CN116019865A - Traditional Chinese veterinary medicine formula and preparation method and application thereof - Google Patents
Traditional Chinese veterinary medicine formula and preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
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Abstract
The invention belongs to the technical field of traditional Chinese veterinary medicines, and relates to a traditional Chinese veterinary medicine formula, a preparation method and application thereof, wherein the traditional Chinese veterinary medicine formula comprises the following raw medicines: the weight portions of the raw materials of geranium, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice are (2-4): (2-4): (1-3): (1-3): 1. the preparation method of the formula comprises the following steps: weighing herba Erodii seu Geranii, herba Violae, atractylodis rhizoma, fructus Vitics Simplicifoliae and Glycyrrhrizae radix according to weight ratio, mixing, reflux extracting, collecting extractive solution, repeating for several times, mixing extractive solutions, vacuum filtering, concentrating filtrate to 1g crude drug/mL, lyophilizing, grinding into fine powder, and sieving. The veterinary drug formula provided by the invention is used for preventing and treating intestinal diseases of livestock and poultry in animal husbandry, particularly bacterial intestinal diseases caused by bacterial infection, and has the functions of resisting bacteria and inflammation and improving immunity.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese veterinary medicines, and is applied to animal husbandry, in particular relates to a traditional Chinese veterinary medicine formula, a preparation method and application thereof.
Background
The traditional Chinese veterinary medicines in the market are mostly powder which directly pulverizes the traditional Chinese medicines, the bioavailability is low, the animal absorption is poor, the research of the traditional Chinese veterinary medicines in the market is mainly focused on the aspects of 'antibiosis, antivirus, antiparasitic, growth promotion' and the like of modern veterinary medicine, the guiding effect of the pharmacology theory of the traditional Chinese veterinary medicine is ignored, and the technical system for researching the traditional Chinese medicines according to the traditional Chinese medicine symptom model is not established yet. Therefore, under the research and development environment of the current technology, the research and application of traditional Chinese veterinary medicines are separated from the traditional Chinese veterinary medicine theory, and the Western traditional Chinese medicines are easily caused.
Veterinarians consider that animal infection diseases are caused by deficiency of healthy qi in the body and deficiency of pathogenic qi in the body or by pathogenic qi in the body defeating healthy qi; the Chinese medicine is used for treating the disease by maintaining healthy qi, removing pathogenic qi, strengthening healthy qi without retaining pathogenic factors, and eliminating pathogenic factors without damaging healthy qi. For example, gastrointestinal diseases caused by bacterial infection are often accompanied with acute symptoms such as vomiting and diarrhea, which can cause deficiency of healthy qi, and the traditional Chinese medicine treatment diseases are supplemented with treatment methods such as tonifying qi and strengthening spleen besides eliminating exogenous evils so as to strengthen healthy qi and promote the recovery of the diseases. The formula is prepared under the guidance of the theory of traditional Chinese medicine, and the development and application of the antibacterial traditional Chinese veterinary medicine are researched.
Disclosure of Invention
The invention aims to solve the problems in the prior art, provides a traditional Chinese veterinary medicine formula, a preparation method and application thereof, provides a traditional Chinese veterinary medicine formula composition comprising five traditional Chinese medicine raw materials and proportions, and simultaneously provides an extraction preparation method of the formula, which is beneficial to full dissolution of active ingredients, and has higher yield and better effect; the formula is a traditional Chinese veterinary medicine formula which has antibacterial and anti-inflammatory effects and can enhance the immunity of livestock and poultry, and can help the livestock and poultry to defend and resist diseases.
The technical scheme of the invention is as follows:
the invention provides a traditional Chinese veterinary medicine formula, which consists of the following raw materials: the weight portions of the raw materials of geranium, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice are (2-4): (2-4): (1-3): (1-3): 1.
further, the weight parts ratio of the geranium wilfordii, the viola philippica, the bighead atractylodes rhizome, the fructus viticis and the liquorice is (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5): 1.
further, the weight parts of the geranium wilfordii, the viola yedoensis, the bighead atractylodes rhizome, the fructus viticis and the liquorice are 3:3:2:2:1.
the invention also protects the application of the traditional Chinese veterinary medicine formula in preparing medicines for treating diseases caused by bacterial infection of livestock and poultry.
The invention protects application of the traditional Chinese veterinary medicine formula in preparing medicines for preventing and treating intestinal diseases of livestock and poultry.
Furthermore, the medicine is used for preventing and treating intestinal diseases of livestock and poultry, wherein the intestinal diseases are bacterial intestinal diseases.
Further, the dosage form of the medicine is powder, decoction, capsule, pill, granule, tablet or solution.
