Water-soluble dimetridazole composition as well as preparation method and application thereof

Abstract

The invention provides a water-soluble dimetridazole composition as well as a preparation method and application thereof, and belongs to the technical field of veterinary drug preparation. The composition comprises 10%-20% of dimetridazole, 2%-4% of tremella oligosaccharide, 2% of vitamin k3, 0.4%-1.2% of hydroxypropyl methylcellulose HPMC E5, 0.1%-0.4% of xanthan gum, 15%-25% of a cosolvent, 5%-10% of a stabilizer, 2% of Tween 20 and the balance of a water-soluble carrier. The composition can be dissolved in 8000 ppm of water and suspended in 12000 ppm, can be uniformly dispersed without layering within 4 hours, and is convenient for drinking water administration. The dimetridazole and the tremella oligosaccharide are combined, so that the prevention and treatment effect on pathogenic bacteria such as escherichia coli and salmonella is enhanced, the use amount of antibiotics and traditional Chinese medicine extracts is reduced, and the bioavailability and efficacy of the medicine are improved. The composition has an exact curative effect on poultry enteritis and salpingitis caused by the pathogenic bacteria, and is suitable for being applied to intensive large-scale farms.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drug preparation, and particularly relates to a water-soluble dimetridazole composition and a preparation method and application thereof. Background Art

[0002] Dimetridazole is an antiprotozoal drug for animals. Since metronidazole was banned, dimetridazole is almost the only choice. It can inhibit the redox reaction of amoeba and break the nitrogen chain; it is effective against protozoa such as Trichomonas, Trichomonas, Balantidium coliforme, Flagellate, Eperythrozoon, etc.; it has a significant inhibitory effect on anaerobic bacteria (Escherichia coli, Streptococcus, Staphylococcus, Necrotic Bacillus, Clostridium, Vibrio), etc., and is also highly effective against spirochetes between protozoa and bacteria. It is an effective drug for the treatment of swine bloody diarrhea. It is often used in the clinic to treat animal anaerobic infections, and can also be used for diseases such as pseudomembranous enterocolitis caused by Clostridium difficile.

[0003] The anti-anaerobic effect of dimetidazole is mainly that in an anaerobic environment, dimetidazole can spontaneously reduce the complex bound to DNA, thereby causing rapid cell death. The unique and broad-spectrum antibacterial mechanism of dimetidazole means that it basically does not have the same or similar antibacterial targets or the same or similar antibacterial mechanisms with other known antibacterial drugs, and there is no cross-resistance with other antibacterial drugs. Therefore, broad-spectrum antibacterial drugs such as dimetidazole are usually used in many bacterial and mycoplasma treatment prescriptions to complement and enhance the efficacy of broad-spectrum antibacterial drugs such as amoxicillin, cephalosporins, ofloxacin, colistin sulfate, sulfonamides, etc. However, the problem of strong drug resistance of pathogens due to the long-term and widespread use of antibacterial drugs cannot be ignored. Under the influence of the "green trend", plant medicine sources have attracted much attention for their characteristics such as being less likely to develop drug resistance, less likely to remain in livestock products for a long time or to be enriched through the food chain, and being relatively safe for humans, animals and the environment. Therefore, the development of a new, efficient and safe demetronidazole preparation will also be an important research direction for veterinary workers.

[0004] Dimetridazole powder is off-white or slightly yellow, in the form of fibrous crystals or crystalline powder, odorless or almost odorless, with a melting point of 138-139°C, and slightly soluble in water. Its water solubility is less than 2000ppm, and its dissolution rate is slow. It is difficult to completely dissolve at room temperature (there are insoluble water particles that need to be stirred for more than 30 minutes to completely dissolve). For insoluble drugs, their bioavailability often depends on the dissolution rate of solid drugs in the preparation. The coarsely dispersed powder obtained by the general mixing method often has a low bioavailability due to its low dissolution rate. Dimetridazole itself is alkaline. Under alkaline conditions, it has poor stability, low solubility, difficulty in dissolution, blackening in light, and easy sublimation at high temperatures. These problems make its utilization rate not high and difficult to store. Therefore, how to improve the stability and water solubility of dimetridazole products is a problem worth exploring. Summary of the invention

