A Chinese veterinary medicine formula and its preparation method and application

Abstract

The present invention belongs to the technical field of traditional Chinese veterinary medicine, and relates to a traditional Chinese veterinary medicine formula, a preparation method and an application thereof. The traditional Chinese veterinary medicine formula is composed of the following raw materials: geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Licorice, and the weight ratio of each raw material is (2-4): (2-4): (1-3): (1-3): 1. The preparation method of the formula comprises the following steps: weighing geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Licorice in weight proportion and mixing them, refluxing extraction, collecting the extract, combining the extracts after repeated multiple times and vacuum filtering, concentrating the filtrate to 1g crude drug / mL, freeze drying, grinding into fine powder and sieving to obtain. The traditional Chinese veterinary medicine formula provided by the present invention is used for the prevention and treatment of intestinal diseases of livestock and poultry in animal husbandry, especially bacterial intestinal diseases caused by bacterial infection, and has antibacterial, anti-inflammatory and immune-enhancing effects.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese veterinary medicines, is applied to animal husbandry, and specifically relates to a traditional Chinese veterinary medicine formula, a preparation method and an application thereof. Background Art

[0002] At present, in line with the national strategic needs of healthy breeding and reducing and replacing antibiotics, the use of antibiotics, especially the use of antibiotics for both humans and animals, is gradually decreasing, and veterinary antibacterial drugs are being developed instead. Currently, most of the Chinese veterinary drugs available on the market are powders directly crushed from Chinese medicine, which have low bioavailability and poor animal absorption. In addition, the research on Chinese veterinary drugs sold on the market mainly focuses on the "antibacterial, antiviral, antiparasitic, and growth-promoting" aspects of modern veterinary medicine, ignoring the guiding role of Chinese veterinary medicine theory, and has not yet established a technical system for studying Chinese medicine according to the TCM symptom model. Therefore, in the current technological research and development environment, the research and application of Chinese veterinary drugs are separated from the theory of Chinese veterinary medicine, which can easily lead to the westernization of Chinese medicine.

[0003] Chinese veterinarians believe that animals are infected with diseases because of insufficient positive energy in the body, which allows evil energy to enter, or because evil energy in the body defeats the positive energy. The principle of Chinese medicine advocates supporting the positive energy and removing the evil energy, supporting the positive energy without leaving the evil energy, and removing the evil energy without hurting the positive energy. For example, gastrointestinal diseases caused by bacterial infection are often accompanied by acute symptoms such as vomiting and diarrhea, which will lead to the deficiency of positive energy. In addition to removing the external evil energy, Chinese medicine also uses methods such as tonifying qi and strengthening the spleen to strengthen the positive energy and promote the recovery of the disease. This formula is formulated under the guidance of Chinese medicine theory to study the development and application of antibacterial Chinese veterinary drugs. Summary of the invention

[0004] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, propose a Chinese veterinary medicine formula and its preparation method and application, provide a Chinese veterinary medicine formula composition including five Chinese medicinal raw materials and proportions, and also provide an extraction and preparation method of the formula, which is conducive to the full dissolution of effective ingredients, with a high yield and good effect; the formula is a Chinese veterinary medicine formula established under the background of "reducing resistance" and "replacing resistance" and has antibacterial and anti-inflammatory effects, and can enhance the immunity of livestock and poultry, which can help livestock and poultry defend and resist diseases.

[0005] The technical solution of the present invention is:

[0006] The present invention protects a Chinese veterinary medicine formula, which is composed of the following raw materials: geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Licorice, and the weight ratio of each raw material is (2-4): (2-4): (1-3): (1-3): 1.

[0007] Furthermore, the weight ratio of the geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Glycyrrhiza uralensis is (2.5-3.5):(2.5-3.5):(1.5-2.5):(1.5-2.5):1.

[0008] Furthermore, the weight ratio of the geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Glycyrrhiza uralensis is 3:3:2:2:1.

[0009] The present invention also protects the use of the Chinese veterinary medicine formula in the preparation of medicines for treating diseases caused by bacterial infections in livestock and poultry.

[0010] The present invention protects the use of the Chinese veterinary medicine formula in preparing medicines for preventing and treating intestinal diseases of livestock and poultry.

[0011] Furthermore, the drug is used to prevent and treat intestinal diseases of livestock and poultry, and the intestinal diseases are bacterial intestinal diseases.

[0012] Furthermore, the dosage form of the drug is powder, decoction, capsule, pill, granule, tablet or solution.