The invention also provides a preparation method of the veterinary drug formula, which comprises the following steps:
weighing and mixing geranium wilfordii, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice according to the weight part ratio, adding a certain amount of water, carrying out reflux extraction for 100-140 min at 90-110 ℃, collecting an extracting solution, continuously adding the same amount of water into the residue, carrying out reflux extraction for 100-140 min at 90-110 ℃, repeating for many times, combining the extracting solutions, carrying out vacuum suction filtration, concentrating filtrate to 1g crude drug/mL at 50-60 ℃, pre-freezing for 100-140 min at-70 to-90 ℃, freeze-drying, grinding into fine powder, and sieving to obtain the medicine.
Further, the water added during reflux extraction is distilled water, and the amount of the added water is 15-25 times of the weight of the raw material medicine.
The reflux extraction step can be performed twice or three times, four times or more according to actual needs.
Further, the preparation method comprises the following steps: weighing and mixing geranium wilfordii, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice according to the weight part ratio, reflux-extracting for 2 hours at 100 ℃ by using 20 times of distilled water, collecting extracting solution, reflux-extracting the residual dregs for 2 hours at 100 ℃ by using 20 times of distilled water, combining the extracting solutions twice, vacuum-filtering, concentrating the filtrate by decompression rotary evaporation at 55 ℃ to obtain extract with the concentration of 1g crude drug/mL, pre-freezing for two hours in a refrigerator at-80 ℃, drying in a freeze dryer, grinding into fine powder, and sieving.
The invention has the beneficial effects that:
(1) The veterinary drug formula provided by the invention is used for preventing and treating intestinal diseases of livestock and poultry in animal husbandry, particularly bacterial intestinal diseases caused by bacterial infection, and has the functions of resisting bacteria and inflammation and improving immunity.
(2) According to the results of in vitro antibacterial experiments, the formula has multiple drug resistance to the adopted strains, and in vitro evaluation of the antibacterial effect shows that the inhibition rate reaches more than 95%, and better in vitro activity is shown; according to the in vivo anti-inflammatory experimental result, the formula reduces the release of inflammatory factors, and meanwhile, the in vivo experiment proves that the formula has the effect of treating colon inflammation pathology and can also improve spleen swelling caused by bacterial infection; the safety evaluation experiment result shows that the veterinary drug formula has no obvious toxicity to mice, the half lethal dose of the formula is more than 40.0g/kg, and the clinical use is safe.
Drawings
Fig. 1 is a bacteriostatic result of the veterinary drug formulation provided in experimental example 1 on clinically isolated multi-drug resistant bacteria;
FIG. 2 is a graph showing spleen index of each group provided in Experimental example 2; significant differences between model and blank groups are identified in the figure (P<0.05 # ；P<0.01 ## ) The method comprises the steps of carrying out a first treatment on the surface of the Significant differences between each drug group and model group (P<0.05*；P<0.01**)；
FIG. 3 shows the IL-6 content and IL-1β content of the inflammatory-related factors provided in Experimental example 2; significant differences between model and blank groups are identified in the figure (P<0.05 # ；P<0.01 ## ) The method comprises the steps of carrying out a first treatment on the surface of the Significant differences between each drug group and model group (P<0.05*；P<0.01**)；
FIG. 4 is an inflammatory pathology analysis of the colon of the multi-drug resistant bacteria infection model provided in Experimental example 2; in the figure, a: blank, no significant inflammatory changes were seen; b: in the model group, a large number of lymphocytes can be gathered in the local submucosa to form lymphoid tissues (shown by black arrows); uneven muscular layer thickness (grey arrow); c: group 1 administration, no significant inflammatory changes were seen; d: group 2 administration, no significant inflammatory changes were seen; e: positive drug groups, no significant inflammatory changes were seen; f: antibiotic group, occasional small inflammatory focal infiltration of lamina propria (shown by black arrow), without other obvious abnormalities;
FIG. 5 is a colon pathology score of a multi-drug resistant bacterial infection model provided in Experimental example 2;
FIG. 6 shows classification results of 38 species used in the antibacterial test of Experimental example 1.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
For a further understanding of the present invention, reference will now be made to the drawings and examples.
The invention provides a traditional Chinese veterinary medicine formula which is prepared from the following raw materials: the weight portions of the raw materials of geranium, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice are (2-4): (2-4): (1-3): (1-3): 1, a step of; preferably, the weight portion ratio of each raw material medicine is (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5): 1.
it is understood that any one of the ranges or any specific value within the above ranges of the respective parts by weight ratio are applicable to the present invention, and may be, for example, 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, etc.
