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] The present invention relates to the technical field of animal medicine, and in particular to a veterinary drug compound preparation, a preparation method and an application thereof. Background Art

[0002] Veterinary medicine, a traditional medical system unique to Tibetan areas, boasts a long history and rich experience, and is commonly used to treat infectious and bacterial infections in animals. Veterinary compound preparations, formulated through the scientific combination of multiple Tibetan medicinal herbs, possess unique pharmacological and therapeutic properties. Currently, research on the in vitro antibacterial effects of veterinary compound preparations is limited, lacking large-sample, multicenter clinical studies. Furthermore, compound preparations contain a wide variety of Chinese medicinal materials, and their efficacy is affected by factors such as their source and processing methods. Therefore, the antibacterial effects and mechanisms of veterinary compound preparations remain to be explored. Summary of the Invention

[0003] To explore the antibacterial effects and mechanisms of veterinary drug compound preparations, the present invention provides a veterinary drug compound preparation, its preparation method, and its application. Antibacterial tests revealed that at a concentration of 1 g / mL, the diameters of the inhibition zones for enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida were all greater than 20 mm, indicating high sensitivity. A mouse diarrhea model test revealed that the veterinary drug compound preparation provided herein had an inhibition rate of 64% against enterotoxigenic Escherichia coli diarrhea, 72% against Salmonella enteritidis diarrhea, and 60% against Pasteurella multocida diarrhea, demonstrating that the veterinary drug compound preparation provided herein can be used to directly kill pathogens and alleviate diarrheal symptoms.

[0004] The invention provides a veterinary compound preparation, which is prepared from the following raw materials in parts by weight: 100-200 parts of wormwood, 100-200 parts of terminalia chebula, 100-200 parts of Tibetan woodruff, 100-200 parts of benzoin, 100-200 parts of pterocephala, 100-200 parts of rabbit ear grass, 100-200 parts of tsaoko, 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 hammer, 200 parts of Terminalia chebula, 200 parts of Tibetan woodruff, 200 parts of benzoin, 200 parts of pterocephala, 200 parts of rabbit ear grass, 200 parts of tsaoko, and 200 parts of rhodiola rosea.

[0006] When the concentration of the veterinary compound preparation provided by the present invention is 1 g / mL, the diameters of the inhibition zones of enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida are all greater than 20 mm, and are judged to be highly sensitive; a mouse diarrhea model test found that the veterinary compound preparation provided by the present invention had an inhibition rate of 64% on enterotoxigenic Escherichia coli diarrhea, an inhibition rate of 72% on Salmonella enteritidis diarrhea, and an inhibition rate of 60% on Pasteurella multocida diarrhea, indicating that the veterinary compound preparation provided by the present invention can be used to directly kill pathogens and alleviate diarrhea symptoms.

[0007] The present invention also provides a method for preparing the above-mentioned veterinary drug compound preparation, which specifically comprises the following steps: Weigh the following raw materials according to parts by weight: tibetan tsaoko, chebula chebula, styrax benzoin, pterygium wilfordii, sedge grass, tsaoko and rhodiola rosea; set aside; The raw materials were washed, dried and crushed, soaked in water and heated to boil for extraction, filtered through gauze, extracted twice, combined the extracts, and centrifuged to obtain the supernatant; The supernatant is heated, concentrated, and then dried to obtain a crude water extract, which is sterilized by ultraviolet light and prepared with sterile distilled water to prepare a water extract of a veterinary drug compound preparation; After centrifugation of the aqueous extract of the veterinary drug compound preparation, the supernatant was collected and diluted 2-fold to obtain veterinary drug compound preparations with different concentration gradients.

[0008] Furthermore, the heating and concentration is to boil and heat the supernatant to concentrate it to 180 mL to 220 mL, pour it into an evaporating dish, and heat it for further concentration.

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

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

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

[0012] Furthermore, the application subjects are diarrhea mice that are gavaged with the pathogenic bacteria.

[0013] Furthermore, the oral administration volume of the veterinary compound preparation does not exceed 0.2 mL / 10 g body weight.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention selects eight Tibetan medicinal herbs, namely, wormwood, terminalia chebula, aconite, benzoin, pterygium wilfordii, sedge, tsaoko, and rhodiola rosea, to form a veterinary compound preparation. The antibacterial range of the preparation on enterotoxigenic coliform bacteria, Salmonella enteritidis, and Pasteurella multocida is 8.67 mm to 23.77 mm, and the inhibition rate on enterotoxigenic Escherichia coli diarrhea, Salmonella enteritidis diarrhea, and Pasteurella multocida diarrhea is 60% to 72%, indicating that the veterinary compound preparation provided by the present invention has a significant inhibitory effect on enterotoxigenic coliform bacteria, Salmonella enteritidis, and Pasteurella multocida, showing potential advantages and possibilities in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

