Veterinary drug compound preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of animal medicines, and particularly discloses a veterinary medicine compound preparation as well as a preparation method and application thereof. The veterinary medicine compound preparation is prepared by boiling, extracting, concentrating and diluting eight Tibetan medicines including aconitum pendulum, fructus chebulae, radix inulae inula, benzoin, pterocephalus hookeri, lagotis lagotis, amomum tsao-ko and rhodiola rosea. The veterinary drug compound preparation provided by the invention has an antibacterial range of 8.67 mm to 23.77 mm on enterotoxic large intestine dried bacteria, salmonella enteritidis and pasteurella multocida, and has an inhibition rate of 60% to 72% on enterotoxigenic escherichia coli diarrhea, salmonella enteritidis diarrhea and pasteurella multocida diarrhea; therefore, the veterinary drug compound preparation provided by the invention has a remarkable inhibition effect on enterotoxic large intestine dry bacteria, salmonella enteritidis and pasteurella multocida.

Description

Technical Field

[0001] This invention relates to the field of animal medicine technology, specifically to a veterinary drug compound preparation, its preparation method, and its application. Background Technology

[0002] Veterinary medicine is a unique traditional medical system in the Tibetan region, with a long history and rich experience, and is commonly used to treat infectious and bacterial infections in animals. Veterinary compound preparations are scientifically formulated from various Tibetan herbs, possessing unique pharmacological effects and therapeutic efficacy. Currently, research on the in vitro antibacterial effects of veterinary compound preparations is relatively limited, lacking large-sample, multi-center clinical studies. Furthermore, the variety of Chinese medicinal materials in compound preparations is vast, and their efficacy can be affected by factors such as the source of the materials and processing methods. Therefore, the antibacterial effects and mechanisms of veterinary compound preparations still need further investigation. Summary of the Invention

[0003] To develop the antibacterial effects and mechanisms of veterinary drug compound preparations, this invention provides a veterinary drug compound preparation, its preparation method, and its applications. Antibacterial tests revealed that at a concentration of 1 g / mL, the diameter of the inhibition zones against enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida were all greater than 20 mm, indicating high sensitivity. Mouse diarrhea model tests showed that the veterinary drug compound preparation provided by this invention exhibited an inhibition rate of 64% against enterotoxigenic Escherichia coli diarrhea, 72% against Salmonella enteritidis diarrhea, and 60% against Pasteurella multocida diarrhea, indicating that the veterinary drug compound preparation provided by this invention can be used to directly kill pathogenic bacteria and alleviate diarrhea symptoms.

[0004] This invention provides a veterinary drug compound preparation, made from the following raw materials in parts by weight: 100-200 parts of *Imperata cylindrica*, 100-200 parts of *Terminalia chebula*, 100-200 parts of *Saussurea costus*, 100-200 parts of *Benzyl benzoin*, 100-200 parts of *Polygonum aviculare*, 100-200 parts of *Hedyotis diffusa*, 100-200 parts of *Amomum tsao-ko*, and 100-200 parts of *Rhodiola rosea*.

[0005] Furthermore, the veterinary compound preparation is made from the following raw materials in parts by weight: 200 parts of iron bark, 200 parts of Terminalia chebula, 200 parts of Tibetan costus root, 200 parts of benzoin, 200 parts of winged head grass, 200 parts of rabbit ear grass, 200 parts of cardamom, and 200 parts of Rhodiola rosea.

[0006] The veterinary compound preparation provided by this invention, at a concentration of 1 g / mL, exhibits inhibition zones greater than 20 mm in diameter for enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida, indicating high sensitivity. Mouse diarrhea model experiments revealed that the veterinary compound preparation provided by this invention exhibits a 64% inhibition rate against enterotoxigenic Escherichia coli diarrhea, a 72% inhibition rate against Salmonella enteritidis diarrhea, and a 60% inhibition rate against Pasteurella multocida diarrhea, demonstrating that the veterinary compound preparation provided by this invention can be used to directly kill pathogenic bacteria and alleviate diarrhea symptoms.

[0007] This invention also provides a method for preparing the above-mentioned veterinary drug compound preparation, specifically comprising the following steps: Weigh out the following ingredients according to their weight proportions: iron bar hammer, Terminalia chebula, Tibetan costus root, benzoin, winged head grass, rabbit ear grass, cardamom, and Rhodiola rosea, and set aside for later use; After washing and drying the raw materials, they were crushed, soaked in water, heated to boiling and extracted, filtered through gauze, and the extraction was repeated twice. The extracts were then combined and centrifuged to obtain the supernatant. The supernatant was heated, concentrated, and dried to obtain a crude aqueous extract. It was then sterilized with ultraviolet light and prepared into an aqueous extract for veterinary drug compound preparations using sterile distilled water. After centrifugation, the supernatant of the aqueous extract of the veterinary drug compound preparation was collected and diluted twice to obtain veterinary drug compound preparations with different concentration gradients.

