A preparation for porcine reproductive and respiratory syndrome and african swine fever and a preparation method thereof

Abstract

The application belongs to the technical field of veterinary drugs and provides a preparation for porcine blue ear disease and African swine fever and a preparation method thereof.The preparation comprises the following raw materials: modified rhizomary peucedani inclusion compound, nano astragalus-houttuynia cordata complex, medium-chain fatty acid glyceride, organic acid sustained-release microcapsule, fermented ammonium glycyrrhizinate, seleniumized isatis indigotica polysaccharide, modified quercetin, modified mannitol and a pharmaceutical carrier.The preparation for porcine blue ear disease and African swine fever provided by the application adds the modified rhizomary peucedani inclusion compound and the modified quercetin, improves the bioavailability through modification and directly inhibits viral replicase;the medium-chain fatty acid glyceride destroys the lipid layer of the viral envelope and blocks the invasion of the virus;the organic acid sustained-release microcapsule continuously releases in the intestinal tract, reduces the pH to inhibit the activity of the virus and adjusts the intestinal flora;after enzymatic nanofication, the nano astragalus-houttuynia cordata complex improves the immune stimulation activity of astragalus polysaccharide and houttuynia cordata flavones and promotes the phagocytosis of macrophages and the secretion of interferon.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug technology, and in particular relates to a preparation for porcine reproductive and respiratory syndrome (PRRS) and African swine fever, and its preparation method. Background Technology

[0002] This disease has been known by various names, including "mysterious swine disease," "new swine disease," "swine epidemic abortion and respiratory syndrome," "swine reproductive and respiratory syndrome," "blue ear disease," and "swine fever." In China, it is classified as a Class II animal disease. Porcine reproductive and respiratory syndrome (PRRS) is highly contagious through contact and exhibits endemic characteristics. The PRRS virus only infects pigs; other animals are not susceptible. Pigs of all ages and breeds can be infected, with piglets under one month old and pregnant sows being the most vulnerable.

[0003] African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boars. It is characterized by a short incubation period, with a mortality rate as high as 100% in the most acute and acute infections. Clinical manifestations include fever (reaching 40–42°C), rapid heartbeat, difficulty breathing, coughing in some cases, serous or mucopurulent discharge from the eyes and nose, cyanosis of the skin, and significant hemorrhage in the lymph nodes, kidneys, and gastrointestinal mucosa. Since the clinical symptoms of ASF are similar to those of classical swine fever (CSF), diagnosis relies solely on laboratory monitoring.

[0004] Porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF) are two devastating diseases in the pig industry, and existing prevention and control measures are inadequate: PRRS vaccines have low protection rates and cannot block ASFV infection; chemical drugs (such as tilmicosin) lead to drug resistance and have high hepatotoxicity and nephrotoxicity; traditional Chinese medicines (such as astragalus polysaccharide) have low bioavailability and weak antiviral effects. Summary of the Invention

[0005] This invention provides a formulation for treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever, aiming to solve the aforementioned problems.

[0006] This invention is achieved as follows: a formulation for treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever, comprising the following raw materials in parts by weight: 10-60 parts of modified polysaccharide inclusion complex, 40-80 parts of nano-Astragalus-Houttuynia cordata complex, 10-30 parts of medium-chain fatty acid glycerides, 8-40 parts of organic acid sustained-release microcapsules, 5-25 parts of fermented glycyrrhizic acid ammonium, 5-20 parts of selenized Isatis indigotica polysaccharide, 10-20 parts of modified quercetin, 5-10 parts of modified mannitol, and 20-100 parts of a pharmaceutical carrier (such as corn starch carrier).

[0007] Preferably, the raw materials include the following in parts by weight: 20-50 parts of modified polysaccharide inclusion complex, 50-70 parts of nano-Astragalus-Houttuynia cordata complex, 15-25 parts of medium-chain fatty acid glycerides, 18-30 parts of organic acid sustained-release microcapsules, 10-20 parts of fermented glycyrrhizic acid ammonium, 10-15 parts of selenized Isatis indigotica polysaccharide, 12-18 parts of modified quercetin, 6-9 parts of modified mannitol, and 40-80 parts of pharmaceutical carrier.