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

[0017] Figure 2 This is a chart showing the minimum bactericidal concentration of veterinary drug compound preparations. DETAILED DESCRIPTION

[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 by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0019] Example 1: A veterinary drug compound preparation and its preparation method and application 1. Test materials and instruments 1. Selection of Tibetan medicine Eight-flavor balance powder was prepared by selecting Pterocephalus spathiphyllum, Herba Cyperi, Amomum villosum, Aucklandia lappa, Trichosanthes kirilowii, Terminalia chebula, Rhodiola rosea, and Benzoin. The eight Tibetan medicines were purchased from Tibet Baoxin Biotechnology Co., Ltd.

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

[0021] 3. Main instruments Cell culture incubator (Thermo Scientific, USA), low-temperature refrigerator MDF-382 (SANYO, Japan), biological safety cabinet HF safe-1500 (Likang Biomedical Technology Co., Ltd.), high-pressure sterilizer HV-85, Chinese medicine decoction machine, freeze dryer, clean bench (Qingdao Haier Special Electrical Co., Ltd.), and pipette (100 μL).

[0022] 2. Test methods 1. Preparation of aqueous extracts of veterinary drug compound preparations 200 g each of eight Tibetan medicinal herbs (Cyperus rotundus, Terminalia chebula, Aucklandia odorifera, Benzoin, Pterocephalus herba, Herba Cyperi, Amomum villosum, and Rhodiola rosea) were collected and cleaned of impurities using an ultrasonic cleaner. The samples were then dried in a 60°C oven for later use. Each sample was crushed and weighed, weighing 125 g. The samples were soaked in an appropriate amount of water for 24 h, boiled for 2 h, filtered through gauze, and extracted twice with an appropriate amount of water. The combined extracts were centrifuged at 5000 rpm for 15 min at 4°C. The supernatant was collected, boiled, concentrated to approximately 200 mL, poured into an evaporating dish, further concentrated by heating, and dried in a 60°C oven to obtain a crude aqueous extract. The extracts were sterilized by ultraviolet irradiation for 30 min, and then prepared with sterile distilled water to prepare a 1 g / mL aqueous extract of the veterinary drug compound preparation. The extracts were stored at 4°C for later use.

[0023] 2. Bacterial activation and bacterial suspension preparation 400 µL of sterile water was injected into freeze-dried tubes of enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida, respectively. The suspension was thoroughly dissolved by gentle pipetting. 200 µL of the suspension was then spread onto LB agar, nutrient agar, and blood plate solid media, respectively. Three replicates were performed for each group. The spreadable solid media were placed in a 37°C incubator for 24 hours to observe the bacterial revival. Single colonies from the three media were inoculated into nutrient broth liquid media. After incubation at 37°C and 200 rpm for 24 hours, the three bacteria were counted using a McFadden turbidimeter, and the concentration of the bacterial solution was adjusted to 1 × 10 using a turbidimeter. 6 CFU / mL is reserved.

[0024] 3. In vitro antibacterial test of veterinary compound preparations After centrifugation of the aqueous extract of the veterinary drug compound preparation, the supernatant was collected and the original 1 g / mL solution was diluted two-fold in 10 mL test tubes to six concentrations of 500 mg / mL, 250 mg / mL, 125 mg / mL, 62.5 mg / mL, 31.25 mg / mL, and 15.625 mg / mL. The solution was sterilized by autoclaving at 121°C for 1 h and stored at 4°C, marked. 0.1 mL of the prepared bacterial suspension was pipetted onto a pre-prepared, autoclaved nutrient agar plate. Spread evenly using a spreader, an Oxford cup was placed, and 0.2 mL of the prepared veterinary drug compound preparation of varying concentrations was added to the Oxford cup using a dropper, in descending order. The plate was then covered with a lid and the nutrient agar plate with the solution was placed horizontally in an incubator and incubated at 37°C for 24 h. The diameter of the inhibition zone was measured using a vernier caliper.

[0025] The results were judged by the sensitivity criteria: the diameter of the inhibition zone was less than 10 mm for resistance, 10 mm to 15 mm for moderate sensitivity, and greater than 15 mm for high sensitivity.

