Application of Chinese tamarisk twigs in preparation of medicine for treating bacterial enteritis of chicken

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly discloses application of Chinese tamarisk twigs in preparation of a medicine for treating chicken bacterial enteritis. And side effects and potential problems caused by long-term use of antibiotics for treatment are effectively solved. A preparation method of the Chinese tamarisk twig extract comprises the following steps: taking a Chinese tamarisk twig medicinal material, adding 50% ethanol which is 10 times of the Chinese tamarisk twig medicinal material to soak for 1 hour, then performing ultrasonic extraction for 1 hour at 50 DEG C, and collecting an extracting solution; then 50% ethanol with the amount 8 times that of the medicinal materials is continuously added into the medicinal materials, ultrasonic extraction is conducted for 1 hour at the temperature of 50 DEG C, and an extracting solution is collected; combining the two extracting solutions, carrying out vacuum suction filtration, carrying out 50 DEG C rotary steam concentration to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain a powdery Chinese tamarisk twig extract; the effective dose of the Chinese tamarisk twig extract is 1g / kg; the Chinese tamarisk twig extract is added into chicken feed as a feed additive. The invention is helpful for reducing the dependence on chemical synthetic drugs and the long-term sustainable development of animal breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and in particular relates to the application of *Salix viminalis* in the preparation of a medicine for treating bacterial enteritis in chickens. Background Technology

[0002] Bacterial enteritis in chickens is a common and serious poultry disease, mainly caused by a variety of pathogens, such as Salmonella, Escherichia coli, and Mycoplasma avianis. These pathogens enter the chicken's intestines through the fecal-oral route, causing enteritis symptoms, including diarrhea, loss of appetite, slow growth, and even death.

[0003] In Traditional Chinese Medicine (TCM) theory, bacterial enteritis in chickens can be considered as caused by "damp-heat" invading the intestines. Damp-heat is a pathological constitution where dampness and heat combine to invade the internal organs, especially the stomach and intestines. Factors such as contaminated feed, poor rearing environment, and unclean drinking water can lead to the generation and accumulation of damp-heat, causing an imbalance in the chicken's intestines and making them susceptible to pathogenic microorganisms, thus triggering enteritis. After damp-heat invades the intestines, it can lead to stagnation of intestinal qi and retention of dampness, resulting in diarrhea and loss of appetite. Bacterial enteritis in chickens causes serious economic losses to the poultry industry because it leads to stunted growth and increased mortality in flocks.

[0004] Currently, bacterial enteritis in chickens is mainly treated with antibiotics. However, long-term use of antibiotics may lead to the following side effects and potential problems: (1) Antibiotic abuse leads to increased drug resistance: Long-term or excessive use of antibiotics may lead to the generation and spread of drug-resistant bacteria in the chicken gut. These drug-resistant strains not only make treatment difficult, but may also spread to other chickens or humans in the breeding environment, increasing the risk of zoonotic infections; (2) Disruption of gut microecological balance: Antibiotics not only kill pathogenic bacteria, but may also destroy the beneficial probiotics in the chicken gut. This microecological imbalance may lead to indigestion, nutrient absorption problems, and even increase the risk of other diseases; (3) Residues and their potential impact on human health: Antibiotics used in feed may remain in chicken meat and enter the human body. Long-term intake of antibiotic residues may affect the human immune system or be related to the health of the human microbiome. Although research in this area is still ongoing, there are potential health risks; (4) Increased cost of antibiotic treatment: Frequent use of antibiotics not only increases breeding costs, but may also further increase the health problems and mortality of chicken flocks due to reduced treatment effectiveness, resulting in higher economic losses.

[0005] Therefore, developing alternative treatment strategies or aquaculture management methods is particularly important. Summary of the Invention

[0006] The purpose of this invention is to provide the application of *Salix viminalis* in the preparation of a drug for treating bacterial enteritis in chickens, effectively solving the side effects and potential problems caused by long-term antibiotic treatment.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of *Salix viminalis* in the preparation of a drug for treating bacterial enteritis in chickens.