[0005] Based on the above problems and in view of the shortcomings and defects of the prior art of dimetridazole preparations, the present invention provides a water-soluble dimetridazole composition and a preparation method and application thereof, which solves the technical problems of poor water solubility of dimetridazole preparations and precipitation after long-term storage; the combination adopts dimetridazole and Tremella oligosaccharides in combination, which enhances the prevention and treatment effect on pathogenic bacteria such as Escherichia coli and Salmonella, and has significant therapeutic effect on livestock and poultry enteritis and salpingitis caused by such pathogenic bacteria.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A water-soluble dimetridazole composition comprises the following components in percentage by weight: 10-20% of dimetridazole, 2-4% of Tremella oligosaccharide, 2% of vitamin K3, 0.4-1.2% of hydroxypropyl methylcellulose HPMC E5, 0.1-0.4% of xanthan gum, 15-25% of cosolvent, 5-10% of stabilizer, 2% of Tween 20 and the balance of a water-soluble carrier.

[0008] According to one embodiment of the present invention, the molecular weight of the Tremella oligosaccharide is 0.6KDa-1.2KDa.

[0009] According to one embodiment of the present invention, the co-solvent is one or more of anhydrous citric acid, tartaric acid, malic acid, succinic acid, and fumaric acid, preferably anhydrous citric acid.

[0010] According to one embodiment of the present invention, the stabilizer is one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium hexametaphosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium tartrate, and sodium tartrate, preferably potassium dihydrogen phosphate.

[0011] According to one embodiment of the present invention, the water-soluble carrier is one or more of anhydrous glucose, lactose, mannitol, sucrose, sodium chloride, water-soluble starch, and anhydrous sodium sulfate.

[0012] Preferably, the dimetridazole composition comprises the following components in weight percentage: dimetridazole 20%, tremella oligosaccharide 4%, vitamin K3 2%, hydroxypropyl methylcellulose HPMC E5 0.4%, xanthan gum 0.4%, anhydrous citric acid 25%, potassium dihydrogen phosphate 10%, Tween 20 3% and the balance anhydrous glucose.

[0013] According to another aspect of the present invention, the present invention also provides a method for preparing a water-soluble dimethylnidazole composition, comprising the following steps:

[0014] (1) The above-mentioned amount of dimetridazole, Tremella oligosaccharide, xanthan gum, and vitamin K3 are micronized by a jet mill to obtain a co-micronized material, wherein the particle size D90 of the co-micronized material is controlled at 3-10 μm; the other solid materials are all crushed to 80 μm and set aside;

[0015] (2) dispersing the co-micronized powder, the cosolvent, the stabilizer and Tween 20 in warm water to obtain a co-micronized powder solution, wherein the temperature of the warm water is controlled at 40° C.-45° C.; dissolving hydroxypropyl methylcellulose HPMC E5 in warm water, and then adding the mixture to the co-micronized powder solution to obtain a material solution, mixing the mixture and spray drying the mixture;

[0016] (3) The dry powder obtained in step (2) is mixed evenly with a water-soluble carrier to obtain the water-soluble dimethylnidazole composition.

[0017] According to one embodiment of the present invention, the air flow pulverization in step (1) is carried out at a temperature of -3°C to 8°C, with an air inlet pressure of 0.9-2.5 MPa and a working pressure of 0.8-2 MPa during pulverization.

[0018] According to one embodiment of the present invention, the material solution in step (2) has a solid content of 17-23%, and the spray drying conditions are: inlet air temperature is 140-160°C, outlet air temperature is 40-60°C, and feed rate is 10-15 mL / min.

[0019] According to another aspect of the present invention, the present invention also provides the use of the above-mentioned dimetridazole composition in the treatment of enteritis / salpingitis in laying hens.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The present invention creatively co-powders dimernidazole with Tremella oligosaccharides, which significantly increases the water solubility of dimernidazole. Tremella oligosaccharides are then compounded with a small amount of xanthan gum to form a low-viscosity hydrosol, which disperses and wraps the dimernidazole. Hydroxypropyl methylcellulose HPMC E5 is a linear polysaccharide substance formed by connecting glucose monomers through β-1,4-glycosidic bonds and has a certain water solubility. Its introduction further binds dimernidazole in an interlocking network structure, thereby enhancing the water solubility of the composition, which can reach 8000ppm. When the concentration is too high, the sample of the composition can be suspended and is stable for a long time without stratification. No additional antioxidant is needed, and the composition can be directly added to feed or drinking water as a veterinary drug for consumption. The composition is easy to use and has a significant therapeutic effect.