[0013] The present invention further protects a method for preparing the Chinese veterinary medicine formulation, comprising the following steps:

[0014] Weigh geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Glycyrrhiza uralensis in proportion by weight and mix them, add a certain amount of water, reflux extract at 90-110° C. for 100-140 min, collect the extract, add the same amount of water to the remaining medicinal residue, reflux extract at 90-110° C. for 100-140 min, repeat for multiple times, combine the extracts and perform vacuum filtration, concentrate the filtrate to 1 g crude drug / mL at 50-60° C., pre-freeze at -70--90° C. for 100-140 min, freeze-dry, grind into fine powder and sieve to obtain the product.

[0015] Furthermore, the water added during reflux extraction is distilled water, and the amount of water added is 15 to 25 times the weight of the raw material drug.

[0016] The above reflux extraction step can be performed two, three, four or more times according to actual needs.

[0017] Furthermore, the preparation method comprises the following steps: weighing geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and licorice in proportion by weight and mixing, extracting with 20 times the amount of distilled water at 100°C for 2 hours, collecting the extract, further extracting with 20 times the amount of distilled water at 100°C for 2 hours on the remaining residue, combining the two extracts and performing vacuum filtration, concentrating the filtrate by reduced pressure rotary evaporation at 55°C to obtain an extract with a concentration of 1 g crude drug / mL, pre-freezing in a -80°C refrigerator for two hours, drying in a freeze dryer, grinding into fine powder, and sieving.

[0018] Beneficial effects of the present invention:

[0019] (1) The Chinese veterinary medicine formula provided by the present invention is used for the prevention and treatment of intestinal diseases of livestock and poultry, especially bacterial intestinal diseases caused by bacterial infection, and has antibacterial, anti-inflammatory and immune-enhancing effects.

[0020] (2) The results of in vitro antibacterial experiments show that the formulation of the present invention has multi-drug resistance to the adopted bacteria, and the in vitro evaluation of its antibacterial effect shows that the inhibition rate reaches more than 95%, showing good in vitro activity; the results of in vivo anti-inflammatory experiments show that the formulation of the present invention reduces the release of inflammatory factors, and at the same time, it is confirmed by in vivo experiments that the formulation has the effect of treating colon inflammation pathology and can also improve spleen swelling caused by bacterial infection; the results of safety evaluation experiments show that the Chinese veterinary medicine formulation of the present invention has no obvious toxicity to mice, and the median lethal dose of the formulation is greater than 40.0 g / kg, which is safe for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The antibacterial results of the Chinese veterinary medicine formula provided in Experimental Example 1 on clinically isolated multidrug-resistant bacteria;

[0022] Figure 2 The spleen index of each group provided in Experimental Example 2; the figure indicates the significant difference between the model group and the blank group (P<0.05 # ; P<0.01 ## ); There were significant differences between each drug group and the model group (P<0.05*; P<0.01**);

[0023] Figure 3 The content of IL-6 and IL-1β, the inflammation-related factors provided in Experimental Example 2; the figure indicates the significant difference between the model group and the blank group (P<0.05 # ; P<0.01 ## ); There were significant differences between each drug group and the model group (P<0.05*; P<0.01**);

[0024] Figure 4 Inflammatory pathological analysis of the colon in the multidrug-resistant bacteria infection model provided for Experimental Example 2; in the figure, A: blank group, no obvious inflammatory changes were observed; B: model group, a large number of lymphocytes were observed to aggregate in the local submucosal layer to form lymphoid tissue (as shown by the black arrow); the thickness of the muscular layer was uneven (gray arrow); C: medication group 1, no obvious inflammatory changes were observed; D: medication group 2, no obvious inflammatory changes were observed; E: positive drug group, no obvious inflammatory changes were observed; F: antibiotic group, occasional focal infiltration of a small amount of inflammatory cells in the lamina propria (as shown by the black arrow), no other obvious abnormalities were observed;

[0025] Figure 5 Colon pathology score of the multidrug-resistant bacteria infection model provided in Experimental Example 2;

[0026] Figure 6 This is the classification result of the 38 bacterial species used in the antibacterial experiment of Experimental Example 1. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0029] The invention provides a Chinese veterinary medicine formula, which is composed of the following raw materials: geranium, viola yedoensis, atractylodes macrocephala, vitex rotundus and liquorice, wherein the weight ratio of each raw material is (2-4): (2-4): (1-3): (1-3): 1; preferably, the weight ratio of each raw material is (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5): 1.

[0030] It can be understood that any range or any specific numerical value within the above-mentioned weight ratio range is applicable to the present invention, for example, it can be any ratio such as 2:2:1:1:1 or 4:4:3:3:1 or 2:4:1:3:1 or 4:2:3:1:1 or 3:2:2:2:1 or 2:2:2:2:1 or 3:3:2:2:1.