[0008] Furthermore, the effective concentration of the aqueous solution of *Salix viminalis* extract is not less than 0.5 mg / mL. The aqueous solution of *Salix viminalis* extract is prepared by dissolving *Salix viminalis* extract in water. The preparation method of *Salix viminalis* extract is as follows: take *Salix viminalis* medicinal material, add 10 times the amount of 50% ethanol and soak for 1 hour, then extract with ultrasound at 50°C for 1 hour, and collect the extract; then add 8 times the amount of 50% ethanol to the medicinal material, extract with ultrasound at 50°C for 1 hour, and collect the extract; combine the two extracts and perform vacuum filtration, concentrate with rotary steam at 50°C to obtain a concentrated solution, and freeze-dry the concentrated solution to obtain *Salix viminalis* extract in powder form.

[0009] Furthermore, the effective dose of the *Salix viminalis* extract is 1 g / kg.

[0010] Furthermore, the extract of *Salix viminalis* was added as a feed additive to chicken feed.

[0011] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention discovers that *Salix viminalis* extract promotes digestive health, helps improve the absorption efficiency of nutrients in animals, and thus improves growth performance and production efficiency. Adding *Salix viminalis* extract as a feed additive to chicken feed, unlike the use of single antibiotics, is beneficial because traditional Chinese medicine is often a compound drug composed of multiple ingredients, which can effectively reduce the risk of antibiotic resistance. Furthermore, traditional Chinese medicine has fewer side effects, helping to reduce dependence on chemically synthesized drugs and promote the long-term sustainable development of animal husbandry. Attached Figure Description

[0012] Figure 1 This study investigated the effect of *Salix viminalis* extract on the viability of RAW264.7 cells.

[0013] Figure 2 The effect of lipopolysaccharide on the activity of RAW264.7 cells was investigated. Compared with the control group, *P<0.05 and **P<0.001 were observed.

[0014] Figure 3 The effect of different concentrations of lipopolysaccharide on NO in the supernatant of RAW264.7 cells was investigated. Compared with the control group, **P<0.001.

[0015] Figure 4The effect of *Salix viminalis* extract on NO in the supernatant of lipopolysaccharide-induced RAW264.7 cells was investigated. Compared with the control group, ##P<0.001; compared with the lipopolysaccharide group, *P<0.05.

[0016] Figure 5 The study investigated the effects of *Salix viminalis* extract on inflammatory factors in the supernatant of RAW264.7 cells induced by lipopolysaccharide (LPS). The results showed that, compared with the control group, ##P<0.001; and compared with the LPS group, **P<0.001.

[0017] Figure 6 This shows the changes in body weight of mice after intervention with *Salix viminalis* extract.

[0018] Figure 7 The effect of *Salix viminalis* extract on serum oxidative stress levels in mice was investigated. Figure A shows MDA concentration, Figure B shows total antioxidant capacity, and Figure C shows SOD activity. *P<0.05 compared with the control group.

[0019] Figure 8 The effects of *Salix viminalis* extract on serum inflammatory factors in mice were investigated. Figure A shows TNF-α, Figure B shows IL-6, and Figure C shows IL-1β. Compared with the control group, *P<0.05.

[0020] Figure 9 The images show the colon morphology of mice fed for 20 days in the control and experimental groups. Figure A represents the control group, Figure B represents the XH group, and Figure C represents the XL group. Detailed Implementation

[0021] To better understand the present invention, it is now further described with reference to embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer. The test strains used in the antibacterial studies were all derived from clinical samples.

[0022] Bacterial enteritis in chickens is a digestive system disease caused by bacterial infection, mainly manifested as intestinal inflammation and related digestive problems. The relationship between antibacterial activity, antioxidant activity, and anti-inflammatory activity and bacterial enteritis can be understood and discussed from the following aspects: (1) Antibacterial activity: The normal flora in the chicken intestine is essential for maintaining intestinal health. Antibacterial activity refers to the ability of a substance or mechanism to fight pathogenic microorganisms. In the chicken intestine, normal antibacterial activity helps to inhibit the growth of harmful bacteria, maintain the balance of intestinal flora, and thus reduce the risk of bacterial enteritis. (2) Antioxidant activity: Antioxidant activity refers to the ability to resist oxidative stress, which can protect cells from damage by neutralizing free radicals and other oxidizing substances. In the case of bacterial enteritis, inflammation and bacterial infection can cause oxidative stress, leading to cell damage and further aggravation of inflammation. Substances with high antioxidant activity (such as vitamins C and E, polyphenolic compounds, etc.) can alleviate this damage and help relieve intestinal inflammation. (3) Anti-inflammatory activity: Anti-inflammatory activity refers to the ability to inhibit inflammatory response. Bacterial enteritis is accompanied by an inflammatory response in intestinal tissue, leading to impaired intestinal function and pain and discomfort. Substances with anti-inflammatory activity (such as active ingredients in some drugs and natural products) can help reduce the degree of inflammation, thereby alleviating the symptoms and progression of bacterial enteritis.