[0022] Second, the water-soluble auxiliary materials of the existing composition are partially hygroscopic, which easily leads to moisture absorption and agglomeration of the product. The addition of Tremella oligosaccharide in the present invention plays an unexpected anti-caking effect, and also cooperates with Tween-20 to eliminate static electricity, further improving the dispersibility of the superfine powder of dimetridazole in water.

[0023] 3. The composition of the present invention adds Tremella oligosaccharides with a preferred molecular weight, which can further enhance the preventive and therapeutic effects on pathogenic bacteria such as Escherichia coli and Salmonella, and has a significant effect on livestock and poultry enteritis and salpingitis caused by such pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of the adhesion of the ultrafine powder of dimethylnidazole alone.

[0025] Figure 2 This is the adhesion diagram of the equipment after co-powdering of dimethoprim-iodide and Tremella oligosaccharide.

[0026] Figure 3 This is the particle size distribution diagram of dimetridazole and Tremella oligosaccharide after co-powdering.

[0027] Figure 4 This is a comparison picture of dirty eggs and blood-spotted eggs in a chicken farm.

[0028] Figure 5 This is a schematic diagram of the color change of clinical eggs in Example 1 over 5-6 days.

[0029] Figure 6 This is a schematic diagram of the color change of clinical eggs in Example 1 over 7-9 days.

[0030] Figure 7 A Venn diagram for species distribution.

[0031] Figure 8 Relative abundance graph of gut microbes sequenced.

[0032] Fig. 9 Heat map of gut microbial sequence functional abundance. DETAILED DESCRIPTION

[0033] In order to make the invention purpose and content of the present application more clearly presented, the applicant further describes the present invention in conjunction with the embodiments below, but the protection scope of the present invention is not limited to these embodiments. Those skilled in the art should understand that the equivalent replacement or corresponding improvement made to the technical features of the present invention still falls within the protection scope of the present invention.

[0034] The traditional Chinese medicine extract Tremella oligosaccharide selected in the present invention is a low-polymerized carbohydrate formed by 2 to 10 monosaccharides connected by glycosidic bonds through dehydration condensation obtained from Tremella through four steps of extraction, degradation, purification and drying, with a molecular weight of 0.6KDa-1.2KDa. It has a shorter sugar molecule chain, is soluble in cold water, is easily absorbed by the body, can be used as an energy source, and is well-known for its prebiotic effect, which means that Tremella oligosaccharide can promote the growth of beneficial intestinal bacteria. It has a positive effect on digestive health and immune function. Tremella oligosaccharide itself has good processing characteristics, and has the effects of moisturizing, oil absorption, anti-oxidation, thickening and suspending, and emulsification and dispersion.

[0035] The Tremella oligosaccharide used in the present invention can be extracted and separated from Tremella fruiting bodies by conventional extraction and purification methods in the field of natural medicinal chemistry, is soluble in cold water, and can also be purchased from commercial products, for example, Shandong Tongyuan Gonghe Biotechnology Co., Ltd.

[0036] The vitamin K3 in the present invention is mainly used as a hemostatic agent and an intestinal protective agent.

[0037] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0038] Example 1

[0039] A water-soluble dimenolide composition comprises the following components in percentage by weight: 20% dimenolide, 4% tremella oligosaccharide, 2% vitamin K3, 0.4% hydroxypropyl methylcellulose HPMC E5, 0.4% xanthan gum, 25% anhydrous citric acid, 10% potassium dihydrogen phosphate, 2% Tween 20 and 36.2% anhydrous glucose.

[0040] The preparation method of the water-soluble dimetridazole composition of this embodiment comprises the following process steps:

[0041] (1) The above-mentioned formula amount of dimetridazole, Tremella oligosaccharide, and xanthan gum are micronized by low-temperature air flow grinding (at a temperature of 8° C., an air inlet pressure of 2.5 MPa, and a grinding pressure of 2 MPa) to obtain a co-micronized material, and the particle size D90 of the material is controlled at 3-10 μm; other solid materials are all ground to 80 μm and set aside;

[0042] (2) Dispersing the co-micronized powder, anhydrous citric acid, potassium dihydrogen phosphate, and Tween 20 in an appropriate amount of warm water at high speed, and controlling the water temperature at 40°C-45°C; dissolving hydroxypropyl methylcellulose HPMC E5 in a small amount of warm water, and then adding it to the co-micronized powder solution, with a solid content of 17%, mixing well, and spray drying, wherein the inlet air temperature is 160°C, the outlet air temperature is 60°C, and the feed rate is 10 mL / min;

[0043] (3) The dry powder obtained in step (2) is mixed evenly with anhydrous glucose to obtain the water-soluble dimetridazole composition.