Most preferably, the weight parts ratio of geranium, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice is 3:3:2:2:1.
the preparation method of the traditional Chinese veterinary medicine formula comprises the following steps:
weighing and mixing geranium wilfordii, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice according to the weight part ratio, adding a certain amount of water, carrying out reflux extraction for 100-140 min at the temperature of 90-110 ℃, collecting an extracting solution, continuously adding the same amount of water into the residue, carrying out reflux extraction for 100-140 min at the temperature of 90-110 ℃, repeating for many times, combining the extracting solutions, carrying out vacuum filtration, concentrating the filtrate at the temperature of 50-60 ℃ to obtain an extract with the crude drug content of 1g crude drug/mL, pre-freezing for 100-140 min at the temperature of-70 to-90 ℃, freeze-drying, grinding into fine powder, and sieving to obtain the drug.
In the above preparation method, the temperature of the reflux extraction is any value in the range of 90 to 110 ℃, and for example, it may be any temperature of 90 ℃, 95 ℃, 98 ℃, 100 ℃, 102 ℃, 105 ℃, 110 ℃ or the like, and other specific temperatures in the temperature range are also applicable.
The extraction time is any value within the range of 100 to 140 minutes, for example, any time of 105 minutes, 110 minutes, 116 minutes, 120 minutes, 124 minutes, 130 minutes, 135 minutes, and the like, and other specific times within the time range can be selected.
The concentration temperature may be any value within the range of 50 to 60 ℃, and may be, for example, any temperature within the range of 50 ℃, 55 ℃, 58 ℃, 60 ℃ and the like, and other specific temperatures within the range of the temperature may be applicable.
The freeze-drying temperature is any value within the range of-70 to-90 ℃, for example, any temperature of-70 ℃, -80 ℃, -75 ℃, -85 ℃, -90 ℃ and the like can be used, and other specific temperatures within the temperature range are also applicable.
The freeze-drying time may be any value within the range of 100 to 140 minutes, and may be any time of, for example, 100 minutes, 110 minutes, 116 minutes, 120 minutes, 124 minutes, 130 minutes, 140 minutes, and the like, and other specific times within the time range may be selected.
The reflux extraction step can be performed for two or three times, four times or more according to actual requirements; the amount of water added in the reflux extraction is 15 to 25 times, preferably 20 times, the amount of water.
The traditional Chinese veterinary medicine formula is used for preparing medicines for treating diseases caused by bacterial infection of livestock and poultry, and can also be used for preparing medicines for preventing and treating intestinal diseases of livestock and poultry; in particular, the medicine is used for preventing and treating intestinal diseases of livestock and poultry, wherein the intestinal diseases are bacterial intestinal diseases.
The application dosage form of the medicine is any one of powder, decoction, capsule, pill, granule, tablet or solution.
Example 1
A traditional Chinese veterinary medicine formula comprises the following components: 15 parts of geranium, 15 parts of viola philippica, 10 parts of bighead atractylodes rhizome, 10 parts of fructus viticis and 5 parts of liquorice.
The preparation method of the traditional Chinese veterinary medicine formula comprises the following steps:
weighing 15 parts of geranium, 15 parts of viola philippica, 10 parts of bighead atractylodes rhizome, 10 parts of fructus viticis and 5 parts of liquorice, mixing, reflux-extracting for 2 hours with 20 times of distilled water at 100 ℃, collecting the extracting solution, reflux-extracting the residual dregs with 20 times of distilled water at 100 ℃ for 2 hours, combining the two extracting solutions, vacuum-filtering, concentrating the filtrate by reduced pressure rotary evaporation at 55 ℃ to obtain extract with the concentration of about 1g crude drug/mL, pre-freezing for two hours in a refrigerator at-80 ℃, drying in a freeze dryer, grinding into fine powder, and sieving.
The related experimental instrument adopted by the preparation method comprises the following steps:
and a reflow device: iron stand, double-mouth clamp, temperature-regulating electric heating sleeve, round bottom flask, spherical condenser tube and rubber tube (for condensed water);
and (3) a suction filtration device: vacuum pump, suction flask, buchner funnel, shan Kongsai (to prevent air leakage), filter paper;
rotary evaporation device: a vacuum pump, a table-type rotary evaporator and a refrigerator;
and (3) a vacuum drying device: -80 ℃ refrigerator, vacuum dryer.
The application of the traditional Chinese veterinary medicine formula is as follows: can be used for preventing and treating bacterial intestinal diseases of livestock and fowl.
Example 2
A traditional Chinese veterinary medicine formula comprises the following components: 20 parts of geranium, 10 parts of herba violae, 15 parts of bighead atractylodes rhizome, 5 parts of fructus viticis and 5 parts of liquorice.