[0023] Overall, antibacterial, antioxidant, and anti-inflammatory activities are of great significance for the prevention and treatment of bacterial enteritis in chickens. By providing antibacterial protection, reducing oxidative damage, and alleviating inflammatory responses, they can effectively improve the gut health of chickens and reduce the occurrence and impact of bacterial enteritis.

[0024] Example 1: The preparation method of the *Salix viminalis* extract used in this example is as follows: *Salix viminalis* medicinal material was taken and soaked in 10 times the amount of 50% ethanol for 1 hour, and then ultrasonically extracted at 50°C for 1 hour, and the extract was collected; then, 8 times the amount of 50% ethanol was added to the medicinal material and ultrasonically extracted at 50°C for 1 hour, and the extract was collected; the two extracts were combined and vacuum filtered, and concentrated by rotary steam at 50°C to obtain a concentrated solution, which was then freeze-dried to obtain *Salix viminalis* extract in powder form.

[0025] (a) Determination of the in vitro antibacterial activity of the extract of *Salix matsudana*.

[0026] (1) Preparation of bacterial suspension: All experimental strains were stored at -20℃. The strains were activated before use. After preparing the culture medium, it was sterilized in an autoclave. 15-20 mL of culture medium was taken from a sterilized petri dish in a laminar flow hood, cooled, and solidified. Four clinically extracted strains of *E. coli* (E. coli 1-E. coli 14) and four strains of *Salmonella* (SAL11-SAL14) were inoculated onto the culture medium using an inoculation loop. The petri dishes were sealed with a sealing strip and placed in a 37℃ constant temperature biochemical incubator for approximately 20 hours. After the strains were activated, colonies were picked up with an inoculation loop and placed in sterile water. After mixing, the mixture was compared with a McFarland turbidimetric tube to prepare a 1×10⁻⁶ suspension. 6 ~1×10 7 CFU•mL -1 The bacterial suspension is ready for use.

[0027] (2) Measurement of inhibition zone diameter by perforation method: Take 100 μL of the prepared bacterial suspension and spread it evenly on a petri dish containing 15–20 mL of 0.04 g / mL Luria-Bertani (LB) medium. Use a pipette tip to make four holes evenly on the medium and remove them with tweezers. Add 100 μL of *Salix viminalis* extract (concentration 200 mg / mL) to each well of the medium, then seal with a sealing strip, label the strain and the name of the traditional Chinese medicine, and incubate at 37°C. Observe the colony growth 18–20 h after inoculation and measure the diameter of the inhibition zone with calipers. Repeat the experiment three times. An inhibition zone diameter ≥20 mm indicates extremely sensitive, an inhibition zone diameter of 15–19 mm indicates highly sensitive, an inhibition zone diameter of 10–14 mm indicates moderately sensitive, an inhibition zone diameter <10 mm indicates low sensitive, and no inhibition zone indicates drug resistance.

[0028] (3) Experimental results: As shown in Table 1, according to the established sensitivity criteria, the extract of *Salix viminalis* showed extremely high sensitivity to *Escherichia coli* (E. coli1, E. coli2) and high sensitivity to *Escherichia coli* (E. coli3, E. coli4) and *Salmonella* (SAL11, SAL12, SAL13, SAL14), indicating that the extract of *Salix viminalis* has good antibacterial activity against both of these types of bacteria.

[0029] Table 1. Results of in vitro antibacterial experiments of *Salix viminalis* extract against *Escherichia coli* and *Salmonella*. (II) Determination of antioxidant activity of *Salix viminalis* extract.

[0030] (I) Determination of the DPPH free radical scavenging ability of the extract of *Salix matsudana*.

[0031] (1) Experimental procedure: The *Salix viminalis* extract and the positive control drug ascorbic acid (VC) were diluted sequentially with sterile water to prepare sample solutions of 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. Following the instructions of the DPPH free radical scavenging kit, the working solution powder was dissolved in 40 mL of anhydrous ethanol to obtain the working solution. 400 μL of sample solution and 600 μL of 80% methanol were added to the control tube; 400 μL of sample solution and 600 μL of working solution were added to the assay tube; and 400 μL of 80% methanol and 600 μL of working solution were added to the blank tube. Each tube was incubated at room temperature (20℃) in the dark for 30 min, centrifuged at 4000 r / min for 5 min, and the absorbance of each tube was measured at a wavelength of 517 nm.