[0044] Example 2

[0045] A water-soluble dimenolide composition comprises the following components in percentage by weight: 15% dimenolide, 3% tremella oligosaccharide, 2% vitamin K3, 0.8% hydroxypropyl methylcellulose HPMC E5, 0.25% xanthan gum, 20% tartaric acid, 8% sodium dihydrogen phosphate, 2% Tween 20 and 48.95% sodium chloride.

[0046] The preparation method of the water-soluble dimetridazole composition of this embodiment comprises the following process steps:

[0047] (1) The formulated amount of dimetridazole, Tremella oligosaccharide, and xanthan gum are subjected to micronization treatment by low-temperature airflow pulverization (at a temperature of 1° C., an air inlet pressure of 1.5 MPa, and a pulverization pressure of 1 MPa) to obtain a co-micronized material, and the particle size D90 of the material is controlled at 3-10 μm; other solid materials are pulverized to 80 μm and set aside;

[0048] (2) Dispersing the co-micronized powder, tartaric acid, sodium dihydrogen phosphate and Tween 20 in an appropriate amount of warm water at high speed, and controlling the water temperature at 40°C-45°C; dissolving hydroxypropyl methylcellulose HPMC E5 in a small amount of warm water, and then adding it to the co-micronized powder solution, with a solid content of 19%, mixing well and spray drying, wherein the inlet air temperature is 145°C, the outlet air temperature is 55°C, and the feed rate is 12 mL / min;

[0049] (3) The dry powder obtained in step (2) is mixed evenly with sodium chloride to obtain the water-soluble dimethylnidazole composition.

[0050] Example 3

[0051] A water-soluble dimenolide composition comprises the following components in percentage by weight: 10% dimenolide, 2% tremella oligosaccharide, 2% vitamin K3, 1.2% hydroxypropyl methylcellulose HPMC E5, 0.1% xanthan gum, 15% malic acid, 5% sodium hexametaphosphate, 2% Tween 20 and 62.7% mannitol.

[0052] The preparation method of the water-soluble dimetridazole composition of this embodiment comprises the following process steps:

[0053] (1) The formulated amount of dimetridazole, Tremella oligosaccharide, and xanthan gum are micronized by low-temperature airflow pulverization (at a temperature of -3°C, an air inlet pressure of 0.9 MPa, and a pulverization pressure of 0.8 MPa) to obtain a co-micronized material, and the particle size D90 of the material is controlled at 3-10 μm; other solid materials are all pulverized to 80 μm and set aside;

[0054] (2) Dispersing the co-micronized powder, malic acid, sodium hexametaphosphate and Tween 20 in an appropriate amount of warm water at high speed, and controlling the water temperature at 40°C-45°C; dissolving hydroxypropyl methylcellulose HPMC E5 in a small amount of warm water, and then adding it to the co-micronized powder solution, with a solid content of 23%, mixing well and spray drying, wherein the inlet air temperature is 140°C, the outlet air temperature is 40°C, and the feed rate is 15 mL / min;

[0055] (3) The dry powder obtained in step (2) is mixed evenly with mannitol to obtain the water-soluble dimetridazole composition.

[0056] Comparative Example 1

[0057] A water-soluble dimenolide composition comprises the following components in percentage by weight: 20% dimenolide, 2% vitamin K3, 0.4% hydroxypropyl methylcellulose HPMC E5, 0.4% xanthan gum, 25% anhydrous citric acid, 10% potassium dihydrogen phosphate and 42.2% anhydrous glucose.

[0058] Since direct ultrafine grinding of raw materials will result in agglomeration, severe adhesion and poor dispersibility, the preparation method of this comparative example is different from that of Example 1 in that the raw materials are not ultrafinely ground, the auxiliary materials are passed through 80 mesh, and then steps (2) and (3) are entered.

[0059] Comparative Example 2

[0060] A water-soluble dimenolide composition comprises the following components in percentage by weight: 20% dimenolide, 4% tremella oligosaccharide, 2% vitamin K3, 25% anhydrous citric acid, 10% potassium dihydrogen phosphate, 2% Tween 20 and 37% anhydrous glucose.

[0061] The preparation method is the same as that of Example 1.

[0062] Comparative Example 3

[0063] A water-soluble dimenolide composition comprises the following components in percentage by weight: 20% dimenolide, 4% tremella oligosaccharide, 2% vitamin K3, 0.4% hydroxypropyl methylcellulose HPMC E5, 0.4% xanthan gum, 2% Tween 20 and 71.2% anhydrous glucose.