[0026] 4. Determination of minimum inhibitory concentration (MIC) The twice-diluted veterinary drug compound preparation was dispensed into test tubes according to the concentration gradient, with 2 mL in each test tube. 100 µL of bacterial solution was then added to each concentration gradient test tube. Three replicates were performed for each sample. At the same time, a positive control tube containing an equal amount of bacterial solution but no veterinary drug compound preparation and a negative control tube containing only veterinary drug compound preparation were set up for each gradient group. The samples were incubated at 37°C for 24 hours. The lowest drug concentration at which no bacterial growth was observed by naked eye was the MIC of the drug.

[0027] 6. Determination of minimum bactericidal concentration (MBC) 100 μL of culture was taken from each test tube with the MIC of each bacteria and the concentration above the MIC, inoculated into nutrient agar medium, and cultured at 37°C for 48 h. The lowest concentration of the compound veterinary drug preparation at which no colonies grew was observed with the naked eye, which was the minimum bactericidal concentration.

[0028] 7. Acute toxicity test of veterinary drug compound preparations in mice after oral administration Forty mice were randomly divided into five groups of eight mice each, half male and half female. All mice were fasted for 12 hours before dosing. Each group was then gavaged with the veterinary drug compound at doses 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 dose was set to no more than 0.2 mL / 10 g per mouse, to determine the dose range that resulted in 0% to 100% mortality in the mice. The mice were fasted for 4 hours after dosing, but water was not withheld. After gavage, the mice were observed for 30 minutes and again 4 hours after gavage, each day in the afternoon and afternoon for 7 consecutive days. The mice's general mental state was observed for the following: eye contact, coat smoothness, sensitivity to stimuli, interest in the environment, feces, respiratory rate, secretions, and any other abnormal behavior. The number of mice that died and survived was recorded in each group.

[0029] 8. Establishment of Mouse Diarrhea Model and Oral Gavage Test of Veterinary Drug Compound Preparation Enterotoxigenic Escherichia coli, Salmonella enteritidis and Pasteurella multocida were diluted with normal saline to 1×10 8 120 mice were randomly divided into four drug groups: high-dose, medium-dose, low-dose, and a control group (the control group received the same dose of normal saline by gavage). Each drug group was further divided into three bacterial solution groups, each of which was gavaged with diluted enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida at a concentration of 0.2 mL / 10g to establish a diarrhea model. Mice were placed in cages lined with filter paper and observed for diarrhea. One hour later, mice in each drug group were gavaged with the corresponding drug dose (high, medium, and low doses were calculated using the median lethal dose) at 0.2 mL / 10g. The filter paper was changed after each diarrhea episode. The number of diarrhea episodes within 6 hours was counted, and the loose stool rate and diarrhea inhibition rate were calculated using the following formula:

[0030] 9. Statistical analysis Data were processed using SPSS 18.0. Data were expressed as "X ± SD." P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference. In the diarrhea model and treatment data, P < 0.05 indicated statistical significance.

[0031] 3. Test results 1. In vitro antibacterial test of veterinary compound preparations Antibacterial testing was conducted on the veterinary drug compound preparation at different concentrations. At the same drug concentration, the test was repeated three times for each pathogenic bacteria bead, and the diameter of the inhibition zone was measured with a vernier caliper. The average value was shown in Table 1. The results showed that the veterinary drug compound preparation had a strong antibacterial effect 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 Antibacterial test results and judgment of veterinary compound preparations 2. Determination of MIC and MBC The in vitro antibacterial results of the veterinary drug compound preparation against enterotoxigenic Escherichia coli, Salmonella enteritidis and Pasteurella multocida showed that the minimum inhibitory concentration (MIC) of the veterinary drug compound preparation 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 concentration (MBC) is 500 mg / mL, 125 mg / mL, 125 mg / mL (see Figure 2 ).

[0033] 3. Acute toxicity test of veterinary drug compound preparations in mice after oral administration After oral administration of the veterinary drug combination at different concentrations (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), mice curled up and remained dormant within 4 hours. After 24 hours of observation, all 40 mice showed normal activity, water intake, and food intake. No mice died after 7 consecutive days of observation. The drug concentration that resulted in 100% mortality was 872 mg / mL, while the concentration that resulted in 0% mortality was 450 mg / mL (see Table 2). No significant abnormalities were observed in the mice's eye expression, coat smoothness, sensitivity to stimuli, interest in the environment, feces, respiratory rate, or other characteristics after administration (see Table 3).