[0008] Preferably, the raw materials include the following in parts by weight: 35 parts of modified polysaccharide inclusion complex, 60 parts of nano-Astragalus-Houttuynia cordata complex, 20 parts of medium-chain fatty acid glycerides, 24 parts of organic acid sustained-release microcapsules, 15 parts of fermented glycyrrhizic acid ammonium, 12.5 parts of selenized Isatis indigotica polysaccharide, 15 parts of modified quercetin, 7.5 parts of modified mannitol, and 60 parts of pharmaceutical carrier.

[0009] Preferably, the modified polydextrin inclusion complex is prepared by mixing polydextrin and β-cyclodextrin at a molar ratio of 1:4-8 and treating with ultrasound at 40-60℃ and 200-500W for 20-40 minutes to improve the solubility of polydextrin through cyclodextrin inclusion.

[0010] Preferably, the preparation method of the nano-Astragalus-Houttuynia cordata complex is as follows:

[0011] Mix Astragalus membranaceus and Houttuynia cordata in a ratio of 2-5:1, add cellulase (enzyme activity ≥5000U / g), and enzymatically hydrolyze at 35-45℃ for 1-3 hours;

[0012] After high-pressure homogenization (1000-2000 bar) followed by liquid nitrogen freezing, the particles are ground to a size of 0.1-2 μm. Enzymatic hydrolysis and nano-sizing are combined to enhance the absorption of traditional Chinese medicine.

[0013] Preferably, the enzymatic hydrolysis uses a composite enzyme system containing cellulase, pectinase, and xylanase (mass ratio 3:1:1); the grinding is carried out in a liquid nitrogen environment at -196℃, and the grinding speed is 300-800 rpm.

[0014] Preferably, the organic acid sustained-release microcapsules are prepared by:

[0015] Dissolve citric acid and fumaric acid in water at a ratio of 1-3:1, and mix with 2-5 wt% sodium alginate;

[0016] The microcapsules are dropped into a coagulation solution containing 1-3 wt% calcium chloride and 0.5-2 wt% chitosan, dried, and then the organic acids are released into the intestine to maintain the antiviral pH.

[0017] Preferably, the preparation method of the fermented ammonium glycyrrhizate is as follows: glycyrrhizic acid and Lactobacillus plantarum are anaerobic fermented at 35-39℃ for 36-60h, and the pH is controlled at 5.5-6.5, so that the microbial fermentation transforms the immune-active components; the preparation method of the selenized Isatis indigotica polysaccharide is as follows: Isatis indigotica polysaccharide and sodium selenite are complexed at 60-80℃ for 2-4h in a mass ratio of 10:1-5:1.

[0018] Preferably, the modified quercetin is prepared by: compounding quercetin with soybean lecithin at a mass ratio of 1:2-5, evaporating the solvent to form a thin film, adding phosphate buffer (pH 7.4) for hydration, and ultrasonically treating to obtain a nanocomposite.

[0019] Preferably, the modified mannitol is prepared by:

[0020] Dissolve mannitol in 5-8 times its mass of isopropanol-water mixed solvent (volume ratio 3-4:1), add 2.0-2.5 times its molar amount of NaOH, and stir the reaction at 60-65℃ for 30 min.

[0021] Add chloroacetic acid in batches (in 3 batches, 20 min apart), controlling the molar ratio of chloroacetic acid to mannitol to be 0.5-0.7:1, and heat to 70-75℃ for 2-3 h.

[0022] Adjust the pH to 6.5-7.5 with glacial acetic acid, add 3 times the volume of ethanol to precipitate, and remove byproducts by ultrafiltration (molecular weight cutoff 1 kDa).

[0023] Spray drying (inlet air temperature 160℃, outlet air temperature 80℃) yields a white powder.

[0024] The present invention also provides a method for preparing an agent targeting porcine reproductive and respiratory syndrome (PRRS) and African swine fever, comprising the following steps:

[0025] Prepare all ingredients according to the formula;

[0026] Mix all raw materials under an inert atmosphere;

[0027] Sift the mixture (e.g., 60 mesh), dispense it into smaller portions, and then seal and store it away from light.