[0064] The preparation method is the same as Example 1.

[0065] Comparative Example 4

[0066] A water-soluble dimenolide composition comprises the following components in percentage by weight: 20% dimenolide, 4% tremella oligosaccharide, 2% vitamin K3, 0.4% hydroxypropyl methylcellulose HPMC E5, 0.4% xanthan gum, 25% anhydrous citric acid, 2% Tween 20 and 46.2% anhydrous glucose.

[0067] The preparation method is the same as Example 1.

[0068] Comparative Example 5

[0069] Commercially available 20% nitropropene premix, Inner Mongolia Huatian Pharmaceutical Co., Ltd., batch number 20240110.

[0070] Comparative Example 6

[0071] The difference from Example 1 is that dimetridazole is not added in the formula, and the other parts are the same, and the preparation method is also the same.

[0072] Figure 1 This is the adhesion diagram of the ultrafine powder equipment of dimetridazole alone. Figure 2 This is the adhesion diagram of the device after the co-powder of dimetridazole and Tremella oligosaccharide. Figure 1 and Figure 2 It is not difficult to see that before co-grinding, some dimethoate adheres to the lower plate of the grinding chamber, a large amount adheres to the discharge port of the upper plate of the grinding chamber, and a large amount adheres to the upper end of the upper plate of the grinding chamber. After co-grinding, almost no adhesion is seen. Figure 3 This is the particle size distribution diagram of dimetridazole and Tremella oligosaccharide after co-powdering.

[0073] Application Test-Solubility Test

[0074] According to the solubility standard of soluble powder in the 2020 edition of the Chinese Veterinary Pharmacopoeia: Take an appropriate amount of each of the 8 samples of Examples 1, 2, 3 and Comparative Examples 1-5, place them in Nessler colorimetric tubes, add water to make 100 ml of a solution of appropriate concentration (in terms of dimetridazole, the concentration is twice the clinical drinking concentration of 100 ppm), turn it upside down 10 times at 23-27°C, the test sample should be completely dissolved, let it stand for 30 minutes, and no turbidity or precipitation should be generated. The results show that all 8 samples are water-soluble.

[0075] At room temperature (10-30°C), weigh an appropriate amount of 7 samples of Examples 1, 2, 3 and Comparative Examples 1, 2, 3 and 4, respectively, add water to make 100 ml of solution, stir for 30 minutes, check the dissolution of the sample during stirring, observe the maximum solubility, and the results are shown in Table 1 below.

[0076] Table 1: Dissolution of each test sample

[0077]

[0078] If the substance dissolves quickly within 5 minutes, please use “√”; otherwise, please use “×”.

[0079] Test conclusion: The example groups are all soluble in water at 8000ppm, the difference is the length of time it takes to dissolve in water, while the comparative examples are all insoluble in water under this condition. This shows that the water solubility and dissolution speed of dimetridazole are improved under the action of ultrafine grinding, water-soluble reticulated glue, cosolvent and dispersant. Comparative Example 5 is a common product on the market, which can only be soluble in water at 500ppm within 5 minutes. After dissolving in water, obvious dimenolide crystal particles can be seen. In about 30 minutes, a concentration of 1000ppm can be slowly dissolved; Comparative Example 1 does not add Tremella oligosaccharides and Tween 20. After dissolving in water, small hard granular substances are found, with poor dispersibility, and the water solubility is slightly better than Comparative Example 5; Comparative Example 3 does not add cosolvents and stabilizers, which reduces the water solubility of dimenolide; Comparative Example 2 does not add xanthan gum and hydroxypropyl methylcellulose HPMC E5, indicating that the water-soluble network glue formed by Tremella oligosaccharides, xanthan gum and hydroxypropyl methylcellulose does improve the solubility of dimenolide. Comparative Example 4 does not add stabilizers, and unstable precipitation occurs after the sample is dissolved in water.

[0080] Application Test 2: Suspension and Homogeneity Test

[0081] The dimetridazole composition examples 1-3 prepared by the present invention and the commercially available sample 5 were prepared into a 12000 ppm suspension. After 4 hours, the stratification of the sample was observed, and samples were slowly taken from the upper, middle and lower positions of the solution. The sample was diluted 50 times to determine the content of dimetridazole, and the method was carried out according to the dimetridazole content method of the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China. The results are shown in Table 2.