[0034] Table 2 Death of mice after oral administration of veterinary drug compound preparations Table 3 Observation items and results of mice after oral administration of veterinary drug compound preparations 4. Administration test of veterinary drug compound preparation in mice with diarrhea As shown in Table 2, the drug concentration that resulted in 100% mortality in mice was 862 mg / mL, the concentration that resulted in 20% mortality was 560 mg / mL, and the concentration that resulted in 0% mortality was 450 mg / mL. When the mortality rate in the lowest dose group was >0% or the mortality rate in the highest dose group was <100%, the probability unit method was used to correct the mortality rate to the theoretical values of 0% and 100%. The corrected minimum mortality rate = 1-e −k P min (P min is the actual minimum mortality rate), the corrected maximum mortality rate = 1−(1−P max / )e m , (P max The actual maximum mortality rate was calculated by converting the adjusted mortality rate into probability units (Probit value) and establishing a linear relationship between the dose (logarithm) and the probability unit. 50 Use weighted least squares method to fit the straight line to reduce the influence of extreme values. Formula: 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 standard error). From the above, the median lethal dose LD can be calculated. 50 =668 mg / mL, rounded to the integer and considering the 95% confidence interval of 1.614 to (-26.113), the median lethal dose LD 50 =700 mg / mL. Based on the median lethal dose (LD50) of 1 / 10, 1 / 20, and 1 / 30, the high dose was 70 mg / mL, the medium dose was 35 mg / mL, and the low dose was 23 mg / mL. Diarrhea indices, as shown in Tables 4, 5, and 6, showed significant differences in the average number of diarrhea episodes and loose stool rates between the high, medium, and low dose groups compared with the control group (P < 0.05), indicating that the mouse diarrhea model was successfully established. Tables 4, 5, and 6 show that the different doses of the Tibetan veterinary medicine compound preparation inhibited diarrhea in mice induced by oral administration of enterotoxigenic Escherichia coli, Salmonella enteritidis, and Pasteurella multocida. The inhibitory rates of the Tibetan veterinary medicine compound preparation on diarrhea in mice were ranked in the order of Salmonella enteritidis > enterotoxigenic E. coli > Pasteurella multocida. The high-dose Tibetan veterinary medicine compound preparation exhibited the greatest inhibitory rate against diarrhea in mice induced by oral administration of Salmonella enteritidis.

[0035] Table 4 Effects of different doses of veterinary drug compound preparations on diarrhea in mice induced by enterotoxigenic Escherichia coli after oral administration Table 5 Effects of different doses of veterinary drug compound preparations on diarrhea in mice administrated with Salmonella enteritidis Table 6 Effects of different doses of veterinary drug compound preparations on diarrhea in mice following oral administration of Pasteurella multocida Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0036] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A veterinary drug compound preparation, characterized in that: The veterinary compound preparation is prepared from the following raw materials in parts by weight: 100-200 parts of iron hammer, 100-200 parts of terminalia chebula, 100-200 parts of Tibetan woodruff, 100-200 parts of benzoin, 100-200 parts of pterocephala, 100-200 parts of rabbit ear grass, 100-200 parts of tsaoko, and 100-200 parts of rhodiola rosea.

2. The veterinary compound preparation according to claim 1, characterized in that: The veterinary compound preparation is prepared from the following raw materials in parts by weight: 200 parts of wormwood, 200 parts of terminalia chebula, 200 parts of Tibetan woodruff, 200 parts of benzoin, 200 parts of pterocephala, 200 parts of sedge, 200 parts of tsaoko, 200 parts of rhodiola rosea, and 200 parts of rhodiola rosea.

3. A method for preparing the veterinary drug compound preparation according to claim 1 or 2, characterized in that: The specific steps include: Weigh the following raw materials according to parts by weight: tibetan tsaoko, chebula chebula, styrax benzoin, pterygium wilfordii, sedge grass, tsaoko and rhodiola rosea; set aside; The raw materials were washed, dried and crushed, soaked in water and heated to boil for extraction, filtered through gauze, extracted twice, combined the extracts, and centrifuged to obtain the supernatant; The supernatant is heated, concentrated, and then dried to obtain a crude water extract, which is sterilized by ultraviolet light and prepared with sterile distilled water to prepare the veterinary drug compound preparation.

4. The method for preparing the veterinary drug compound preparation according to claim 3, wherein: 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 is to boil and heat the supernatant to concentrate to 180 mL to 220 mL, pour it into an evaporating dish, and heat it for further concentration.

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

7. Use of the veterinary 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, and the pathogenic bacteria are enterotoxigenic Escherichia coli, Salmonella enteritidis or Pasteurella multocida.

8. Use of the veterinary compound preparation according to claim 7 in the preparation of antibacterial drugs, characterized in that: The application subjects are diarrhea mice that are gavaged with the pathogenic bacteria.

9. Use of the veterinary compound preparation according to claim 1 in inhibiting pathogenic bacteria, characterized in that: The oral administration volume of the veterinary drug compound preparation shall not exceed 0.2 mL / 10 g body weight.

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