[0028] Compared with the prior art, the embodiments of this application have the following main advantages:

[0029] The formulations for porcine reproductive and respiratory syndrome (PRRS) and African swine fever provided by this invention incorporate modified polysaccharide inclusion complex and modified quercetin. These modifications enhance bioavailability and directly inhibit viral replication enzymes. Medium-chain fatty acid glycerides disrupt the viral envelope lipid layer, blocking viral invasion. Organic acid sustained-release microcapsules continuously release nutrients in the intestine, lowering pH to inhibit viral activity and simultaneously regulating intestinal flora. The nano-Astragalus-Houttuynia cordata complex, after enzymatic hydrolysis and nano-sizing, exhibits enhanced immunostimulatory activity of Astragalus polysaccharides and Houttuynia cordata flavonoids, promoting macrophage phagocytosis and interferon secretion. Selenized Isatis indigotica polysaccharides enhance antioxidant capacity through selenium. The formula works synergistically with the antiviral components of Isatis indigotica to enhance lymphocyte proliferation rate; fermented glycyrrhizic acid ammonium, after microbial transformation, has increased aglycone content, inhibiting excessive activation of the NF-κB pathway and reducing cytokine storm; the chitosan-sodium alginate coating of organic acid microcapsules enables colon-targeted release, avoiding gastric acid degradation; the addition of modified mannitol forms a hydrophilic-hydrophobic balance structure, enhancing mucosal adhesion, prolonging drug retention time in the intestine, and synergistically with modified quercetin, modified polysaccharide inclusion complex, and other components to improve virus adsorption and blocking, promote transmembrane transport, and enhance antioxidant and antiviral effects. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the preparation process of an agent for porcine reproductive and respiratory syndrome (PRRS) and African swine fever provided by this invention. Detailed Implementation

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0032] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0033] Example 1

[0034] This invention provides a formulation for treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF), comprising the following raw materials in parts by weight: 10 parts modified polysaccharide inclusion complex, 40 parts nano-Astragalus-Houttuynia cordata complex, 10 parts medium-chain fatty acid glycerides, 8 parts organic acid sustained-release microcapsules, 5 parts fermented glycyrrhizic acid ammonium, 5 parts selenized Isatis indigotica polysaccharide, 10 parts modified quercetin, 5 parts modified mannitol, and 20 parts pharmaceutical carrier (e.g., corn starch carrier). The preparation method of the formulation for PRRS and ASF described in this embodiment is as follows: Figure 1 As shown, it includes the following steps:

[0035] 1) Prepare all raw materials according to the formula;

[0036] The modified polydextrin inclusion complex is prepared by mixing polydextrin and β-cyclodextrin at a molar ratio of 1:4 and treating with ultrasound at 40°C and 200W for 20 minutes to improve the solubility of polydextrin through cyclodextrin inclusion.

[0037] The preparation method of the nano-Astragalus and Houttuynia cordata complex is as follows:

[0038] Astragalus and Houttuynia cordata were mixed in a 2:1 ratio, and cellulase (enzyme activity ≥5000U / g) was added and enzymatically hydrolyzed at 35℃ for 1 hour. The enzymatic hydrolysis used a complex enzyme system containing cellulase, pectinase and xylanase (mass ratio 3:1:1).

[0039] After high-pressure homogenization (1000 bar) and liquid nitrogen freezing, the particles were ground to a particle size of 0.1 μm. The grinding was carried out in a liquid nitrogen environment at -196℃ and the grinding speed was 300 rpm. The absorption of traditional Chinese medicine was enhanced by combining enzymatic hydrolysis with nano-processing.

[0040] The method for preparing the organic acid sustained-release microcapsules is as follows:

[0041] Dissolve citric acid and fumaric acid in water at a 1:1 ratio, and mix with 2wt% sodium alginate;

[0042] The microcapsules were dropped into a coagulation solution containing 1 wt% calcium chloride and 0.5 wt% chitosan and dried to obtain microcapsules. The organic acid was released into the intestines to maintain the antiviral pH.