[0082] Table 2 Results of content determination at different positions of suspension

[0083] Sampling location Concentration 1 (μg / mL) Concentration 2 (μg / mL) Mean concentration (μg / mL) superior 234.12 235.38 234.75 Example 1 middle 237.27 238.46 237.87 Down 241.73 241.54 241.64 superior 235.25 235.41 235.33 Example 2 middle 238.67 239.12 238.90 Down 243.32 242.83 243.08 superior 229.16 230.42 229.79 Example 3 middle 235.24 234.96 235.10 Down 240.18 239.94 240.06

[0084] From the results in the table, it can be seen that after standing for 4 hours, samples were taken from the top, middle and bottom of the suspension, and the concentration gradually increased from top to bottom, and the difference in the single overall content was not obvious, indicating that the sample prepared by the present invention can basically achieve stable suspension within 4 hours under the condition of 12000ppm concentration. Comparative Example 5 completely sank to the bottom in 4 hours, and the upper solution was clear, so no sampling was taken for measurement.

[0085] Application Test 3 Stability Test

[0086] The products of Examples 1-3 and Comparative Examples 1-4 were subjected to high temperature stability tests and accelerated tests respectively to examine the stability of the samples of the present invention, confirm whether discoloration, caking, deliquescence and the like occur, whether the content is stable, and check whether the water solubility is qualified. The water solubility detection method is the same as that of application test 1.

[0087] 1. High temperature stability test

[0088] The samples were placed in an open container at 60°C for 10 days and then returned to room temperature for observation. The results showed that the appearance of Examples 1-3 and Comparative Examples 2-4 did not change significantly, and no discoloration or caking occurred. The water solubility and content did not change significantly compared with the initial state, indicating that Examples 1-3 and Comparative Examples 2-4 had good stability at high temperatures. Comparative Example 1 showed high-temperature caking, indicating that the sample was prone to high-temperature caking without the protection of Tremella oligosaccharides.

[0089] 2. Accelerated test

[0090] The samples were packaged for sale, placed in an accelerated box at a temperature of 40°C and a relative humidity of 75% for 6 months, and sampled and observed every 30 days. The results showed that the appearance of Examples 1-3 and Comparative Examples 2-4 did not change significantly, and no discoloration, caking, deliquescence, etc. occurred. The water solubility and content did not change significantly compared with the initial state, indicating that the samples of Examples 1-3 and Comparative Examples 2-4 had good stability. Comparative Example 1 showed a phenomenon of moisture absorption and caking, indicating that the sample was prone to moisture absorption and caking without the moisture-proof protection of Tremella oligosaccharides.

[0091] Application Test IV Clinical Efficacy Trial

[0092] Observation on the therapeutic effect of enteritis / salpingitis in laying hens

[0093] Location: A laying hen breeding company in Gansu

[0094] Test subjects: About 48,014 laying hens aged 320 days. Recently, the number of dirty eggs, broken eggs, and white-shelled eggs in the flock has increased, the feces are poorly formed, and autopsy showed inflammation of the intestines and bleeding spots in the fallopian tubes. The diagnosis was that there were complications of enteritis and salpingitis caused by infections such as Escherichia coli and Salmonella.

[0095] Trial Groups:

[0096] 1) Blank group: no drugs were used, 8,000 laying hens;

[0097] 2) Drug control group 1: using the sample of comparative example 5, 8005 laying hens;

[0098] Drug control group 2: using the sample of comparative example 6, 8000 laying hens;

[0099] 3) Experiment 1-3 groups: using samples of Examples 1-3, with 8002, 8004 and 8003 laying hens respectively;

[0100] Experimental method: The experimental groups 1 to 3 and the drug control group were all administered with drinking water. The concentration of dimetridazole in drinking water was 100 ppm, calculated according to the total daily drinking water volume. Each group was administered with the drug for 5 consecutive days, and observed for 10 consecutive days from the start of drug administration. The drinking water, feeding, feces, egg production rate, qualified egg rate, mortality rate and other indicators of the laying hens were recorded.

[0101] (1) Statistics of water intake

[0102] The statistics of water drinking during the treatment period showed no significant difference among the groups (P>0.05), which proved that the drugs in each group would not affect the palatability of the chickens.

[0103] (2) Feed intake statistics

[0104] There was no significant difference in food intake among the groups during the treatment period (P>0.05), which proved that the drugs in each group had no effect on food intake.