[0031] Most preferably, the weight ratio of geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Glycyrrhiza uralensis is 3:3:2:2:1.

[0032] The preparation method of the Chinese veterinary medicine formula comprises the following steps:

[0033] Weigh geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Glycyrrhiza uralensis in the above weight proportion and mix them, add a certain amount of water, reflux extract at 90-110° C. for 100-140 min, collect the extract, add the same amount of water to the remaining medicinal residue, reflux extract at 90-110° C. for 100-140 min, repeat several times, combine the extracts and perform vacuum filtration, concentrate the filtrate at 50-60° C. to obtain an extract with a crude drug amount of 1 g crude drug / mL, pre-freeze at -70--90° C. for 100-140 min, freeze-dry, grind into fine powder and sieve to obtain the extract.

[0034] In the above preparation method, the temperature of reflux extraction is any value in the range of 90-110°C, for example, it can be any temperature of 90°C, 95°C, 98°C, 100°C, 102°C, 105°C, 110°C, etc. Other specific temperatures within this temperature range are also applicable.

[0035] The extraction time is any value within the range of 100 to 140 minutes, for example, it can be any time such as 105 minutes, 110 minutes, 116 minutes, 120 minutes, 124 minutes, 130 minutes, 135 minutes, etc. Other specific times within the time range can also be selected.

[0036] The concentration temperature is any value within the range of 50 to 60° C., for example, it can be any temperature of 50° C., 55° C., 58° C., 60° C., etc. Other specific temperatures within this temperature range are also applicable.

[0037] The freeze-drying temperature is any value within the range of -70 to -90°C, for example, it can be any temperature of -70°C, -80°C, -75°C, -85°C, -90°C, etc. Other specific temperatures within this temperature range are also applicable.

[0038] The freeze-drying time is any value within the range of 100 to 140 minutes, for example, it can be any time of 100 minutes, 110 minutes, 116 minutes, 120 minutes, 124 minutes, 130 minutes, 140 minutes, etc. Other specific times within the time range can also be selected.

[0039] The above reflux extraction step can be performed twice, three times, four times or more according to actual needs; the amount of water added in the reflux extraction is 15 to 25 times, preferably 20 times.

[0040] The above-mentioned Chinese veterinary medicine formula is used to prepare medicines for treating diseases caused by bacterial infections in livestock and poultry, and can also be used to prepare medicines for preventing and treating intestinal diseases in livestock and poultry; specifically, the medicine is used to prevent and treat intestinal diseases in livestock and poultry, and the intestinal diseases are bacterial intestinal diseases.

[0041] The drug can be in the form of powder, decoction, capsule, pill, granule, tablet or solution.

[0042] Example 1

[0043] A Chinese veterinary medicine formula comprises: 15 parts of geranium, 15 parts of Viola yedoensis, 10 parts of Atractylodes macrocephala, 10 parts of Vitex rotundifolia and 5 parts of Licorice.

[0044] The steps of the method for preparing a Chinese veterinary medicine formula are as follows:

[0045] Weigh 15 parts of Geranium herb, 15 parts of Viola yedoensis, 10 parts of Atractylodes macrocephala, 10 parts of Vitex rotundifolia, and 5 parts of Glycyrrhiza uralensis, mix them, use 20 times the amount of distilled water to reflux extract at 100°C for 2 h, collect the extract, and continue to use 20 times the amount of distilled water to reflux extract at 100°C for 2 h on the remaining residues. Combine the two extracts and perform vacuum filtration. The filtrate is concentrated by rotary evaporation at 55°C under reduced pressure to obtain an extract with a concentration of about 1 g of crude drug / mL. Pre-freeze it in a -80°C refrigerator for two hours, then put it into a freeze dryer for drying, grind it into fine powder, and sieve it.

[0046] The relevant experimental instruments used in the above preparation method include:

[0047] Reflux device: iron stand and double-mouth clamp, temperature-adjustable electric heating jacket, round-bottom flask, spherical condenser, rubber hose (for condensed water);

[0048] Filtration device: vacuum pump, filtration bottle, Buchner funnel, single-hole plug (to prevent air leakage), filter paper;

[0049] Rotary evaporation device: vacuum pump, desktop rotary evaporator, refrigerator;

[0050] Vacuum drying device: -80℃ refrigerator, vacuum dryer.

[0051] The application of the above-mentioned Chinese veterinary medicine formula is to prevent and treat bacterial intestinal diseases of livestock and poultry.