[0043] The preparation method of the fermented ammonium glycyrrhizate is as follows: glycyrrhizic acid and Lactobacillus plantarum are anaerobic fermented at 35°C for 36 hours, and the pH is controlled at 5.5. The microbial fermentation transforms the immune-active components. The preparation method of the selenized Isatis indigotica polysaccharide is as follows: Isatis indigotica polysaccharide and sodium selenite are complexed at 60°C for 2 hours at a mass ratio of 10:1:1.

[0044] The modified quercetin is prepared by: compounding quercetin and soybean lecithin in a mass ratio of 1:2, evaporating the solvent to form a thin film, adding phosphate buffer (pH 7.4) for hydration, and ultrasonic treatment to obtain a nanocomposite.

[0045] The modified mannitol is prepared by:

[0046] Dissolve mannitol in 5 times its mass of isopropanol-water mixed solvent (volume ratio 3:1), add 2.0 times its molar amount of NaOH, and stir the reaction at 60℃ for 30 min.

[0047] Chloroacetic acid was added in batches (in 3 batches, 20 min apart), and the molar ratio of chloroacetic acid to mannitol was controlled at 0.5:1. The temperature was raised to 70°C and the reaction was carried out for 2 h.

[0048] Adjust the pH to 6.5 with glacial acetic acid, add 3 times the volume of ethanol to precipitate, and remove byproducts by ultrafiltration (molecular weight cutoff 1 kDa).

[0049] Spray drying (inlet air temperature 160℃, outlet air temperature 80℃) yields a white powder.

[0050] 2) Mix all raw materials under an inert atmosphere;

[0051] 3) Sieve the mixture (e.g., 60 mesh), dispense it into smaller portions, and then seal and store it away from light.

[0052] Example 2

[0053] This invention provides a formulation for treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF), comprising the following raw materials in parts by weight: 20 parts modified polysaccharide inclusion complex, 50 parts nano-Astragalus-Houttuynia cordata complex, 15 parts medium-chain fatty acid glycerides, 18 parts organic acid sustained-release microcapsules, 10 parts fermented glycyrrhizic acid ammonium, 10 parts selenized Isatis indigotica polysaccharide, 12 parts modified quercetin, 6 parts modified mannitol, and 40 parts pharmaceutical carrier (e.g., corn starch carrier). The preparation method of the formulation for PRRS and ASF described in this embodiment is as follows: Figure 1 As shown, it includes the following steps:

[0054] 1) Prepare all raw materials according to the formula;

[0055] The modified polydextrin inclusion complex is prepared by mixing polydextrin and β-cyclodextrin at a molar ratio of 1:4 and treating with ultrasound at 40°C and 200W for 20 minutes to improve the solubility of polydextrin through cyclodextrin inclusion.

[0056] The preparation method of the nano-Astragalus and Houttuynia cordata complex is as follows:

[0057] Astragalus and Houttuynia cordata were mixed in a 2:1 ratio, and cellulase (enzyme activity ≥5000U / g) was added and enzymatically hydrolyzed at 35℃ for 1 hour. The enzymatic hydrolysis used a complex enzyme system containing cellulase, pectinase and xylanase (mass ratio 3:1:1).

[0058] After high-pressure homogenization (1000 bar) and liquid nitrogen freezing, the particles were ground to a particle size of 0.1 μm. The grinding was carried out in a liquid nitrogen environment at -196℃ and the grinding speed was 300 rpm. The absorption of traditional Chinese medicine was enhanced by combining enzymatic hydrolysis with nano-processing.

[0059] The method for preparing the organic acid sustained-release microcapsules is as follows:

[0060] Dissolve citric acid and fumaric acid in water at a 1:1 ratio, and mix with 2wt% sodium alginate;

[0061] The microcapsules were dropped into a coagulation solution containing 1 wt% calcium chloride and 0.5 wt% chitosan and dried to obtain microcapsules. The organic acid was released into the intestines to maintain the antiviral pH.

[0062] The preparation method of fermented ammonium glycyrrhizate is as follows: glycyrrhizic acid and Lactobacillus plantarum are anaerobic fermented at 35°C for 36 hours, and the pH is controlled at 5.5. The microbial fermentation transforms the immune-active components. The preparation method of selenized Isatis indigotica polysaccharide is as follows: Isatis indigotica polysaccharide and sodium selenite are complexed at 60°C for 2 hours at a mass ratio of 10:1:1.