[0032] , where A 测定管 A 对照管 A 空白管 These represent the absorbance values ​​of the test tube, control tube, and blank tube, respectively.

[0033] (2) Experimental results: As shown in Table 2, when the concentration of the extract of *Salix viminalis* is greater than 0.25 mg / mL, the DPPH free radical scavenging rate is greater than 89%, showing a high DPPH free radical scavenging rate.

[0034] Table 2. DPPH free radical scavenging rates of *Salix viminalis* extract and ascorbic acid. (II) Determination of the ability of *Salix viminalis* extract to scavenge ABTS free radicals.

[0035] (1) Experimental procedure: The *Salix viminalis* extract and the positive control drug ascorbic acid (VC) were diluted with sterile water to solutions of 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, respectively. After treatment according to the instructions of the Total Antioxidant Capacity (T-AOC) assay kit, the solutions were reacted at room temperature for 6 min at a wavelength of 405 nm, and the OD values ​​were read by an ELISA reader. A standard curve was plotted, and the curve formula was obtained using an Excel spreadsheet. The measured OD values ​​were substituted into the calculation formula to obtain the results.

[0036] (2) Experimental results: As shown in Table 3, the extract of *Salix viminalis* showed good antioxidant activity at concentrations of 1 mg / mL and 0.5 mg / mL.

[0037] Table 3 Total antioxidant capacity of *Salix viminalis* extract and ascorbic acid (III) Determination of the in vitro anti-inflammatory activity of the extract of *Salix viminalis*.

[0038] (1) Experimental methods.

[0039] ① The effect of lipopolysaccharide (LPS) on the viability of RAW264.7 cells was detected by MTT assay: RAW264.7 cells in logarithmic growth phase were used to prepare a single-cell suspension in complete culture medium containing a mixture of 10% FBS and 1% streptomycin, and the concentration was adjusted to 5 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell adhesion, different concentrations of *Salix viminalis* extract were added. Two experimental groups were set up: a control group and a lipopolysaccharide (LPS) group. The control group received 100 μL of blank culture medium. The LPS concentrations in the LPS groups were 200 ng / mL, 100 ng / mL, and 50 ng / mL. Each group had six replicates, with 100 μL of LPS added to each well. After incubation for 24 h and 48 h, the supernatant was discarded, and 100 μL of 10% MTT solution was added to each well. After incubation for 4 h, the supernatant was discarded, and 150 μL of dimethyl sulfoxide was added. The mixture was thoroughly shaken, and the absorbance was measured at 490 nm using a microplate reader. A cell viability of 95% or higher was used as the screening criterion for no cytotoxicity, and the highest concentration with a cell viability exceeding 95% was used as the modeling concentration of LPS.

[0040] ② MTT assay for the effect of *Salix viminalis* extract on the viability of RAW264.7 cells: Logarithmic growth phase RAW264.7 cells were used to prepare a single-cell suspension in complete culture medium containing a mixture of 10% FBS and 1% streptomycin, and the concentration was adjusted to 5 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell adhesion, different concentrations of *Salix viminalis* extract were added. The experiment included a control group and a *Salix viminalis* extract group. The control group received 100 μL of blank culture medium. In the *Salix viminalis* extract group, the concentrations of *Salix viminalis* extract were prepared using blank culture medium at concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, and 0.78125 μg / mL. Each group had 6 replicates, with 100 μL of the extract administered to each well. After incubating in a cell culture incubator for 24 hours, the supernatant was discarded, and 100 μL of 10% MTT solution was added to each well. After incubating in a cell culture incubator for 4 hours, the supernatant was discarded, and 150 μL of dimethyl sulfoxide was added. After thorough shaking, the absorbance value was measured using an ELISA reader at a wavelength of 490 nm. The effects of different concentrations of *Salix viminalis* extract and the blank group on the survival rate of RAW264.7 cells were compared. A cell survival rate of 95% or higher was used as the screening criterion for no cytotoxicity, and the highest concentration with a cell survival rate exceeding 95% was used as the administration concentration of *Salix viminalis* extract.