[0052] Example 2

[0053] A Chinese veterinary medicine formula comprises: 20 parts of geranium, 10 parts of Viola yedoensis, 15 parts of Atractylodes macrocephala, 5 parts of Vitex rotundifolia and 5 parts of Licorice.

[0054] The steps of the method for preparing a Chinese veterinary medicine formula are as follows:

[0055] Weigh 20 parts of Geranium, 10 parts of Viola yedoensis, 15 parts of Atractylodes macrocephala, 5 parts of Vitex rotundifolia, and 5 parts of Glycyrrhiza uralensis, mix them, use 20 times the amount of distilled water to reflux extract at 100°C for 2h, collect the extract, and continue to use 20 times the amount of distilled water to reflux extract at 100°C for 2h on the remaining residues. Repeat the extraction three times, combine the three extracts and perform vacuum filtration. The filtrate is concentrated by reduced pressure rotary evaporation at 55°C to obtain an extract with a concentration of about 1g crude drug / mL. Pre-freeze it in a -80°C refrigerator for two hours, then put it into a freeze dryer for drying, grind it into fine powder, and sieve it.

[0056] The application of the above-mentioned Chinese veterinary medicine formula is to prevent and treat bacterial intestinal diseases of livestock and poultry.

[0057] Example 3

[0058] A Chinese veterinary medicine formula comprises: 20 parts of geranium, 20 parts of Viola yedoensis, 15 parts of Atractylodes macrocephala, 15 parts of Vitex rotundifolia and 5 parts of Licorice.

[0059] The steps of the method for preparing a Chinese veterinary medicine formula are as follows:

[0060] Weigh 20 parts of Geranium Herb, 20 parts of Viola Yedoensis, 15 parts of Atractylodes Macrocephala, 15 parts of Vitex Marigoldensis, and 5 parts of Licorice, mix them, use 20 times the amount of distilled water to reflux extract at 95°C for 130 min, collect the extract, and continue to use 20 times the amount of distilled water to reflux extract at 100°C for 110 min on the remaining residues. Combine the two extracts and perform vacuum filtration. The filtrate is concentrated by rotary evaporation at 60°C under reduced pressure to obtain an extract with a concentration of about 1 g of crude drug / mL. Pre-freeze it in a -80°C refrigerator for two hours, then put it into a freeze dryer for drying, grind it into fine powder, and sieve it.

[0061] The application of the above-mentioned Chinese veterinary medicine formula is to prevent and treat bacterial intestinal diseases of livestock and poultry.

[0062] Example 4

[0063] A Chinese veterinary medicine formula comprises: 20 parts of geranium, 20 parts of Viola yedoensis, 20 parts of Atractylodes macrocephala, 20 parts of Vitex rotundifolia and 10 parts of Licorice.

[0064] The steps of the method for preparing a Chinese veterinary medicine formula are as follows:

[0065] Weigh 20 parts of Geranium, 20 parts of Viola yedoensis, 20 parts of Atractylodes macrocephala, 20 parts of Vitex rotundifolia, and 10 parts of Glycyrrhiza uralensis, mix them, use 20 times the amount of distilled water to reflux extract at 100°C for 2 h, collect the extract, and continue to use 20 times the amount of distilled water to reflux extract at 100°C for 2 h on the remaining residues. Combine the two extracts and perform vacuum filtration. The filtrate is concentrated by rotary evaporation at 55°C under reduced pressure to obtain an extract with a concentration of about 1 g of crude drug / mL. Pre-freeze at -90°C for 100 min, put into a freeze dryer for drying, grind into fine powder, and sieve.

[0066] The application of the above-mentioned Chinese veterinary medicine formula is to prevent and treat bacterial intestinal diseases of livestock and poultry.

[0067] Experimental Example 1 In vitro antibacterial test

[0068] The traditional Chinese medicines with antibacterial effects in the formula have all undergone in vitro antibacterial screening, and the bacterial species used are all clinically isolated species, which have been tested to have multidrug resistance. The formula targets resistance genes to ensure clinical effectiveness.

[0069] The specific experimental process is as follows:

[0070] 1.1 Experimental strains

[0071] All the strains to be tested in the experiment were stored at -20℃. The strains were activated before use. After the culture medium was prepared, it was placed in a sterilizer for high temperature and high pressure sterilization. 15-20mL of culture medium was drawn into a sterilized culture dish in a clean bench. After cooling and solidification, the culture liquid was inoculated into the culture medium with an inoculation loop. The culture dish was sealed with a sealing strip and placed in a 37℃ constant temperature biochemical incubator for about 20 hours. After all the strains to be tested in the experiment were activated, each colony was picked up with an inoculation loop and placed in a transparent test tube filled with sterile water. After shaking well, it was configured into a No. 1.0 McFarland turbidimetric tube (3×10 8 ) and dilute to 1×10 8 , and then diluted 1:1000 to 1×10 5 CFU·mL-1. 38 kinds of bacterial suspensions were prepared for use. The classification results of these 38 kinds of bacteria are as follows Figure 6 shown.