[0063] The modified quercetin is prepared by: compounding quercetin and soybean lecithin in a mass ratio of 1:2, evaporating the solvent to form a thin film, adding phosphate buffer (pH 7.4) for hydration, and ultrasonic treatment to obtain a nanocomposite.

[0064] The modified mannitol is prepared by:

[0065] Dissolve mannitol in 5 times its mass of isopropanol-water mixed solvent (volume ratio 3:1), add 2.0 times its molar amount of NaOH, and stir the reaction at 60℃ for 30 min.

[0066] Chloroacetic acid was added in batches (in 3 batches, 20 min apart), and the molar ratio of chloroacetic acid to mannitol was controlled at 0.5:1. The temperature was raised to 70°C and the reaction was carried out for 2 h.

[0067] Adjust the pH to 6.5 with glacial acetic acid, add 3 times the volume of ethanol to precipitate, and remove byproducts by ultrafiltration (molecular weight cutoff 1 kDa).

[0068] Spray drying (inlet air temperature 160℃, outlet air temperature 80℃) yields a white powder.

[0069] 2) Mix all raw materials under an inert atmosphere;

[0070] 3) Sieve the mixture (e.g., 60 mesh), dispense it into smaller portions, and then seal and store it away from light.

[0071] Example 3

[0072] This invention provides a formulation for treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF), comprising the following raw materials in parts by weight: 35 parts modified polysaccharide inclusion complex, 60 parts nano-Astragalus-Houttuynia cordata complex, 20 parts medium-chain fatty acid glycerides, 24 parts organic acid sustained-release microcapsules, 15 parts fermented glycyrrhizic acid ammonium, 12.5 parts selenized Isatis indigotica polysaccharide, 15 parts modified quercetin, 7.5 parts modified mannitol, and 60 parts pharmaceutical carrier (e.g., corn starch carrier). The preparation method of the formulation for PRRS and ASF described in this embodiment is as follows: Figure 1 As shown, it includes the following steps:

[0073] 1) Prepare all raw materials according to the formula;

[0074] The modified polydextrin inclusion complex is prepared by mixing polydextrin and β-cyclodextrin at a molar ratio of 1:4-8 and treating with ultrasound at 50°C and 350W for 30 minutes to improve the solubility of polydextrin through cyclodextrin inclusion.

[0075] The preparation method of the nano-Astragalus-Houttuynia cordata complex is as follows:

[0076] Astragalus and Houttuynia cordata were mixed at a ratio of 3.5:1, and cellulase (enzyme activity ≥5000U / g) was added and enzymatically hydrolyzed at 40℃ for 2 hours. The enzymatic hydrolysis used a complex enzyme system containing cellulase, pectinase and xylanase (mass ratio 3:1:1).

[0077] After high-pressure homogenization (1500 bar) and liquid nitrogen freezing, the particles were ground to a particle size of 0.15 μm. The grinding was carried out in a liquid nitrogen environment at -196℃ and the grinding speed was 550 rpm. The absorption of traditional Chinese medicine was enhanced by combining enzymatic hydrolysis and nano-sizing.

[0078] The method for preparing the organic acid sustained-release microcapsules is as follows:

[0079] Dissolve citric acid and fumaric acid in water at a ratio of 2:1, and mix with 3.5 wt% sodium alginate;

[0080] The microcapsules were dropped into a coagulation solution containing 2 wt% calcium chloride and 1.25 wt% chitosan and dried to obtain microcapsules. The organic acid was released into the intestines to maintain the antiviral pH.

[0081] The preparation method of the fermented ammonium glycyrrhizate is as follows: glycyrrhizic acid and Lactobacillus plantarum are anaerobic fermented at 37°C for 48 hours, and the pH is controlled at 6.0. The microbial fermentation transforms the immune-active components. The preparation method of the selenized Isatis indigotica polysaccharide is as follows: Isatis indigotica polysaccharide and sodium selenite are complexed at 70°C for 3 hours at a mass ratio of 10:3:1.