The preparation method of the traditional Chinese veterinary medicine formula comprises the following steps:
weighing 20 parts of geranium, 10 parts of viola philippica, 15 parts of bighead atractylodes rhizome, 5 parts of fructus viticis and 5 parts of liquorice, mixing, reflux-extracting for 2 hours with 20 times of distilled water at 100 ℃, collecting the extracting solution, reflux-extracting the residual dregs with 20 times of distilled water at 100 ℃ for 2 hours, repeatedly extracting for three times, combining the three extracting solutions, vacuum-filtering, concentrating the filtrate by vacuum rotary evaporation at 55 ℃ to obtain extract with the concentration of about 1g crude drug/mL, pre-freezing for two hours in a refrigerator at-80 ℃, drying in a freeze dryer, grinding into fine powder, and sieving.
The application of the traditional Chinese veterinary medicine formula is as follows: can be used for preventing and treating bacterial intestinal diseases of livestock and fowl.
Example 3
A traditional Chinese veterinary medicine formula comprises the following components: 20 parts of geranium, 20 parts of viola philippica, 15 parts of bighead atractylodes rhizome, 15 parts of fructus viticis and 5 parts of liquorice.
The preparation method of the traditional Chinese veterinary medicine formula comprises the following steps:
weighing 20 parts of geranium, 20 parts of viola philippica, 15 parts of bighead atractylodes rhizome, 15 parts of fructus viticis and 5 parts of liquorice, mixing, reflux-extracting with 20 times of distilled water at 95 ℃ for 130min, collecting the extracting solution, reflux-extracting the residual dregs with 20 times of distilled water at 100 ℃ for 110min, combining the two extracting solutions, vacuum-filtering, concentrating the filtrate by reduced pressure rotary evaporation at 60 ℃ to obtain extract with the concentration of about 1g crude drug/mL, pre-freezing for two hours in a refrigerator at-80 ℃, drying in a freeze dryer, grinding into fine powder, and sieving.
The application of the traditional Chinese veterinary medicine formula is as follows: can be used for preventing and treating bacterial intestinal diseases of livestock and fowl.
Example 4
A traditional Chinese veterinary medicine formula comprises the following components: 20 parts of geranium, 20 parts of viola philippica, 20 parts of bighead atractylodes rhizome, 20 parts of fructus viticis and 10 parts of liquorice.
The preparation method of the traditional Chinese veterinary medicine formula comprises the following steps:
weighing 20 parts of geranium, 20 parts of viola philippica, 20 parts of bighead atractylodes rhizome, 20 parts of fructus viticis and 10 parts of liquorice, mixing, reflux-extracting for 2 hours with 20 times of distilled water at 100 ℃, collecting the extracting solution, reflux-extracting the residual dregs with 20 times of distilled water at 100 ℃ for 2 hours, combining the two extracting solutions, vacuum-filtering, concentrating the filtrate by reduced pressure rotary evaporation at 55 ℃ to obtain extract with the concentration of about 1g crude drug/mL, pre-freezing for 100 minutes at the temperature of minus 90 ℃, drying in a freeze dryer, grinding into fine powder, and sieving.
The application of the traditional Chinese veterinary medicine formula is as follows: can be used for preventing and treating bacterial intestinal diseases of livestock and fowl.
Experimental example 1 in vitro antibacterial experiment
The traditional Chinese medicines with antibacterial effect in the formula are subjected to in-vitro antibacterial screening, and the adopted strains are clinically separated strains, and have multi-drug resistance through detection, and the group Fang Ba is directed against resistance genes, so that clinical effectiveness is ensured.
The specific experimental process is as follows:
1.1 Experimental strains
The strains to be tested are all preserved at-20 ℃. The strain is activated before use. Placing the culture medium in a sterilizing pot for high-temperature high-pressure sterilization, sucking 15-20 mL of culture medium in a super clean bench, cooling and solidifying, inoculating bacterial liquid in the culture medium by using an inoculating loop, sealing the culture dish by using a sealing strip, and placing the culture dish in a constant-temperature biochemical incubator at 37 ℃ for culturing for about 20 hours. Activating all the strains to be tested, picking each colony with an inoculating loop in a transparent test tube filled with sterile water, shaking, and preparing into No. 1.0 Mirabilitum tube (3×10) 8 ) Is diluted 1 to 3 to 1X 10 8 Then 1:1000 dilution is carried out to obtain 1X 10 5 CFU.mL-1. Preparing 38 bacterial suspensions for standby, 3The classification results of the class of 8 strains are shown in FIG. 6.