[0072] The solid culture medium used in the experiment was configured as follows: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 20 g agar per 1 L of culture medium. The liquid culture medium was configured as follows: 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride per 1 L of culture medium. Solid culture medium was used in the culture dishes, and liquid culture medium was used in the 96-well plates.

[0073] 1.2 Experimental methods

[0074] Determination of inhibition zone: The diameter of the inhibition zone is determined using the Oxford cup method. Before the experiment, the culture dish is placed in a clean bench for ultraviolet irradiation sterilization. Next, pour the autoclaved culture medium and wait for it to cool and solidify. Take 100μL of the prepared bacterial suspension and use a coating rod to evenly apply it on the solid culture medium. Press the Oxford cup to fit it with the culture dish, add 140μL of the drug solution to the Oxford cup, and add water extract to the upper and lower holes of the four Oxford cup holes in each culture dish, and add alcohol extract to the left and right holes. After adding the drug, seal it with a sealing strip, mark the strain and the name of the traditional Chinese medicine, place it in a 37℃ constant temperature biochemical incubator for 18 to 20 hours, and observe the results. If an inhibition zone appears, measure its diameter and record it.

[0075] Minimum inhibitory concentration (MIC): two-fold dilution method: Take a 96-well plate, set the first to eighth columns as drug groups, the ninth column as blank control, and the tenth column as bacterial solution control. The drug stock solution concentration is 200mg / ml, and three replicate wells are set. The specific operation steps are as follows: First, add culture solution. Add 50μL of culture solution to each well in the first to eighth columns, and add 25μL to the eighth and ninth columns. Next, add drugs to the drug group and dilute twice. Add 50μL of drug stock solution to the first column, mix evenly, and then dilute to the eighth column in turn (that is, after the first column is mixed evenly, draw 50μL from the first column and add it to the second column, mix evenly again, draw 50μL from the second column and add it to the third column, and continue to dilute down like this), draw 50μL from the eighth column and discard it to ensure that the total liquid volume is the same. The concentrations of the first to eighth columns after dilution are 200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625mg / ml, respectively. Add 25 μL of sterile water to the ninth and tenth columns. Finally, add the bacterial solution. Add 50 μL of the prepared bacterial solution to the first to eighth and tenth columns. Add 50 μL of sterile water to the ninth column. After the operation, cover the 96-well plate, shake and mix, and culture at 37°C for 16 to 20 hours to observe whether the bacteria grow at each concentration of the drug solution. If there is bacterial growth, the wells will be turbid. If there is no bacterial growth, the wells will be clear. The concentration of the last well with clear color is recorded as the minimum inhibitory concentration.

[0076] Minimum bactericidal concentration (MBC): After the MIC is measured by the two-fold dilution method, the mixed solution in the small hole without turbidity is sucked out and injected into the culture dish. Use a coating rod to spread it evenly and seal it. The name of the strain and the drug concentration of the culture dish are recorded in detail on the cover of the culture dish. Place it in a 37℃ incubator and culture it for 24 hours. Observe whether there is colony growth in the plate, and the minimum concentration at which there is no colony growth in each strain is regarded as the minimum bactericidal concentration (MBC) of the Chinese medicine extract.

[0077] 1.3 Experimental Results

[0078] The experiment used the agar punch method to determine the diameter of the inhibition zone, and the two-fold dilution method to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) to evaluate the antibacterial effect of the formula. The in vitro evaluation of the antibacterial effect of the formula showed that the inhibition rate reached more than 95%. Figure 1 As shown, the size of the inhibition zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the formula against clinically isolated pathogenic bacteria demonstrate good in vitro activity and ensure bioavailability.

[0079] Experimental Example 2 In vivo anti-inflammatory experiment

[0080] Shigella infection leads to rapid death of macrophages, accompanied by the release of a large number of inflammatory factors, and its clinical symptoms are acute inflammatory bowel disease, with the main pathological manifestations in the colon, with a large number of inflammatory cell infiltration and tissue edema, and even infiltration and destruction of the intestinal epithelium. In vivo experimental results have shown that the prescription of the present invention has the effect of reducing the release of colon inflammatory factors and improving the pathological state of the colon. In addition, the prescription can improve spleen swelling caused by bacterial infection.