[0105] (3) Stool status

[0106] Before medication (Day 1), the overall feces formation of the farm was poor, with a proportion of loose feces of about 50%, and obvious feed feces of about 20%. On the third day of medication (Day 4), each experimental group showed significant improvement compared with the blank group, with loose feces and feed feces decreasing by nearly 15%, especially in Experimental Groups 1-3, where loose feces were controlled at 25%, which was better than the other experimental groups. On the fifth day of medication (Day 6), the proportion of abnormal feces in Experimental Groups 1-3 was controlled at about 20%, which was better than the 35% of Comparative Example 5, and the feces improved significantly. Continued follow-up to the fifth day after medication (Day 11), in Experimental Groups 1-3, loose feces were maintained at 5%, and feed feces were controlled at 5%, while the loose feces of the Comparative Group were maintained at 10%, and feed feces were controlled at 10%. This shows that Example 1-3 ensures effective regulation of the intestinal tract of the chickens and has a good prevention and control effect. See Table 3 below for details:

[0107] Table 3 Feces of each test group

[0108]

[0109] (4) Mortality rate

[0110] During the test, the number of daily deaths in each group was counted, and the results are shown in Table 4. Each group had different degrees of death every day, and the total death results for 10 days showed that the number of deaths in the test groups was reduced compared with the blank group, especially in the Example 1-3 groups, the death rates were reduced by 75%, 63% and 63%, respectively, the Comparative Example 6 was reduced by 12%, and the Comparative Example 5 was reduced by 25%. The Example group was significantly reduced and was better than the other test groups.

[0111] Table 4 Death rate of each experimental group

[0112] Group Mortality rate (‰) Blank Group 1 Comparative Example 6 0.88 Comparative Example 5 0.75 Example 1 0.25 Example 2 0.37 Example 3 0.37

[0113] (5) Egg production

[0114] During the test, the good egg rate of each group was statistically calculated, and the results are shown in Table 5. The egg production rate after 10 days showed that the test group and the comparative group were improved compared with the blank group, especially the groups of Examples 1-3, the good egg rate increased from 75% to about 90%, the dirty egg rate decreased from about 7.5% to about 2%, and the blood egg rate decreased from 6% to about 1.5%. There was a significant improvement in egg production. Among them, the comparison of dirty eggs and bloody eggs in the blank group and Example 1 group is shown in Table 5. Figure 4 .

[0115] In addition, during the test, it was found that the color of the eggs in the blank group and the comparative example 6 group was lighter, and there were white shell eggs. The eggshell color of the comparative example 5 group was improved, but worse than that of the example group. Figure 5 , 7 to 9 days eggshell changes see Figure 6 .

[0116] Table 5 Egg laying situation of each experimental group

[0117]

[0118]

[0119] (6) Impact on intestinal microbial diversity

[0120] Since the blank group and comparative example 6 were similar in the above test conditions, the experiment selectively selected 50g of feces from the chicken farms of comparative example 6 (KB2), comparative example 5 (KB1), embodiment 1 (STK1), and implementation 2 (STK2) for 16S-rDNA sequencing. The species distribution Venn diagram is shown in Figure 7 , relative abundance diagram is shown in Figure 8 ,The function heat map is shown in 9.

[0121] Figure 7 It can be seen that the STK group of the embodiment completely covers the KB control group of the comparative example, and has 272 more species, which is more species diverse.

[0122] Figure 8 In the relative abundance diagram, the abundance of beneficial bacteria in the STK group of the embodiment increased, which is more conducive to maintaining intestinal health. The control group of Comparative Example 6 has more Proteobacteria, mainly pathogenic bacteria. It shows that the addition of a small amount of Tremella oligosaccharides has little effect on the intestine. The proportion of Proteobacteria in Comparative Example 5 is about 20% lower than that in Comparative Example 6, indicating that dimetridazole still has an effect on pathogenic bacteria. The proportion of Proteobacteria in the STK group has dropped significantly, falling below 10%, and Example 1 has dropped to about 5%. It shows that a small amount of Tremella oligosaccharides combined with dimetridazole increases the effect of the product on Proteobacteria and enhances the abundance of intestinal microorganisms in laying hens.

[0123] Proteobacteria is the largest phylum of bacteria, including many pathogens, such as Escherichia coli, Salmonella, Vibrio cholerae, Helicobacter pylori and other well-known species.

[0124] Firmicutes (Firmicutes) mainly decompose complex nutrients such as cellulose, starch and protein, and produce beneficial metabolites. Many members are beneficial bacteria, such as Lactobacillus, Bacillus, Eubacterium, and Roseburia.