The solid medium used in the experiment was prepared in a proportion of 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 20g of agar per 1L of medium. The liquid medium was prepared in such a ratio that 10g of tryptone, 5g of yeast extract and 10g of sodium chloride were used per 1L of medium. Wherein, the culture dishes are all used with solid culture medium, and the 96-well plates are all used with liquid culture medium.
1.2 Experimental methods
Determination of the zone of inhibition: the diameter of the inhibition zone is measured by using an oxford cup method, a culture dish is placed in an ultra-clean bench for ultraviolet irradiation sterilization before an experiment, and then an autoclaved culture medium is poured into the culture dish for cooling and solidification. 100. Mu.L of the prepared bacterial suspension was aspirated and each of the suspensions was uniformly spread on a solid medium using a spreading bar. Pressing oxford cup to attach to the culture dish, adding 140 μl of liquid medicine into oxford cup, adding water extractive solution into upper and lower holes of each of four oxford cup holes, and extracting with water Kong Jiachun. Sealing with sealing strips after adding medicines, marking the names of strains and traditional Chinese medicines, placing the strains and the traditional Chinese medicines in a biochemical incubator with constant temperature of 37 ℃ for culturing for 18-20 hours, observing results, measuring the diameter of the strains and the traditional Chinese medicines when a bacteriostasis zone appears, and recording the diameters.
Minimum Inhibitory Concentration (MIC): double dilution method: taking a 96-well plate, setting a first column to an eighth column as a drug group, setting a ninth column as a blank control, and setting a tenth column as a bacterial liquid control. The concentration of the stock solution of the medicine is 200mg/ml, and three compound holes are arranged. The specific operation steps are as follows: first, a culture solution is added. 50. Mu.L of the culture solution was added to each of the first to eighth columns, and 25. Mu.L was added to each of the eighth and ninth columns. Next, the drug was added to the drug group and double diluted. Adding 50 mu L of the stock solution of the medicine into the first column, uniformly mixing, sequentially performing multiple dilution to the eighth column (namely, sucking 50 mu L of the first column into the second column after uniformly mixing the first column, sucking 50 mu L of the second column into the third column after uniformly mixing the second column again, and continuously downwards diluting the mixture), and discarding 50 mu L of the eighth column to ensure that the total liquid amount is the same. The first to eighth column concentrations after dilution were 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625mg/ml, respectively. The ninth and tenth columns were filled with 25. Mu.L of sterile water. And finally, adding the bacterial liquid. The bacterial liquid was added to 50. Mu.L of each of the first to eighth and tenth columns. Sterile water 50 μl was added to column nine. After the operation is finished, a 96-well plate cover is covered, shaking and mixing are carried out, the culture is carried out for 16-20 h at 37 ℃, whether bacteria grow under each liquid medicine concentration is observed, if bacteria grow, the inside of the hole is turbid, if bacteria grow in a sterile mode, the inside of the hole is clear, and the concentration of the last hole with clear color is recorded to be the minimum antibacterial concentration.
Minimum Bactericidal Concentration (MBC): after the MIC was measured by the double dilution method, the mixed solution in the small hole where no turbidity was observed was sucked out, poured into a petri dish, uniformly coated with a coating rod, and sealed. The strain name and the drug concentration of the culture dish are recorded in detail on the culture dish cover. Placing in a 37 ℃ incubator, and culturing for 24 hours. The minimum concentration of each bacteria at which no colony grows was regarded as the Minimum Bactericidal Concentration (MBC) of the herbal extract, as observed whether colonies grew in the plate.
1.3 experimental results
The diameter of the inhibition zone was measured by an agar perforation method, and the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) were measured by a double dilution method, thereby evaluating the antibacterial effect of the formulation. The in vitro evaluation of the antibacterial effect of the formula shows that the inhibition rate reaches more than 95%. As shown in figure 1, the formula shows better in-vitro activity on the size of a bacteriostasis zone, the minimum bacteriostasis concentration (MIC) and the Minimum Bacteriostasis Concentration (MBC) of clinically isolated pathogenic bacteria, and ensures bioavailability.
Experimental example 2 in vivo anti-inflammatory experiment
Shigella infection can lead to rapid death of macrophages, and is accompanied by release of a large amount of inflammatory factors, the clinical symptoms of the shigella infection are acute inflammatory bowel diseases, the main pathological manifestations are colon, and a large amount of inflammatory cells infiltrate and tissue edema, and even infiltration and destruction of intestinal epithelium. The in vivo experiment results prove that the formula has the effects of reducing the release of colonitis factors and improving the pathological state of colon, and in addition, the formula can improve spleen swelling caused by bacterial infection.