[0081] The specific experimental process is as follows:

[0082] 2.1 Experimental animals

[0083] The experiment used 30 BALB / c mice, weighing 20±2g. The animals were fed adaptively for 4 days. After observation, healthy mice were randomly divided into groups for the experiment. The mice were divided into 6 groups, including blank group, model group, amikacin positive drug group (antibiotic group), positive drug group (Liancan Zhili Granule group), low concentration group (drug group 1), and high concentration group (drug group 2), with 5 mice in each group, and each group was raised separately. They were fed with standard feed and high-temperature sterilized distilled water.

[0084] 2.2 Experimental methods

[0085] The experimental period was 7 days, and the medication time was 7 days. The medication regimen was as follows: the dosage of group 1 was 1g / kg of the formula; the dosage of group 2 was 2g / kg of the formula; the dosage of the Liancan Zhili Granule group was 4g / kg of Liancan Zhili Granule; the dosage of the antibiotic group was 2mg / kg of amikacin; the blank group and the model group were given an equal amount of normal saline.

[0086] All groups were gavaged at a dose of 0.1 mL / 10 g and fasted for 12 h before gavage. On the third day, the intestinal inflammation model caused by pathogenic bacteria was established by intraperitoneal injection of 0.1 mL / 10 g bacterial suspension (1×10 7 After the experiment ended on the 8th day, the mice were killed after anesthesia and blood was taken, and samples were taken. Chloral hydrate solution was used for anesthesia during sampling. Preparation of chloral hydrate (400 mg / kg): Dissolved into chloral hydrate solution at a concentration of 100 mg / mL, and the intraperitoneal injection volume was 40 μL / 10g.

[0087] Processing of test results: (1) Determination of cytokine levels: After blood is collected from the abdominal aorta, let it stand at room temperature for 1 hour, centrifuge it for 10 minutes (4°C, 2000rpm), and obtain serum; use ELISA kit to determine the serum cytokine content; finally, use an enzyme marker to measure the absorbance value at 490nm to determine the cytokine level. (2) Calculation of spleen index: After the experiment, immediately remove the spleen and weigh it, and calculate the organ index (%) according to the following formula: Index = organ weight (mg) / body weight (g). (3) Detection of pathological sections: The sample is fixed with 4% paraformaldehyde. After the fixation is in good condition, it is trimmed, dehydrated, embedded, sliced, stained, sealed, and finally the qualified samples are examined under a microscope.

[0088] 2.3 Experimental Results

[0089] 2.3.1 Spleen index and cytokine detection

[0090] Pathogenic bacteria infection can lead to lesions and damage to multiple organs. The spleen index can reflect the bleeding and swelling of the spleen. As an immune organ, the spleen can indirectly reflect the impact on the body's immunity. Figure 2 As shown, the spleen index increased significantly after modeling, and this situation was improved after drug administration, which indicated that the formula could improve spleen damage caused by bacterial infection.

[0091] Pathogenic bacteria infection can lead to the secretion of inflammatory cytokines IL-1β and IL-6. ELISA detection of inflammatory cytokines in serum showed that compared with the blank group, the content of inflammatory factors in the model group was higher than the normal level, and the release of inflammatory factors was reduced after administration. The content of inflammatory-related factors IL-6 and IL-1β in each group is shown in Figure 2. Figure 3 shown.

[0092] 2.3.2 Colon pathological histological observation

[0093] Colon pathological analysis of the multidrug-resistant bacteria infection model showed that the Chinese veterinary medicine formula of the present invention had a significant therapeutic effect on it, and the pathological scores were as follows: Figure 5 shown. Figure 4 The pathological section results are shown, and the specific pathological manifestations are as follows:

[0094] The results of the blank group showed (see Figure 4 A): The structures of each layer of colon tissue are clear, the single-layer columnar epithelial cells of the mucosa have regular morphology, the intestinal glands in the lamina propria are closely arranged, the goblet cells are abundant, and no obvious folds are observed; the connective tissue of the submucosal layer is fine and dense; the muscular layer is thin, and there is no obvious abnormality in the muscle fibers, and no obvious inflammatory changes are observed.

[0095] The results of the model group showed (see Figure 4B): The structures of each layer of colon tissue are clear, the single-layer columnar epithelial cells of the mucosa have regular morphology, the intestinal glands in the lamina propria are closely arranged, the goblet cells are abundant, the local mucosal layer bulges into the intestinal cavity to form folds; the connective tissue of the submucosal layer is dense, and a large number of lymphocytes can be seen gathering in the local submucosal layer to form lymphoid tissue; the thickness of the muscle layer is uneven.