[0008] Furthermore, the heating and concentration involves boiling the supernatant and concentrating it to 180 mL to 220 mL, then pouring it into an evaporating dish and heating it for further concentration.

[0009] Furthermore, the soaking time is 24 hours, and the boiling extraction time is 2 hours.

[0010] Furthermore, the concentration of the veterinary drug compound preparation is 15.625 mg / mL to 2120 mg / mL.

[0011] The present invention also provides the application of the aforementioned veterinary drug compound preparation in the preparation of antibacterial drugs, wherein the antibacterial drugs are used to inhibit pathogenic bacteria, wherein the pathogenic bacteria are enterotoxigenic Escherichia coli, Salmonella enteritidis, or Pasteurella multocida.

[0012] Furthermore, the application is applied to diarrheal mice that have been administered the pathogenic bacteria via gavage.

[0013] Furthermore, the volume of the veterinary drug compound preparation administered by gavage shall not exceed 0.2 mL / 10g body weight.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention selects eight Tibetan medicinal herbs—Iron Bark Hammer, Terminalia chebula, Tibetan Costus Root, Benzoin, Winged Grass, Rabbit Ear Grass, Amomum tsao-ko, and Rhodiola Rosea—to form a veterinary compound preparation. The inhibitory range against enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida is 8.67 mm to 23.77 mm. The inhibition rate against enterotoxigenic Escherichia coli diarrhea, Salmonella enteritidis diarrhea, and Pasteurella multocida diarrhea is 60% to 72%. This indicates that the veterinary compound preparation provided by this invention has a significant inhibitory effect on enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida, demonstrating its potential advantages and possibilities in clinical application. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a graph showing the minimum inhibitory concentration of compound veterinary drug preparations.

[0017] Figure 2 This is a graph showing the minimum bactericidal concentration for compound veterinary drug preparations. Detailed Implementation

[0018] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0019] Example 1: A veterinary drug compound preparation, its preparation method and application I. Experimental Materials and Instruments 1. Selection of Tibetan Medicine The following herbs were selected to make an eight-ingredient balancing powder: Spoonleaf Winged Head Grass, Rabbit Ear Grass, Amomum tsao-ko, Tibetan Costus Root, Iron Hammer, Terminalia chebula, Rhodiola Rosea, and Benzoin. The eight Tibetan herbs were purchased from Tibet Baoxin Biotechnology Co., Ltd.

[0020] 2. Strains selection Enterotoxigenic Escherichia coli (ETEC, strain number: BNCC186736), Salmonella Enteritidis (strain number: BNCC103134), and Pasteurella multocida subsp. multocida (strain number: BNCC270568) were all purchased from the National Veterinary Microbiology Culture Collection Center.

[0021] 3. Main Instruments Cell culture incubator (Thermo Scientific, USA), MDF-382 low-temperature freezer (SANYO, Japan), HF safe-1500 biosafety cabinet (Likang Biomedical Technology Co., Ltd.), HV-85 autoclave, traditional Chinese medicine decoction machine, freeze dryer, clean bench (Qingdao Haier Special Electric Appliance Co., Ltd.), pipette (100 µL).

[0022] II. Test Methods 1. Preparation of aqueous extracts of compound veterinary drug formulations Take 200g each of eight Tibetan medicinal herbs: *Iron Hammer*, *Terminalia chebula*, *Saussurea costus*, *Benzyl benzoin*, *Polygonum aviculare*, *Corydalis yanhusuo*, *Amomum villosum*, and *Rhodiola rosea*. Clean the herbs with an ultrasonic cleaner to remove impurities and dry them in a 60℃ oven for later use. Crush each sample separately and weigh 125g. Soak each sample in an appropriate amount of water for 24 hours, boil for 2 hours, filter with gauze, and extract twice with an appropriate amount of water. Combine the extracts and centrifuge at 5000 rpm for 15 minutes at 4℃. Take the supernatant, boil and concentrate to about 200 mL, pour into an evaporating dish, heat for further concentration, and dry in a 60℃ oven to obtain a crude aqueous extract. Sterilize by ultraviolet irradiation for 30 minutes. Prepare a veterinary drug compound preparation aqueous extract with sterile distilled water at 1 g / mL and store at 4℃ for later use.