[0041] ③ Griess method for detecting NO levels: Take a 96-well plate and configure cells in the logarithmic growth phase into 10 wells. 5Cell suspension was prepared at 100 µL / mL, and 96-well plates were pre-cultured at 37°C with 5% CO2 for 8 h. The old culture medium was then discarded. For the *Salix viminalis* extract group, 100 µL of 200 ng / mL LPS and 100 µL of 100 μg / mL *Salix viminalis* extract were added; for the dexamethasone group, 100 µL of 200 ng / mL LPS and 100 µL of 20 μg / mL dexamethasone were added; for the lipopolysaccharide group, 200 µL of 100 ng / mL LPS was added; and for the blank group, 200 µL of blank culture medium was added. Each group was divided into six replicates. After incubating the different culture plates for 24 h, the culture medium from each well was removed, centrifuged at 1000 rpm for 10 min, and 50 µL of the supernatant was collected. The NO level in the cell supernatant was measured using a nitric oxide kit, and the absorbance was measured at 540 nm.

[0042] ④ ELISA method for detecting inflammatory factor levels: Take a 96-well plate and configure cells in logarithmic growth phase into 10 wells. 5 Cell suspension was added at a density of 100 µL / mL to each well of a 96-well plate. The plates were then pre-cultured at 37°C with 5% CO2 for 8 hours, after which the old culture medium was discarded. For the *Salix viminalis* extract group, 100 µL of 200 ng / mL LPS and 100 µL of 100 μg / mL *Salix viminalis* extract were added; for the dexamethasone group, 100 µL of 200 ng / mL LPS and 100 µL of 20 μg / mL dexamethasone were added; for the lipopolysaccharide group, 200 µL of 100 ng / mL LPS was added; and for the blank group, 200 µL of blank culture medium was added. Each group had six replicates. After incubating the different culture plates for 24 hours, the culture medium from each well was removed and centrifuged at 1000 rpm for 10 minutes. The inflammatory factors TNF-α, IL-1β, and IL-6 were then detected according to the enzyme-linked immunosorbent assay (ELISA) kit instructions at a wavelength of 450 nm.

[0043] (2) Experimental results: According to Figure 1 The data obtained were selected from the highest concentrations of each *Salix viminalis* extract that showed activity of 95% or higher against RAW264.7 cells. Figure 2 It can be seen that after adding different concentrations of lipopolysaccharide for 24 hours, cell viability did not change significantly, but after 48 hours, cell viability decreased significantly. Figure 3It was found that lipopolysaccharide (LPS) at concentrations of 200 ng / mL, 100 ng / mL, and 50 ng / mL all significantly increased the NO content in the cell supernatant (P < 0.001). Among these, the increase in NO in the cell supernatant was relatively small after treatment with 50 ng / mL LPS, while the increases in NO were similar for 100 ng / mL and 200 ng / mL LPS, indicating that 100 ng / mL LPS was sufficient to significantly increase the NO level in the cell supernatant. Furthermore, after 24 hours of treatment, the cell survival rate was highest with 100 ng / mL LPS, and the cell survival rate significantly decreased after 48 hours of treatment with all LPS concentrations. Therefore, in this embodiment, 24 hours was chosen as the modeling time, and a final concentration of 100 ng / mL was selected as the modeling concentration of LPS.

[0044] Depend on Figure 4 It was found that the extract of *Salix viminalis* showed significant anti-inflammatory activity in the supernatant of RAW264.7 cells induced by lipopolysaccharide (P < 0.001). Figure 5 It was found that the extract of *Salix viminalis* significantly reduced the levels of TNF-α (P < 0.001), IL-1β (P < 0.001), and IL-6 (P < 0.001) in the supernatant of RAW264.7 cells.

[0045] Example 2: Effects of *Salix viminalis* extract on growth performance and intestinal health in mice. The preparation method of the *Salix viminalis* extract used in this example is the same as that in Example 1.

[0046] (1) Experimental methods: KM mice were housed in an environment that met the experimental requirements, with environmental conditions controlled at a temperature of 21–25℃, relative humidity of 50%–60%, and a light / dark cycle of 12h. They were acclimatized for 7 days, during which their health status was observed, and healthy individuals were selected for random grouping. Each group consisted of 8 mice, which were housed independently. All mice were fed standard feed and provided with high-temperature sterilized distilled water.