[0082] The modified quercetin is prepared by: compounding quercetin with soybean lecithin at a mass ratio of 1:3.5, evaporating the solvent to form a thin film, adding phosphate buffer (pH 7.4) for hydration, and ultrasonic treatment to obtain a nanocomposite.

[0083] The modified mannitol is prepared by:

[0084] Dissolve mannitol in 6.5 times its mass of isopropanol-water mixed solvent (volume ratio 3.5:1), add 2.25 times its molar amount of NaOH, and stir the reaction at 62.5℃ for 30 min.

[0085] Chloroacetic acid was added in batches (in 3 batches, 20 min apart), with the molar ratio of chloroacetic acid to mannitol controlled at 0.6:1, and the temperature was raised to 72.5℃ and the reaction was carried out for 2.5 h.

[0086] Adjust the pH to 7.0 with glacial acetic acid, add 3 times the volume of ethanol to precipitate, and remove byproducts by ultrafiltration (molecular weight cutoff 1 kDa).

[0087] Spray drying (inlet air temperature 160℃, outlet air temperature 80℃) yields a white powder.

[0088] 2) Mix all raw materials under an inert atmosphere;

[0089] 3) Sieve the mixture (e.g., 60 mesh), dispense it into smaller portions, and then seal and store it away from light.

[0090] Example 4

[0091] This invention provides a formulation for treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF), comprising the following raw materials in parts by weight: 50 parts modified polysaccharide inclusion complex, 70 parts nano-Astragalus-Houttuynia cordata complex, 25 parts medium-chain fatty acid glycerides, 30 parts organic acid sustained-release microcapsules, 20 parts fermented glycyrrhizic acid ammonium, 15 parts selenized Isatis indigotica polysaccharide, 18 parts modified quercetin, 9 parts modified mannitol, and 80 parts pharmaceutical carrier (e.g., corn starch carrier). The preparation method of the formulation for PRRS and ASF described in this embodiment is as follows: Figure 1 As shown, it includes the following steps:

[0092] 1) Prepare all raw materials according to the formula;

[0093] The modified polydextrin inclusion complex is prepared by mixing polydextrin and β-cyclodextrin at a molar ratio of 1:8 and treating with ultrasound at 60°C and 500W for 40 minutes to improve the solubility of polydextrin through cyclodextrin inclusion.

[0094] The preparation method of the nano-Astragalus and Houttuynia cordata complex is as follows:

[0095] Astragalus and Houttuynia cordata were mixed in a 5:1 ratio, and cellulase (enzyme activity ≥5000U / g) was added and enzymatically hydrolyzed at 45℃ for 3 hours. The enzymatic hydrolysis used a complex enzyme system containing cellulase, pectinase and xylanase (mass ratio 3:1:1).

[0096] After high-pressure homogenization (2000 bar) and liquid nitrogen freezing, the particles were ground to a size of 0.2 μm. The grinding was carried out in a liquid nitrogen environment at -196℃ and the grinding speed was 800 rpm. The absorption of traditional Chinese medicine was enhanced by combining enzymatic hydrolysis with nano-processing.

[0097] The method for preparing the organic acid sustained-release microcapsules is as follows:

[0098] Dissolve citric acid and fumaric acid in water at a ratio of 3:1, and mix with 5wt% sodium alginate;

[0099] The microcapsules were dropped into a coagulation solution containing 3 wt% calcium chloride and 0.2 wt% chitosan and dried to obtain microcapsules. Organic acids were released into the intestines to maintain the antiviral pH.

[0100] The preparation method of fermented ammonium glycyrrhizate is as follows: glycyrrhizic acid and Lactobacillus plantarum are anaerobic fermented at 39°C for 60 h, and the pH is controlled at 6.5. The microbial fermentation transforms the immune-active components. The preparation method of selenized Isatis indigotica polysaccharide is as follows: Isatis indigotica polysaccharide and sodium selenite are complexed at 80°C for 4 h at a mass ratio of 10:5:1.

[0101] The modified quercetin is prepared by: compounding quercetin and soybean lecithin at a mass ratio of 1:5, evaporating the solvent to form a thin film, adding phosphate buffer (pH 7.4) for hydration, and ultrasonic treatment to obtain a nanocomposite.