[0096] The results of group 1 showed (see Figure 4 C): The structures of each layer of colon tissue are clear, the single-layer columnar epithelial cells of the mucosa have regular morphology, the intestinal glands in the lamina propria are closely arranged, the goblet cells are abundant, the local mucosal layer bulges into the intestinal cavity to form folds; the connective tissue of the submucosal layer is fine and dense; the thickness of the muscle layer is uneven, and no obvious inflammatory changes are observed.

[0097] The results of the two groups showed that (see Figure 4 D): The structures of each layer of colon tissue are clear, the single-layer columnar epithelial cells of the mucosa have regular morphology, the intestinal glands in the lamina propria are closely arranged, the goblet cells are abundant, the local mucosal layer bulges into the intestinal cavity to form folds; the connective tissue of the submucosal layer is fine and dense; no obvious inflammatory changes are observed.

[0098] The results of the positive drug group showed (see Figure 4 E): The structures of each layer of colon tissue are clear, the single-layer columnar epithelial cells of the mucosa have regular morphology, the intestinal glands in the lamina propria are closely arranged, the goblet cells are abundant, the local mucosal layer bulges into the intestinal cavity to form folds; the connective tissue of the submucosal layer is fine and dense; no obvious inflammatory changes are observed.

[0099] The results of the antibiotic group showed (see Figure 4 F): Occasionally, a small amount of inflammatory cell focal infiltration was observed in the lamina propria of colon tissue, but no other obvious abnormalities were observed.

[0100] Experimental Example 3 Safety Evaluation Experiment

[0101] There is a saying among the people that "medicine is three-point poisonous". In order to dispel this concern, more accurately evaluate the safety of this Chinese veterinary medicine formula, and provide experimental basis for the efficacy and safe use in animal husbandry in the future, an acute toxicity test was conducted in this experimental example. The observation results showed that no mice died in each medication group, the mental state of each group of mice was good, and there was no significant difference between the medication group and the blank group (Table 1). The anatomical results showed that the morphology and color of each organ were normal, and there was no obvious toxic change. Therefore, the median lethal dose of this prescription is greater than 40.0g / kg, and it is safe for clinical use.

[0102] The specific process of acute toxicity test is as follows:

[0103] 3.1 Experimental animals

[0104] The test used SPF grade Kunming mice, weighing 18.0-22.0 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0011. The mice were fed in separate cages, fed with standard feed and high-temperature sterilized distilled water, and adaptively fed for 7 days. After observation, healthy mice were selected for acute toxicity tests.

[0105] 3.2 Test methods

[0106] 3.2.1 Determination of LD50 of Chinese veterinary drug formula in mice

[0107] The experiment set up 5 groups, namely the drug test groups which were gavaged with 40.0g / kg (drug test group 1), 20.0g / kg (drug test group 2), 10.0g / kg (drug test group 3), 5.0g / kg (drug test group 4) and the blank group which was gavaged with 0.8mL / 20g normal saline.

[0108] Take 50 SPF Kunming mice (18g-22g), half male and half female. Fast for 12h before administration but not water overnight. On the day of the experiment, the drug test group was gavaged with the corresponding concentration of the formula, and the maximum dose of 0.4mL / 10g mouse was gavaged, and the blank group was gavaged with 0.8mL of normal saline. Fast for 3-4 hours after gavage, feed normally after the drug is fully absorbed, and continue fasting for 12h before the next gavage. Observe the death of mice and abnormal changes in the physiological activities of mice for 7 consecutive days.

[0109] Observation index: During the administration period, observe the mice for poisoning at any time and record the clinical manifestations of the mice in time. If there are dead mice, record the number of dead mice and the corresponding drug concentration and perform dissection in time to observe whether there are changes in various organs. The experimental results refer to the guidelines for acute toxicity tests of veterinary drugs (LD50 determination) and determine the LD50 value based on the death of mice. If mice die, the LD50 is obtained according to the experimental results; if there is no death or other abnormalities in the mice in each group after 7 days, and the LD50 cannot be obtained, the maximum tolerance test is carried out.

[0110] 3.2.2 Determination of the maximum tolerated dose (MTD) of Chinese veterinary drug formulations in mice

[0111] Twenty SPF Kunming mice (weight 18-22g) were randomly divided into two groups, drug test group and blank group, with 10 mice in each group, half male and half female. Animals were fasted but not watered for 12 hours before the experiment. The drug test group was gavaged twice at 40g / kg per mouse within 24 hours, with an interval of 10 hours (9:00 and 19:00). Mice in the blank group were gavaged with an equal volume of normal saline. After administration, they were free to eat and drink, and were observed for 7 consecutive days.

