine Co., Ltd.

[0067] Anaerobic meat and liver soup culture medium, Beijing Aoboxing Biotechnology Co., Ltd., 250g / bottle, prepared according to the instructions, freshly prepared for laboratory use.

[0068] Anaerobic meat and liver soup culture medium (×2) (nutrient content twice the normal amount), prepared in-house and used fresh in the laboratory.

[0069] Physiological saline solution, homemade.

[0070] 4. Test methods

[0071] 4.1 Preparation of the test bacterial suspension

[0072] After opening the freeze-dried bacterial culture, it was enriched on anaerobic meat liver soup medium and then streaked onto blood agar medium. After overnight incubation at 37°C in a CO2 incubator, typical single colonies were selected and inoculated onto anaerobic meat liver soup medium. After incubation at 37°C for 24 hours, viable cell counts were performed, and then the culture was diluted with physiological saline to a bacterial count of 10⁻⁶. 4 ~10 5 cfu / ml was used as the test bacterial solution.

[0073] 4.2 Test Operation Procedures

[0074] MIC measurement

[0075] Arrange eight medium-sized test tubes on a test tube rack and label them. Add 2.5 mL of physiological saline to each tube. Add 2.5 mL of the test sample to the first tube and mix well. Transfer 2.5 mL from the first tube to the second tube and mix well. Transfer 2.5 mL from the second tube to the third tube, and so on, until the sixth tube. After mixing well, discard 2.5 mL. The seventh tube serves as a negative control, and the eighth tube contains bacterial culture as a positive control. Then, add anaerobic meat and liver broth culture medium (×2) to tubes 1-8. Add 20 μL of the test bacterial culture to tubes 1-6 and 8 respectively, shake well, and incubate at 37°C in a carbon dioxide incubator for 24 hours. Observe the experimental results.

[0076] 5. Results

[0077] The minimum inhibitory concentrations against Clostridium perfringens in vitro for each sample are shown in Table 1.

[0078]

[0079] Note: "-" indicates no bacterial growth. "+" indicates bacterial growth, resulting in a cloudy test tube.

[0080] As shown in Table 1, the best in vitro antibacterial effect against Clostridium perfringens was achieved with the extract of dried plum (Example 3), while the worst effect was achieved with the extract of creeping juniper (Example 4).

[0081] II. The preventive and therapeutic effects of various drugs on enteritis caused by Clostridium perfringens infection in chickens.

[0082] 1. Experimental Objective

[0083] The various embodiments demonstrate the alleviating effect on enteritis symptoms caused by Clostridium perfringens infection in chickens.

[0084] 2. Experimental Materials

[0085] 2.1 Test strains

[0086] Clostridium perfringens toxin type A, CVCC2030, was purchased from the China Institute of Veterinary Drug Control and the China Veterinary Microbial Culture Collection Center (CVCC).

[0087] 2.2 Test Drugs

[0088] Test drugs: Examples 1-5, self-made, medicinal materials purchased from Anguo Jufu Chinese Herbal Medicine Co., Ltd.

[0089] Control drug: 5% lincomycin hydrochloride solution, the raw material of which was purchased from Shandong Yakang Pharmaceutical Co., Ltd. and prepared into a liquid for oral administration.

[0090] 2.3 Experimental animals and feed

[0091] Experimental chickens: 1-day-old broilers. 161Q corn-fed broiler complete feed, containing no antibiotics, specially formulated by the feed mill.

[0092] 3. Test Methods

[0093] 3.1 Experimental Design and Grouping

[0094] When the chicks were 7 days old, they were grouped. Each chick was weighed, and weak or overweight chicks were removed. Healthy chicks with an individual weight difference of less than 30g were selected and randomly divided into 8 groups of 20 chicks each. Groups 1-5 were the drug groups of Examples 1-5, group 6 was the control drug lincomycin hydrochloride group, group 7 was the model group with Clostridium infection but no drug treatment, and group 8 was the blank control group without infection and no drug treatment.

[0095] Each drug administration group (groups 1-6) was administered the drug orally from day 13 until the end of the experiment; groups 1-7 were administered 7.5 × 10⁻⁶ mg / day orally from day 15-19. 8 Clostridium perfringens CFU / mL.

[0096] The experimental chickens were fed a complete corn-based broiler feed throughout the entire experiment. They had free access to water and feed throughout the experiment, which lasted until the end of the 20th day of age, as detailed in Table 2.

[0097]

[0098] 3.2 Evaluation Standards for Enteritis and Production Performance Indicators

[0099] 3.2.1 Intestinal Lesion Scoring Criteria

[0100] At 20 days of age, the intestines (duodenum) of chickens in each group were collected for lesion scoring. The scoring criteria were as follows: 0 = no obvious lesions, 1 = intestinal mucosal congestion, 2 = focal necrosis or ulceration (1-5 lesions), 3 = focal necrosis or ulceration (6-15 lesions), and 4 = focal necrosis or ulceration (16 or more lesions).

[0101] Lesion score (0-40) = Average lesion score (0-4) × 10 for each experimental group.