The specific experimental process is as follows:
2.1 laboratory animals
The experiment uses 30 BALB/c mice, the weight is 20+/-2 g, the animals are adaptively fed for 4 days, and healthy mice are selected for random grouping development experiment through observation. Mice were divided into 6 groups of a blank group, a model group, amikacin positive drug group (antibiotic group), positive drug group (continuous ginseng anti-diarrhea granule group), low concentration group of formula (administration 1 group), and high concentration group of formula (administration 2 group), 5 animals per group, and each group was fed separately. Feeding standard feed and distilled water sterilized at high temperature.
2.2 Experimental methods
The experimental period was 7 days and the administration time was 7 days. The dosing regimen was as follows: the administration dosage of the administration group 1 is 1g/kg formula; the administration dosage of the administration group 2 is 2g/kg formula; the administration scheme of the continuous ginseng dysentery stopping granule group is 4g/kg continuous ginseng dysentery stopping granule; the antibiotic group dosing regimen was 2mg/kg amikacin; the blank group and model group were given an equal amount of physiological saline.
All groups were gavaged according to a gavage dose of 0.1mL/10g, with a 12h fasted prior to gavage. On day 3, modeling of intestinal inflammation model caused by pathogenic bacteria was performed by intraperitoneal injection of 0.1mL/10g of bacterial suspension (1×10) 7 and/mL). At the end of the day 8 experiment, mice were sacrificed after anesthesia and blood sampling. The samples were anesthetized with an aqueous chloral solution. Preparation of chloral hydrate (400 mg/kg): dissolved as an aqueous chloral solution at a concentration of 100mg/mL, and the intraperitoneal injection dose was 40. Mu.L/10 g.
Treatment of test results: (1) determination of cytokine levels: after the main abdominal vein blood collection is finished, standing for 1 hour at room temperature, and centrifuging for 10min (4 ℃ C., 2000 rpm) to obtain serum; measuring the serum cytokine content by ELISA kit; finally, the cytokine level is measured by measuring the absorbance value at 490nm by an enzyme-labeled instrument. (2) calculation of spleen index: immediately after the end of the experiment, the spleen was taken out and weighed, and the organ index (%) was calculated according to the following formula: index = organ weight (mg)/body weight (g). (3) detection of pathological sections: and fixing the sample by 4% paraformaldehyde, and after the fixing state is good, trimming, dehydrating, embedding, slicing, dyeing, sealing the sample, and finally microscopic examination to obtain the qualified sample.
2.3 experimental results
2.3.1 spleen index and cytokine detection
After pathogenic bacteria infection, the pathological changes and injuries of various organs can be caused, spleen indexes can reflect the bleeding and swelling conditions of the spleen, and the spleen is used as an immune organ and can reflect the influence on the immunity of the organism laterally. As shown in fig. 2, the spleen index increased significantly after molding, and this was improved after administration, indicating that the formulation could improve spleen damage caused after bacterial infection.
The phenomenon of the secretion of inflammatory cytokines IL-1 beta and IL-6 in large quantities can be caused after the infection of pathogenic bacteria. Detection of inflammatory cytokines in serum by ELISA showed that the model group had higher levels of inflammatory factors than normal compared to the blank group, and that the release of inflammatory factors was reduced after administration. The amounts of the inflammatory-related factors IL-6 and IL-1β in each group are shown in FIG. 3.
2.3.2 colonography histopathological observations
Colon pathology analysis of the multi-drug resistant bacteria infection model shows that the traditional Chinese veterinary medicine formula has obvious treatment effect, and the pathology score is shown in figure 5. Fig. 4 shows pathological section results, and the specific pathology is as follows:
blank results display (see fig. 4A): the structure of each layer of colon tissue is clear, the morphology of a mucous membrane monolayer columnar epithelial cell is regular, intestinal glands in the lamina propria are closely arranged, the number of goblet cells is rich, and obvious folds are not seen; submucosal connective tissue is dense; the myometrium is thin, the myofibers are not obviously abnormal, and the inflammatory changes are not obviously seen.
Model set results display (see fig. 4B): the structure of each layer of colon tissue is clear, the shape of a mucous membrane monolayer columnar epithelial cell is regular, intestinal glands in the lamina propria are closely arranged, the number of goblet cells is rich, and a local mucous membrane layer protrudes into an intestinal cavity to form folds; the submucosa connective tissue is dense, and a large number of lymphocytes can be gathered in the local submucosa to form lymphoid tissues; the muscular layer is uneven in thickness.