[0112] Observation indicators: record the clinical symptoms of mice after administration, and observe whether there are any abnormalities in the eyes, stool and autonomous activities of mice. If mice die within 7 days, perform autopsy in time and record the autopsy results. The mice were killed on the 7th day of the experiment, and their main organs such as heart, liver, spleen, lung, and kidney were observed by dissection. The drug test group was compared with the blank group to observe whether there were any abnormalities in the organs of mice in the drug test group. According to the "Technical Guidelines for Acute Toxicity of Chemical Drugs", if the mice are still alive, the entire acute toxicity test can be terminated.

[0113] 3.2.3 Test results

[0114] Five groups of mice were given a single oral administration every day and observed for seven days. The observation results within seven days showed that no mice died in each administration group, the mental state of each group of mice was good, and there was no significant difference between the administration group and the blank group. The results are shown in Table 1 below. The autopsy results after seven days showed that the morphology and color of each organ were normal, and there was no obvious toxic change. Therefore, the median lethal dose of this Chinese veterinary drug formula is greater than 40.0g / kg.

[0115] Table 1 Mortality of mice in acute toxicity test of the formula

[0116]

[0117] In the maximum tolerated dose experiment, both the mice in the drug test group and the blank group were normal and there was no death, and there were no abnormalities in physiological activities and various signs (pupils, eyes, skin color, breathing, secretion, etc.). After the experiment, the mice were weighed, and the results are shown in Table 2 below. The t-test was performed on the weight of male and female mice using SPSS software. The results showed that there was no significant difference between the female drug test group and the blank group, and there was no significant difference between the male drug test group and the blank group.

[0118] Table 2 Body weight of mice on day 8 of the maximum tolerated dose test

[0119] Group gender Number of mice Mouse weight Drug trial group ♂ 5 26.42±2.94 Drug experimental group ♀ 5 25.23±2.91 Blank Group ♂ 5 26.09±0.89 Blank Group ♀ 5 23.43±2.70

[0120] The results show that the LD50 and MTD concentration of the Chinese veterinary medicine formula of the present invention for mice were 40g / kg and there was no death. The LD50 and MTD of the formula were both greater than 50mg / 10g. Therefore, it can be determined that the Chinese veterinary medicine formula is non-toxic.

[0121] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Chinese veterinary medicine formula, characterized in that: The Chinese veterinary medicine formula is composed of the following raw materials: geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Licorice, and the weight ratio of each raw material is (2~4):(2~4):(1~3):(1~3):

1.

2. The Chinese veterinary medicine formulation according to claim 1, characterized in that: The weight ratio of the geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Licorice is (2.5-3.5):(2.5-3.5):(1.5-2.5):(1.5-2.5):

1.

3. The Chinese veterinary medicine formulation according to claim 1, characterized in that: The weight ratio of the geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Licorice is 3:3:2:2:

1.

4. Use of the Chinese veterinary medicine formula according to any one of claims 1 to 3 in the preparation of medicines for preventing and treating bacterial intestinal diseases of livestock and poultry.

5. The use according to claim 4, characterized in that: The dosage form of the medicine is powder, decoction, capsule, pill, granule or tablet.

6. A method for preparing the Chinese veterinary medicine formulation according to any one of claims 1 to 3, characterized in that: The following steps are involved: Weigh geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Glycyrrhiza uralensis in proportion by weight and mix them, add a certain amount of water and reflux extract at 90-110°C for 100-140 min, collect the extract, add the same amount of water to the remaining medicinal residue and reflux extract at 90-110°C for 100-140 min, repeat several times, combine the extracts and perform vacuum filtration, concentrate the filtrate at 50-60°C to 1 g crude drug / mL, pre-freeze at -70- -90°C for 100-140 min, freeze-dry, grind into fine powder and sieve to obtain the product.

7. The preparation method according to claim 6, characterized in that: The water added during reflux extraction is distilled water, and the amount of water added is 15 to 25 times the weight of the raw material.

8. The preparation method according to claim 7, characterized in that: The following steps are involved: Weigh geranium, Viola yedoensis, Atractylodes macrocephala, Vitex rotundifolia and Glycyrrhiza uralensis in proportion by weight and mix them, use 20 times amount of distilled water to reflux extract at 100℃ for 2 h, collect the extract, and continue to use 20 times amount of distilled water to reflux extract at 100℃ for 2 h on the remaining residues. Combine the two extracts and perform vacuum filtration. The filtrate is concentrated by rotary evaporation at 55℃ under reduced pressure to obtain an extract with a concentration of 1 g crude drug / mL. Pre-freeze it in a -80℃ refrigerator for two hours, then put it into a freeze dryer for drying, grind it into fine powder, and sieve it.

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