[0125] The main function of Bacteroidetes is to produce enzymes to decompose food and help digest nutrients such as carbohydrates, proteins and fats. It can accelerate intestinal mucosal angiogenesis, improve host immunity and maintain the balance of intestinal microecology.

[0126] Fusobacteria is a small group of Gram-negative bacteria. They can produce some metabolites that are beneficial to the body, such as pyruvate and butyrate. Fusobacteria may be involved in the homeostasis and metabolic function of the intestinal flora.

[0127] Fig. 9 In the function heat map, the color represents the function abundance value. The redder the color, the higher the abundance, and the greener the color. The results show that the color of the STK group is darker than that of the KB group, with STK1 being the darkest and KB1 being the greenest. This indicates that the STK group has increased the abundance of beneficial bacteria.

[0128] In summary, the effects of Examples 1-3 are significant in clinical application for enteritis and salpingitis caused by infections such as Escherichia coli and Salmonella, increase the abundance of beneficial intestinal microbial flora, improve loose stools and feed stools caused by diarrhea; reduce the mortality rate; increase the egg production rate, and reduce dirty eggs and blood-stained eggs.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, which are also considered as the protection scope of the present invention.

Claims

1. A water-soluble dimetridazole composition, characterized in that: The invention comprises the following components in percentage by weight: 10-20% of dimetridazole, 2-4% of tremella oligosaccharide, 2% of vitamin K3, 0.4-1.2% of hydroxypropyl methylcellulose HPMC E5, 0.1-0.4% of xanthan gum, 15-25% of cosolvent, 5-10% of stabilizer, 2% of Tween 20 and the balance of water-soluble carrier.

2. A water-soluble dimetridazole composition according to claim 1, characterized in that: The molecular weight of the Tremella oligosaccharide is 0.6KDa-1.2KDa.

3. A water-soluble dimetridazole composition according to claim 1, characterized in that: The co-solvent is one or more of anhydrous citric acid, tartaric acid, malic acid, succinic acid, and fumaric acid, preferably anhydrous citric acid.

4. A water-soluble dimetridazole composition according to claim 1, characterized in that: The stabilizer is one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium hexametaphosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium tartrate, and sodium tartrate, preferably potassium dihydrogen phosphate.

5. A water-soluble dimetridazole composition according to claim 1, characterized in that: The water-soluble carrier is one or more of anhydrous glucose, lactose, mannitol, sucrose, sodium chloride, water-soluble starch, and anhydrous sodium sulfate.

6. A water-soluble dimetridazole composition according to claim 5, characterized in that: The dimetridazole composition comprises the following components in percentage by weight: 20% of dimetridazole, 4% of Tremella oligosaccharide, 2% of vitamin K3, 0.4% of hydroxypropyl methylcellulose HPMC E5, 0.4% of xanthan gum, 25% of anhydrous citric acid, 10% of potassium dihydrogen phosphate, 2% of Tween 20 and the balance of anhydrous glucose.

7. A method for preparing a water-soluble dimetridazole composition according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) The above-mentioned amount of dimetridazole, Tremella oligosaccharide, xanthan gum, and vitamin K3 are micronized by a jet mill to obtain a co-micronized material, wherein the particle size D90 of the co-micronized material is controlled at 3-10 μm; the other solid materials are all crushed to 80 μm and set aside; (2) Dispersing the co-micronized powder, cosolvent, stabilizer and Tween 20 in warm water, wherein the temperature of the warm water is controlled at 40° C.-45° C.; dissolving HPMC E5 in warm water, and then adding the mixture to the co-micronized powder solution, mixing well and spray drying; (3) The dry powder obtained in step (2) is mixed evenly with a water-soluble carrier to obtain the water-soluble dimethylnidazole composition.

8. The preparation method according to claim 7, characterized in that: The air flow pulverization in step (1) is carried out at a temperature of -3°C to 8°C, an air inlet pressure of 0.9-2.5MPa, and a working pressure of 0.8-2MPa during pulverization.

9. The preparation method according to claim 7, characterized in that: The material solution described in step (2) has a solid content of 17-23%, and the spray drying conditions are: an inlet air temperature of 140-160° C., an outlet air temperature of 40-60° C., and a feed rate of 10-15 mL / min.

10. Use of the dimetridazole composition according to any one of claims 1 to 6 in the treatment of enteritis / salpingitis in laying hens.

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