[0047] The experiment lasted for 20 days, with administration occurring once daily. The specific administration regimens were as follows: the high-dose group (XH group) of *Salix viminalis* extract was administered 2 g / kg (i.e., 2 g of *Salix viminalis* extract per 1 kg of mouse); the low-dose group (XL group) was administered 1 g / kg (i.e., 1 g of *Salix viminalis* extract per 1 kg of mouse); the control group received an equal volume of saline. All experimental groups were administered the drug via gavage at a dose of 0.1 mL / 10 g body weight, and mice were fasted for 12 hours prior to gavage. During the experiment, mice were allowed free access to food and water. Weight was measured every 5 days during the administration period, and the gavage dose was adjusted accordingly to ensure accuracy. On day 20, mice were fasted for 12 hours, but water was permitted. Mice were weighed, euthanized by cervical dislocation, and samples were quickly collected for measurement and analysis of relevant indicators.

[0048] ① Changes in mouse body weight and determination of Lee's index.

[0049] The method for measuring weight change was as follows: the weight of mice was measured every five days, and the effect of the *Salix viminalis* extract on the weight gain of mice was calculated.

[0050] The Lee's index was determined by recording the mouse's weight and body length (from the tip of the mouse's nose to its anus) after the last administration. .

[0051] ② Mouse organ index determination: Take the liver, thymus, and spleen of mice, and calculate according to the formula: .

[0052] ③ Measurement of oxidative stress and inflammatory factor indicators.

[0053] Mouse serum was collected, and oxidative stress-related indicators were measured using a total antioxidant capacity assay kit, a total SOD activity assay kit, and a lipid peroxidation (MDA) assay kit. Inflammatory factors TNF-α, IL-1β, and IL-6 were detected according to the ELISA kit instructions at a wavelength of 450 nm.

[0054] ④ Determination of fecal moisture content and pH value.

[0055] Dilute mouse colon contents with distilled water at a ratio of 1:9 (m / V) and centrifuge at 4000 rpm for 5 min. After centrifugation, measure the pH of the supernatant. Weigh the colon contents and dry them in a 100℃ oven until constant weight. Record the mass change and calculate the fecal water content using the formula: Each sample group was repeated three times.

[0056] ⑤ Colonic tissue morphology examination: After harvesting and longitudinally dissecting the distal colon, the intestinal contents were rinsed with physiological saline to ensure cleanliness. Excess moisture was absorbed with filter paper, and the colon was longitudinally cut into two parts. One part was fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned. During sectioning, the sections were first immersed in hematoxylin solution for staining for about 5 minutes, then placed in ammonia solution for 5 seconds for separation, followed by rinsing with purified water for 1 hour to remove excess dye. Subsequently, the sections were dehydrated in 70% and 90% alcohol, 10 minutes each time. Next, they were stained with eosin solution for 2 minutes, then dehydrated with anhydrous ethanol, and cleared in xylene. Finally, the sections were covered with coverslips and mounted with resin. After the resin was completely dry, the tissue sections were observed under a microscope, and the results were photographed and recorded.

[0057] (2) Experimental results.

[0058] ① Effects of West Willow Extract on Mouse Condition.

[0059] At the end of the 20-day feeding period, the mice were in good health, with no deaths or illnesses. There were no significant differences in appearance between the drug experimental group and the control group, and all physiological activities and other signs were normal. Figure 6 As shown, there were no significant differences in body weight among the control group, the high-dose group of *Salix viminalis* extract (XH group), and the low-dose group of *Salix viminalis* extract (XL group) throughout the entire experimental period, and no individual mice experienced a sudden decrease in body weight.

[0060] ② Effects of West Willow Extract on Lee's Index and Organ Coefficient.

[0061] As shown in Table 4, compared with the blank group, the Lee's index of the high-dose group of *Salix viminalis* extract was significantly reduced; the intake of the low-dose group of *Salix viminalis* extract did not have a significant effect on the liver, thymus and spleen indices of mice, indicating that the 20-day low-dose drug intervention did not cause damage to the major organs of mice.

[0062] Table 4. Effects of the experimental group and the control group on Lee's index and organ coefficient. Note: * P < 0.05 compared to the control group.

[0063] ③ Effects of *Salix viminalis* extract on serum oxidative stress levels and inflammatory factors in mice.

[0064] like Figure 7As shown, compared with the blank group, the serum MDA concentration in the XH group was significantly increased (P<0.05) and the T-AOC level was significantly decreased (P<0.05), indicating that the body's antioxidant capacity was insufficient and the body's oxidative stress damage was more obvious. The XL group was able to significantly increase the serum T-AOC level in mice (P<0.05) and significantly decrease the serum MDA concentration (P<0.05), indicating that the XL group (administration regimen of 1g / kg) can enhance the body's total antioxidant capacity, reduce the degree of cell damage by clearing MDA, and thus have a positive effect on delaying the aging of the body.