Results for dosing group 1 are shown (see fig. 4C): the structure of each layer of colon tissue is clear, the shape of a mucous membrane monolayer columnar epithelial cell is regular, intestinal glands in the lamina propria are closely arranged, the number of goblet cells is rich, and a local mucous membrane layer protrudes into an intestinal cavity to form folds; submucosal connective tissue is dense; the muscular layer was not uniform in thickness and no significant inflammatory changes were seen.
Results for group 2 administration are shown (see fig. 4D): the structure of each layer of colon tissue is clear, the shape of a mucous membrane monolayer columnar epithelial cell is regular, intestinal glands in the lamina propria are closely arranged, the number of goblet cells is rich, and a local mucous membrane layer protrudes into an intestinal cavity to form folds; submucosal connective tissue is dense; no significant inflammatory changes were seen.
Positive drug group results show (see fig. 4E): the structure of each layer of colon tissue is clear, the shape of a mucous membrane monolayer columnar epithelial cell is regular, intestinal glands in the lamina propria are closely arranged, the number of goblet cells is rich, and a local mucous membrane layer protrudes into an intestinal cavity to form folds; submucosal connective tissue is dense; no significant inflammatory changes were seen.
The antibiotic group results show (see fig. 4F): colonic tissue occasionally showed small inflammatory focal infiltrates of the lamina propria, and no other obvious abnormalities were seen.
Experimental example 3 safety evaluation experiment
In order to more accurately evaluate the safety of the veterinary drug formulation, an experimental basis is provided for drug effect and future safe use in animal husbandry, and an acute toxicity test is performed in the experimental example. The observations showed that no death of mice occurred in each of the dosing groups, the mice in each group had better mental status, and the dosing groups were not significantly different from the blank groups (table 1). The anatomical result shows that each organ has normal morphology and color and no obvious toxicity change. Therefore, the half lethal dose of the prescription is more than 40.0g/kg, and the clinical use is safe.
The acute toxicity test comprises the following specific procedures:
3.1 test animals
SPF-grade Kunming mice, weighing 18.0-22.0 g, purchased from Peking Violet laboratory animal technologies Co., ltd., license number: SCXK (Beijing) 2021-0011. Mice were fed in separate cages, fed with standard feed and autoclaved distilled water, fed adaptively for 7 days, and healthy mice were selected for acute toxicity test by observation.
3.2 test methods
3.2.1 determination of LD50 in veterinary drug formulations
The test set consisted of 5 groups, namely, a drug test group with a lavage of 40.0g/kg (drug test 1 group), 20.0g/kg (drug test 2 group), 10.0g/kg (drug test 3 group), 5.0g/kg (drug test 4 group) and a blank group with a lavage of 0.8mL/20g of physiological saline.
50 SPF-class Kunming mice (18 g-22 g) were taken, and the male and female were half-bred. The administration is carried out for 12 hours before feeding, water is not forbidden for overnight, on the test day, the drug test group is infused with the corresponding concentration formula, the administration of the maximum dose of 0.4mL/10g mice is carried out, and the blank group is infused with 0.8mL of normal saline. After the stomach is filled, the medicine is fasted for 3 to 4 hours, and is fed normally after being fully absorbed, and the fasted medicine is continued for 12 hours before the next stomach filling. Mice were observed for mortality and abnormal changes in physiological activity for 7 days.
And (3) observing the indexes: the mice are observed at any time during the administration period, the poisoning condition of the mice is recorded in time, if the mice which die are appeared, the death number of the mice and the corresponding administration concentration are recorded, the mice are dissected in time, and the change of each viscera is observed. Experimental results LD50 values were determined according to the death status of mice with reference to guidelines for acute toxicity testing of veterinary drugs (LD 50 assay). If the mice die, obtaining LD50 according to the experimental result; if no death or other abnormality occurred in each group of mice after 7d, LD50 could not be obtained, a maximum tolerance test was performed.
3.2.2 determination of Maximum Tolerance (MTD) of veterinary drug formulation to mice
20 SPF-grade Kunming mice (body weight 18-22 g) were randomly divided into two groups, a drug test group and a blank group, each group of 10, male and female halves. Animals were fasted and not water-inhibited for 12h prior to the test. Drug test groups were perfused 2 times per mouse at 40g/kg over 24 hours, 10h apart (9:00 and 19:00). The mice in the blank group were perfused with an equal volume of physiological saline. After administration, the patients can eat and drink water freely, and the patients can observe for 7 days continuously.
And (3) observing the indexes: the clinical symptoms of the mice after administration are recorded, and the eyes, stool and autonomous activities of the mice are observed. If the mice die within 7 days, the mice are examined in time and the examination result is recorded. Mice were sacrificed on day 7 of the test, the heart, liver, spleen, lung, kidney and other major organs were dissected and observed, the drug test group was compared with the blank group, and the presence or absence of abnormalities in the organs of the mice in the drug test group was observed. According to the guidelines for acute toxicity technology of chemical drugs, if the mice still do not die, the whole acute toxicity test can be ended.