[0102] The modified mannitol is prepared by:

[0103] Dissolve mannitol in 8 times its mass of isopropanol-water mixed solvent (volume ratio 4:1), add 2.5 times its molar amount of NaOH, and stir the reaction at 65℃ for 30 min.

[0104] Chloroacetic acid was added in batches (in 3 batches, 20 min apart), and the molar ratio of chloroacetic acid to mannitol was controlled at 0.7:1. The temperature was raised to 75°C and the reaction was carried out for 3 h.

[0105] Adjust the pH to 7.5 with glacial acetic acid, add 3 times the volume of ethanol to precipitate, and remove byproducts by ultrafiltration (molecular weight cutoff 1 kDa).

[0106] Spray drying (inlet air temperature 160℃, outlet air temperature 80℃) yields a white powder.

[0107] 2) Mix all raw materials under an inert atmosphere;

[0108] 3) Sieve the mixture (e.g., 60 mesh), dispense it into smaller portions, and then seal and store it away from light.

[0109] Example 5

[0110] This invention provides a formulation for treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF), comprising the following raw materials in parts by weight: 60 parts modified polysaccharide inclusion complex, 80 parts nano-Astragalus-Houttuynia cordata complex, 30 parts medium-chain fatty acid glycerides, 40 parts organic acid sustained-release microcapsules, 25 parts fermented glycyrrhizic acid ammonium, 20 parts selenized Isatis indigotica polysaccharide, 20 parts modified quercetin, 10 parts modified mannitol, and 100 parts pharmaceutical carrier (e.g., corn starch carrier). The preparation method of the formulation for PRRS and ASF described in this embodiment is as follows: Figure 1 As shown, it includes the following steps:

[0111] 1) Prepare all raw materials according to the formula;

[0112] The modified polydextrin inclusion complex is prepared by mixing polydextrin and β-cyclodextrin at a molar ratio of 1:8 and treating with ultrasound at 60°C and 500W for 40 minutes to improve the solubility of polydextrin through cyclodextrin inclusion.

[0113] The preparation method of the nano-Astragalus and Houttuynia cordata complex is as follows:

[0114] Astragalus and Houttuynia cordata were mixed in a 5:1 ratio, and cellulase (enzyme activity ≥5000U / g) was added and enzymatically hydrolyzed at 45℃ for 3 hours. The enzymatic hydrolysis used a complex enzyme system containing cellulase, pectinase and xylanase (mass ratio 3:1:1).

[0115] After high-pressure homogenization (2000 bar) and liquid nitrogen freezing, the particles were ground to a size of 0.2 μm. The grinding was carried out in a liquid nitrogen environment at -196℃ and the grinding speed was 800 rpm. The absorption of traditional Chinese medicine was enhanced by combining enzymatic hydrolysis with nano-processing.

[0116] The method for preparing the organic acid sustained-release microcapsules is as follows:

[0117] Dissolve citric acid and fumaric acid in water at a ratio of 3:1, and mix with 5wt% sodium alginate;

[0118] The microcapsules were dropped into a coagulation solution containing 3 wt% calcium chloride and 0.2 wt% chitosan and dried to obtain microcapsules. Organic acids were released into the intestines to maintain the antiviral pH.

[0119] The preparation method of fermented ammonium glycyrrhizate is as follows: glycyrrhizic acid and Lactobacillus plantarum are anaerobic fermented at 39°C for 60 h, and the pH is controlled at 6.5. The microbial fermentation transforms the immune-active components. The preparation method of selenized Isatis indigotica polysaccharide is as follows: Isatis indigotica polysaccharide and sodium selenite are complexed at 80°C for 4 h at a mass ratio of 10:5:1.

[0120] The modified quercetin is prepared by: compounding quercetin and soybean lecithin at a mass ratio of 1:5, evaporating the solvent to form a thin film, adding phosphate buffer (pH 7.4) for hydration, and ultrasonic treatment to obtain a nanocomposite.

[0121] The modified mannitol is prepared by:

[0122] Dissolve mannitol in 8 times its mass of isopropanol-water mixed solvent (volume ratio 4:1), add 2.5 times its molar amount of NaOH, and stir the reaction at 65℃ for 30 min.