[0023] 2. Activation of bacterial strains and preparation of bacterial suspensions 400 µL of sterile water was injected into freeze-dried tubes containing enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida, respectively. The tubes were gently pipetted to fully dissolve the bacteria into suspensions. Then, 200 µL of each suspension was spread onto LB agar, nutrient agar, and blood agar plates, with three replicates per group. The spread plates were incubated at 37°C for 24 h to observe the bacterial reactivation. Single colonies from each of the three media were inoculated into nutrient broth and incubated at 37°C and 200 rpm for 24 h. The bacteria were then counted using a McFarland turbidimeter, and the bacterial concentration was adjusted to 1 × 10⁻⁶ using a turbidimeter. 6 CFU / mL available for use.

[0024] 3. In vitro antibacterial test of veterinary drug compound preparations After centrifugation, the supernatant of the aqueous extract of the veterinary drug compound preparation was collected. The original drug solution concentration of 1 g / mL was serially diluted twice in 10 mL test tubes to obtain six concentrations of veterinary drug compound preparation: 500 mg / mL, 250 mg / mL, 125 mg / mL, 62.5 mg / mL, 31.25 mg / mL, and 15.625 mg / mL. The solutions were then autoclaved at 121℃ for 1 h and stored at 4℃ with labels. 0.1 mL of the prepared bacterial suspension was added to a pre-prepared autoclaved nutrient agar culture dish, spread evenly with a spreader, and placed in an Oxford cup. Then, 0.2 mL of the prepared veterinary drug compound preparation of different concentrations was added to the Oxford cup in descending order of concentration using a dropper. The culture dish was then capped, and the nutrient agar culture dish containing the drug solution was placed horizontally in an incubator and incubated at 37℃ for 24 h. The diameter of the inhibition zone was measured using calipers.

[0025] Results judgment criteria: Sensitivity is defined as follows: inhibition zone diameter less than 10 mm indicates drug resistance, 10 mm to 15 mm indicates moderate sensitivity, and greater than 15 mm indicates high sensitivity.

[0026] 4. Determination of minimum inhibitory concentration (MIC) The veterinary drug compound preparation, which has been serially diluted twice, was dispensed into test tubes in sequence according to the concentration gradient, 2 mL per tube. Then, 100 µL of bacterial suspension was added to each concentration gradient tube. Each sample was performed in triplicate. At the same time, each gradient group had one positive control tube containing only an equal amount of bacterial suspension and one negative control tube containing only the veterinary drug compound preparation and no bacterial suspension. The tubes were incubated at 37℃ for 24 h. The lowest drug concentration at which no bacterial growth was observed by the naked eye was the MIC of the drug.

[0027] 6. Determination of Minimum Bacterial Concentration (MBC) Take 100 µL of culture from each test tube containing the MIC of each bacterium and concentrations above the MIC, inoculate them onto nutrient agar medium, and incubate at 37°C for 48 h. The lowest concentration of the compound veterinary drug preparation at which no colonies grow can be observed with the naked eye is the minimum bactericidal concentration.

[0028] 7. Acute toxicity test of compound veterinary drug preparation administered to mice by gavage Forty mice were randomly divided into five groups of eight each, with half males and half females. Before administration, the mice were fasted for 12 hours. Each group was then administered the compound veterinary drug via gavage at concentrations of 2120 mg / mL, 1700 mg / mL, 1360 mg / mL, 1090 mg / mL, 872 mg / mL, 698 mg / mL, 560 mg / mL, and 450 mg / mL, respectively. The dosage was set at no more than 0.2 mL / 10g per mouse to determine the range of 0%–100% mortality. The mice were fasted for four hours after administration, but water was permitted. Observations were taken 30 minutes after gavage and again 4 hours later, twice daily in the afternoon for seven consecutive days. The main observations of the mice's overall mental state included: eye expression, coat smoothness, sensitivity to stimuli, interest in the environment, feces, respiration and frequency, secretions, and any other abnormal behaviors. The number of deaths and survivors in each group was recorded.