3.2.3 test results
The 5 groups of mice were given a single intragastric administration daily and observed for seven days. The observation results within seven days showed that no death of mice occurred in each of the administration groups, the mental status of the mice in each group was good, and the administration groups were not significantly different from the blank groups, and the results are shown in table 1 below. The anatomical results after seven days show that each organ is normal in morphology and color and has no obvious toxicity change. Therefore, the half lethal dose of the veterinary drug formula is more than 40.0g/kg.
Table 1 acute toxicity test of formulations mortality in mice
The mice in the drug test group and the mice in the blank group in the maximum tolerance test are not dead normally, and physiological activities and various signs (pupil, eyes, skin color, respiration, secretion and the like) are not abnormal. The body weight of the mice was weighed after the test was completed, and the results are shown in the following table 2, and the body weight of the male and female mice were respectively t-tested by using the spss software, and the results show that the female mice had no significant difference from the blank group, and the male mice had no significant difference from the blank group.
Table 2 maximum tolerance test day 8 mouse body weight
| Group of | Sex (sex) | Number of mice | Body weight of mice |
| Drug test group | ♂ | 5 | 26.42±2.94 |
| Pharmaceutical experimental group | ♀ | 5 | 25.23±2.91 |
| Blank group | ♂ | 5 | 26.09±0.89 |
| Blank group | ♀ | 5 | 23.43±2.70 |
As a result, the veterinary drug formulation of the present invention was not dead when 40g/kg was used for both LD50 and MTD concentrations in mice. Both LD50 and MTD of the formulation are greater than 50mg/10g. Therefore, the veterinary medicine formula is nontoxic.
The foregoing description is only a preferred embodiment of the present invention and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, variation, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The traditional Chinese veterinary medicine formula is characterized by comprising the following raw materials: the weight portions of the raw materials of geranium, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice are (2-4): (2-4): (1-3): (1-3): 1.
2. the veterinary drug formula according to claim 1, wherein the weight parts ratio of geranium wilfordii, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice is (2.5-3.5): (2.5-3.5): (1.5-2.5): (1.5-2.5): 1.
3. the veterinary drug formula as claimed in claim 1, wherein the weight parts ratio of geranium wilfordii, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice is 3:3:2:2:1.
4. use of a veterinary formulation according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of a disease caused by bacterial infection in livestock and poultry.
5. Use of a veterinary drug formulation according to any one of claims 1 to 3 for the manufacture of a medicament for the prevention and treatment of intestinal diseases in livestock and poultry.
6. The use according to claim 5, wherein the medicament is for the prevention and treatment of intestinal diseases of livestock and poultry, the intestinal diseases being bacterial intestinal diseases.
7. The use according to claim 5, wherein the medicament is in the form of a powder, decoction, capsule, pill, granule, tablet or solution.
8. A process for preparing a veterinary formulation as claimed in any one of claims 1 to 3, comprising the steps of:
weighing and mixing geranium wilfordii, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice according to the weight part ratio, adding a certain amount of water, carrying out reflux extraction for 100-140 min at 90-110 ℃, collecting an extracting solution, continuously adding the same amount of water into the residue, carrying out reflux extraction for 100-140 min at 90-110 ℃, repeating for many times, combining the extracting solutions, carrying out vacuum suction filtration, concentrating filtrate to 1g crude drug/mL at 50-60 ℃, pre-freezing for 100-140 min at-70 to-90 ℃, freeze-drying, grinding into fine powder, and sieving to obtain the medicine.
9. The method according to claim 8, wherein the water added during the reflux extraction is distilled water, and the amount of the added water is 15 to 25 times the weight of the crude drug.
10. The method of manufacturing according to claim 9, comprising the steps of:
weighing and mixing geranium wilfordii, herba violae, bighead atractylodes rhizome, fructus viticis and liquorice according to the weight part ratio, reflux-extracting for 2 hours at 100 ℃ by using 20 times of distilled water, collecting extracting solution, reflux-extracting the residual dregs for 2 hours at 100 ℃ by using 20 times of distilled water, combining the extracting solutions twice, vacuum-filtering, concentrating the filtrate by decompression rotary evaporation at 55 ℃ to obtain extract with the concentration of 1g crude drug/mL, pre-freezing for two hours in a refrigerator at-80 ℃, drying in a freeze dryer, grinding into fine powder, and sieving.
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