[0102] 3.2.2 Relative weight gain rate

[0103] Weigh the chickens at 20 days of age and calculate the average weight gain and relative weight gain rate. Relative weight gain rate = (average weight gain of the experimental group / average weight gain of the control group) × 100%.

[0104] 3.2.3 Survival rate

[0105] Record the number of dead chickens in each group, perform necropsy to determine the cause of death, and calculate the survival rate. Survival rate = (number of surviving chickens at the end of the experiment / number of chickens in the experimental group) × 100%.

[0106] 4. Experimental Results

[0107] 4.1 Clinical symptoms

[0108] After infection, the experimental chickens showed symptoms such as decreased feed intake, poor mental state, and emaciation. On the 3rd day after infection, the model group and groups 2-5 of Examples all had varying degrees of loose stools and reduced water intake; the control group had normal feed intake and water intake.

[0109] 4.2 Intestinal lesions

[0110] After 5 days of bacterial challenge (days 15-19), the duodenum and jejunum of the experimental chickens in each group were eviscerated and observed. The results showed that the duodenum of the model group and groups 2-5 of Examples showed varying degrees of damage (see...). Figure 1-8 ).

[0111] 4.3 Intestinal lesion scoring results

[0112] The experimental results showed that, regarding duodenal lesions, the blank control group had the lowest lesion value, while the model group had the highest lesion value. In addition, the lesions in the Example 1 group showed significant improvement compared to the model group, while the lesions in Examples 2-5 showed no significant difference compared to the model group. In terms of relative weight gain, the effect of the Example 1 group was better than that of the Example 2-5 groups.

[0113]

[0114] Note: Different letters indicate significant differences.

[0115] 5. Conclusion

[0116] Based on the results of intestinal lesions, it can be concluded that: Example 1 has a therapeutic effect on enteritis caused by Clostridium perfringens; Examples 2-5 have no significant therapeutic effect on enteritis caused by Clostridium perfringens, and the in vitro antibacterial effect does not necessarily have an in vivo effect, indicating that Hemp tree has a prominent and unexpected effect in preventing and treating necrotic enteritis in chickens.

[0117] III. Regional Clinical Prevention and Treatment Effects of Examples 1 and 6 on Necrotic Enteritis in Chickens

[0118] 1. Test Drugs

[0119] Examples 1 and 6, and lincomycin hydrochloride soluble powder (5%), were all provided by Beijing Shengtai Technology Co., Ltd.

[0120] 2. Experimental site and flock conditions

[0121] At a poultry farm in Tai'an County, 50,000 70-day-old laying hens developed necrotic enteritis. Necropsy revealed symptoms of necrotic enteritis (see...). Figure 9 Symptoms include intestinal mucosal shedding, uneaten feed, and tomato-like droppings in the flock (see...). Figure 10 The chicken was diagnosed with necrotizing enteritis by a veterinarian.

[0122] 3. Test Methods

[0123] By simply modifying the water supply lines, the chickens in the same building were divided into four groups. The first group was a non-medicated group (to minimize economic losses, the chickens in the first non-medicated group were placed at the top of one row of chickens). Two other rows of chicken cages were selected. The water supply line of one row was modified separately, and the chickens were given medication using the 1:500 dosage method (Example 1). The chickens in the other row were given medication mixed with feed (Example 6), with a dosage of 4 kg of feed mixed with 1 ton of feed. The water supply line of the group in Example 6 was also modified, and they were given only blank drinking water. The remaining rows of laying hens were treated with conventional antibiotics, including lincomycin hydrochloride soluble powder (dosage: 3g per 1L of drinking water, for 5 consecutive days).

[0124] 4 Key Observation Indicators

[0125] The efficacy of a drug is evaluated by observing changes in the amount of tomato-like stool within a specific area.

[0126] 5 Results

[0127] The results are shown in Table 4.

[0128]

[0129] Before use, the chicken droppings in each group were mostly tomato-like. After use, the amount of tomato-like droppings in the same area of ​​Examples 1 and 6 was greatly reduced, and the amount in the lincomycin hydrochloride group was also reduced. The effects of the two groups in Examples 1 and 6 and the antibiotic group were similar, while there was no significant change in the amount of tomato-like droppings in the same area of ​​the blank control group.

[0130] The above cases illustrate that tree hemp has a significant and prominent effect in preventing and treating necrotizing enteritis in chickens.

[0131] 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. Use of Cannabis sativa L. as the only active ingredient in the preparation of a medicament for the prevention and / or treatment of chicken necrotic enteritis disease.

2. Use according to claim 1, characterized in that: The chicken necrotic enteritis disease is caused by Clostridium perfringens.

3. Use according to claim 1 or 2, characterized in that: The Cannabis sativa L. is medicinal material powder or Cannabis sativa L. extract.

4. Use according to claim 3, characterized in that: The Cannabis sativa L. extract is water extract of Cannabis sativa L.

5. Use according to claim 1 or 2, characterized in that: The medicament further comprises pharmaceutically acceptable adjuvants.

6. Use of Cannabis sativa L. as the only active ingredient in the preparation of a product for inhibiting and / or killing Clostridium perfringens.

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