[0029] 8. Establishment of a mouse diarrhea model and gavage test of compound veterinary drug preparation Enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida were diluted with physiological saline to a concentration of 1×10⁻⁶. 8 120 mice were randomly divided into four drug groups: a high-dose group, a medium-dose group, a low-dose group, and a control group (the control group received the same dose of physiological saline via gavage). Each drug group was further divided into three bacterial suspension groups, which were administered diluted enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida via gavage at a bacterial suspension concentration of 0.2 mL / 10g, respectively, to establish a diarrhea model. Mice were placed in cages lined with filter paper, and their diarrhea was observed. One hour later, mice in each drug group were administered the corresponding dose of drug via gavage at 0.2 mL / 10g (high, medium, and low doses were calculated using the median lethal dose). The filter paper was changed after each episode of diarrhea. The number of diarrhea episodes within 6 hours was recorded, and the rate of loose stools and the diarrhea inhibition rate were calculated using the following formulas:

[0030] 9. Statistical Analysis Data processing was performed using SPSS 18.0 software. Data measurements are expressed as "X±SD". A p-value < 0.05 was considered statistically significant; a p-value < 0.01 was considered extremely significant. In both the diarrhea model and treatment data, p < 0.05 indicated statistical significance.

[0031] III. Test Results 1. In vitro antibacterial test of veterinary drug compound preparations Antibacterial tests were conducted on veterinary drug compound preparations at different concentrations. At the same drug concentration, each pathogenic bacterial droplet was tested three times, and the diameter of the inhibition zone was measured using calipers. The average value was recorded, as shown in Table 1. The results showed that the veterinary drug compound preparations had strong antibacterial activity against all three pathogens. When the drug concentration was adjusted to 1 g / mL, the diameter of the inhibition zone for all three pathogens was greater than 20 mm. The diameter of the inhibition zone for *Salmonella enteritidis* was higher than that for *Enterotoxigenic Escherichia coli* and *Pasteurella multocida* at the same drug concentration.

[0032] Table 1. Results and Interpretation of Antibacterial Tests for Compound Veterinary Drug Preparations 2. Measurement of MIC and MBC In vitro antibacterial results of veterinary compound preparations against enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida showed that the minimum inhibitory concentrations (MICs) of the veterinary compound preparations against the three diarrheal bacteria were 125 mg / mL, 62.5 mg / mL, and 62.5 mg / mL, respectively (see...). Figure 1 The minimum bactericidal concentrations (MBCs) are 500 mg / mL, 125 mg / mL, and 125 mg / mL, respectively (see...). Figure 2 ).

[0033] 3. Acute toxicity test of compound veterinary drug preparation administered to mice by gavage Mice were administered veterinary drug compound preparations orally via gavage at concentrations of 2120 mg / mL, 1700 mg / mL, 1360 mg / mL, 1090 mg / mL, 872 mg / mL, 698 mg / mL, 560 mg / mL, and 450 mg / mL. Within 4 hours, the mice remained curled up and in a dormant state. After 24 hours of observation, all 40 mice showed normal activity, water intake, and food consumption. After 7 days of continuous observation, no mice died. The drug concentration at which 100% mortality occurred was 872 mg / mL, while the concentration at which 0% mortality occurred was 450 mg / mL (see Table 2). No significant abnormalities were observed in the mice's eyes, coat gloss, stimuli sensitivity, interest in the environment, feces, respiration and frequency, or other aspects after administration (see Table 3).

[0034] Table 2. Mortality of mice after gavage administration of veterinary drug compound preparations Table 3. Observation items and results of mice after gavage administration of veterinary drug compound preparations. 4. Dosing test of compound veterinary drug preparation in mice with diarrhea Table 2 shows that the drug concentration at which mouse mortality is 100% is 862 mg / mL, the concentration at which mortality is 20% is 560 mg / mL, and the concentration at which mortality is 0% is 450 mg / mL. When the mortality rate in the lowest dose group is >0% or the mortality rate in the highest dose group is <100%, the probability unit method is used to correct for theoretical 0% and 100%, respectively. The corrected minimum mortality rate = 1 − e −k P min (P) min (actual minimum mortality rate), corrected maximum mortality rate = 1 − (1 − P) max / )e m (P) max To determine the actual maximum mortality rate, the corrected mortality rate is converted to probability units (Probit value), and a linear relationship between the dose (logarithm) and the probability units is established. Weighted regression is then used to calculate LD. 50 We use weighted least squares to fit the straight line to reduce the influence of extreme values. The formula is: LD 50 =log −1 (a / b), where a and b are regression coefficients (Y = a + b × log(dose)). Calculate LD. 50 and confidence interval LD 50 =10 −a / b 95% confidence interval: log −1 (log(LD 50 (±1.96×SE) (SE is the standard error). From the above, the median lethal dose (LD50) can be calculated. 50 =668 mg / mL, rounded to the nearest integer and considering a 95% confidence interval of 1.614 ~ (-26.113), the median lethal dose (LD50) can be considered as 668 mg / mL. 50 =700 mg / mL. The dosage was determined according to 1 / 10, 1 / 20, and 1 / 30 of the median lethal dose: high dose 70 mg / mL, medium dose 35 mg / mL, and low dose 23 mg / mL. Diarrhea indicators, as shown in Tables 4, 5, and 6, showed that the average number of diarrhea episodes and the rate of loose stools in the high, medium, and low dose groups were significantly different from the control group (P<0.05), indicating that the mouse diarrhea model was successfully established. Tables 4, 5, and 6 also showed that different doses of the Tibetan veterinary medicine compound preparation inhibited diarrhea in mice subjected to gavage with enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida. The inhibition rate of diarrhea in mice was in the order of Salmonella enteritidis > enterotoxigenic Escherichia coli > Pasteurella multocida, with the high-dose group showing the greatest inhibition rate against Salmonella enteritidis diarrhea.