[0065] Depend on Figure 8 It can be seen that, compared with the blank group, the XL group significantly reduced the mass concentrations of TNF-α, IL-6 and IL-1β in mouse serum, but the XH group had no significant effect on the levels of the three inflammatory factors in mouse serum.

[0066] ④ The water content and pH value of the colon contents.

[0067] As shown in Table 5, the water content of the colon contents of mice in the XL group was significantly increased compared with that in the control group. This result indicates that the XL group can give the colon contents of mice good water-holding capacity, which helps the colon contents carry metabolic waste out of the body and contributes to the intestinal health of mice.

[0068] The pH value of the colon contents of mice in the XL group was significantly lower than that in the control group. The decrease in pH value helps to inhibit the growth of harmful pathogens, reduce the incidence of colon diseases, and maintain the intestinal ecological balance. Therefore, the XL group (administration regimen of 1g / kg) has a positive effect on the intestinal health of mice.

[0069] Table 5. Effects of *Salix viminalis* extract on the moisture content and pH of mouse colon contents. Note: * P < 0.05 compared to the control group.

[0070] ⑤ Observation of colon tissue morphology.

[0071] Colon tissue morphology as Figure 9 As shown, the specific findings are as follows: In the blank control group, the intestinal mucosa protruded into the intestinal lumen, forming significant folds, and the number of folds was relatively abundant. The mucosal epithelium was mainly composed of columnar epithelial cells and a small number of goblet cells; some epithelial cells showed edema, with loose and lightly stained cytoplasm, and the nuclei of a few cells were shrunken. The intestinal glands in the lamina propria were densely arranged in tubular shapes, and goblet cells were abundant. The connective tissue of the submucosa was relatively dense, the muscular layer structure was intact, the muscle fibers showed no obvious abnormalities, and no significant inflammatory response was observed.

[0072] The results of the XH group showed that the intestinal tissue had small areas of erosion, a decrease in the number of intestinal glands, which were replaced by more proliferating connective tissue, a small number of new blood vessels, and a small number of inflammatory cells, mainly lymphocytes, infiltrating. A small number of mucosal epithelial cells had shrunken nuclei, increased cell acidity, and a large number of mucosal epithelial cells were sloughed off.

[0073] Results in group XL showed that the intestinal mucosa protruded into the intestinal lumen, forming significant folds in a relatively abundant number; the mucosal epithelium was mainly composed of columnar epithelial cells and a small number of goblet cells, with normal morphology and structure; the intestinal glands in the lamina propria were arranged tubularly and densely distributed, with a large number of goblet cells. The submucosa had a compact connective tissue structure, the muscular layer was structurally intact, the muscle fibers showed no obvious abnormalities, and no obvious inflammatory response was observed.

[0074] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. Application of *Salix viminalis* in the preparation of drugs for treating bacterial enteritis in chickens.

2. The application of the *Salix viminalis* according to claim 1 in the preparation of a medicament for treating bacterial enteritis in chickens, characterized in that... The effective concentration of the aqueous solution of *Salix viminalis* extract is not less than 0.5 mg / mL; The aqueous solution of *Salix viminalis* extract is prepared by dissolving *Salix viminalis* extract in water. The preparation method of *Salix viminalis* extract is as follows: take *Salix viminalis* medicinal material, add 10 times the amount of 50% ethanol and soak for 1 hour, then extract with ultrasound at 50°C for 1 hour, and collect the extract; then add 8 times the amount of 50% ethanol to the medicinal material, extract with ultrasound at 50°C for 1 hour, and collect the extract; combine the two extracts and perform vacuum filtration, concentrate with rotary steam at 50°C to obtain a concentrated solution, and freeze-dry the concentrated solution to obtain *Salix viminalis* extract in powder form.

3. The application of the *Salix viminalis* according to claim 2 in the preparation of a medicament for treating bacterial enteritis in chickens, characterized in that... The effective dose of the extract of *Salix matsudana* is 1 g / kg.

4. The use of the *Salix viminalis* according to claim 3 in the preparation of a medicament for treating bacterial enteritis in chickens, characterized in that... West River Willow Extract was added to chicken feed as a feed additive.

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