[0035] Table 4. Effects of different doses of veterinary drug compound preparations on diarrhea in mice infected with enterotoxigenic Escherichia coli via gavage. Table 5. Effects of different doses of veterinary drug compound preparations on diarrhea in mice treated with Salmonella enteritidis via gavage. Table 6. Effects of different doses of veterinary drug compound preparations on diarrhea in mice infected with Pasteurella multocida via gavage. Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0036] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A compound veterinary drug preparation, characterized in that, The veterinary compound preparation is made from the following raw materials in parts by weight: 100-200 parts of *Imperata cylindrica*, 100-200 parts of *Terminalia chebula*, 100-200 parts of *Saussurea costus*, 100-200 parts of *Benzyl benzoin*, 100-200 parts of *Polygonum aviculare*, 100-200 parts of *Hedyotis diffusa*, 100-200 parts of *Amomum tsao-ko*, and 100-200 parts of *Rhodiola rosea*.

2. The veterinary drug compound preparation according to claim 1, characterized in that, The veterinary compound preparation is made from the following raw materials in parts by weight: 200 parts of iron bark, 200 parts of Terminalia chebula, 200 parts of Tibetan costus root, 200 parts of benzoin, 200 parts of winged head grass, 200 parts of rabbit ear grass, 200 parts of cardamom, and 200 parts of Rhodiola rosea.

3. A method for preparing a veterinary drug compound preparation according to claim 1 or 2, characterized in that, Specifically, it includes the following steps: Weigh out the following ingredients according to their weight proportions: iron bar hammer, Terminalia chebula, Tibetan costus root, benzoin, winged head grass, rabbit ear grass, cardamom, and Rhodiola rosea, and set aside for later use; After washing and drying the raw materials, they were crushed, soaked in water, heated to boiling and extracted, filtered through gauze, and the extraction was repeated twice. The extracts were then combined and centrifuged to obtain the supernatant. The supernatant was heated, concentrated, and dried to obtain a crude aqueous extract, which was then sterilized by ultraviolet light and prepared into the aforementioned veterinary drug compound preparation using sterile distilled water.

4. The method for preparing the veterinary drug compound preparation according to claim 3, characterized in that, The soaking time is 24 hours, and the boiling extraction time is 2 hours.

5. The method for preparing the veterinary drug compound preparation according to claim 3, characterized in that, The heating and concentration process involves boiling the supernatant and concentrating it to 180 mL to 220 mL, then pouring it into an evaporating dish and heating it for further concentration.

6. The method for preparing the veterinary drug compound preparation according to claim 3, characterized in that, The concentration of the veterinary drug compound preparation is 15.625 mg / mL to 2120 mg / mL.

7. The use of the veterinary drug compound preparation according to claim 1 or 2 in the preparation of antibacterial drugs, characterized in that, The antibacterial drug is used to inhibit pathogenic bacteria, which are enterotoxigenic Escherichia coli, Salmonella enteritidis, or Pasteurella multocida.

8. The use of the veterinary drug compound preparation according to claim 7 in the preparation of antibacterial drugs, characterized in that, The application was performed on diarrheal mice that were given the pathogenic bacteria by gavage.

9. The application of the veterinary drug compound preparation according to claim 1 in inhibiting pathogenic bacteria, characterized in that, The volume of the veterinary drug compound preparation administered by gavage shall not exceed 0.2 mL / 10g body weight.

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