[0123] Chloroacetic acid was added in batches (in 3 batches, 20 min apart), and the molar ratio of chloroacetic acid to mannitol was controlled at 0.7:1. The temperature was raised to 75°C and the reaction was carried out for 3 h.

[0124] Adjust the pH to 7.5 with glacial acetic acid, add 3 times the volume of ethanol to precipitate, and remove byproducts by ultrafiltration (molecular weight cutoff 1 kDa).

[0125] Spray drying (inlet air temperature 160℃, outlet air temperature 80℃) yields a white powder.

[0126] 2) Mix all raw materials under an inert atmosphere;

[0127] 3) Sieve the mixture (e.g., 60 mesh), dispense it into smaller portions, and then seal and store it away from light.

[0128] Comparative Example 1: Compared with Example 3, the modified polysaccharide inclusion complex was missing (replaced with an equal amount of pharmaceutical carrier).

[0129] Comparative Example 2: Compared with Example 3, modified quercetin was missing (replaced with an equal amount of pharmaceutical carrier).

[0130] Comparative Example 3: Compared with Example 3, modified mannitol was missing (replaced with an equal amount of pharmaceutical carrier).

[0131] Comparative Example 4: Compared with Example 3, the modified polysaccharide inclusion complex, modified quercetin, and modified mannitol were missing (replaced with an equal amount of pharmaceutical carrier).

[0132] Comparative Example 5: Compared with Example 3, the nano-Astragalus-Houttuynia cordata complex was missing (replaced with an equal amount of pharmaceutical carrier).

[0133] Comparative Example 6: Compared with Example 3, ordinary polygalactoside (unmodified) was used instead of the modified polygalactoside inclusion complex.

[0134] Comparative Example 7: Compared with Example 3, ordinary quercetin (unmodified) was used instead of modified quercetin.

[0135] Comparative Example 8: Compared with Example 3, ordinary mannitol (unmodified) was used instead of modified mannitol.

[0136] Comparative Example 9: A commercially available Chinese herbal medicine compound (Astragalus membranaceus + Isatis indigotica).

[0137] Experimental Test

[0138] Animal model: 60kg healthy sows (PRRSV / ASFV double negative), 10 sows per group;

[0139] Challenge method: PRRSV (JXA1 strain, 10 5 TCID 50 )+ASFV (Georgia 2007 strain, 10 4 HAD 50 Combined intramuscular injection;

[0140] Dosage regimen: Add 0.3% of the formulation to the feed for 7 consecutive days (starting 3 days before the challenge).

[0141] Testing time: 7 days after infection.

[0142] The antiviral test results are shown in Table 1 below:

[0143] Table 1. Antiviral Test Results

[0144] Group PRRSV inhibition rate (%) ASFV inactivation rate (%) Survival rate (%) PRRSV load in lung tissue (log) Lymph node ASFV load (log) Example 1 92.5 94.3 80 3.8 3.0 Example 2 95.8 96.7 90 2.9 2.3 Example 3 98.7 99.1 95 2.1 1.8 Example 4 97.2 98.0 90 2.5 2.0 Example 5 96.0 97.5 85 3.0 2.5 Comparative Example 1 61.3 85.2 40 5.6 4.8 Comparative Example 2 83.7 88.9 60 4.2 3.7 Comparative Example 3 76.4 82.1 50 4.8 4.2 Comparative Example 4 42.1 68.5 20 6.5 5.9 Comparative Example 5 71.6 80.3 55 4.5 4.0 Comparative Example 6 52.8 70.5 30 6.1 5.3 Comparative Example 7 68.9 75.3 35 5.4 4.7 Comparative Example 8 74.2 79.6 45 4.9 4.1 Comparative Example 9 47.5 - 10 7.2 6.5

[0145] The results above show that the formulation prepared in this invention has good preventive and therapeutic effects against porcine reproductive and respiratory syndrome (PRRS) and African swine fever.

[0146] The results of the immune regulation and safety tests are shown in Table 2 below:

[0147] Table 2 Results of Immune Regulation and Safety Tests

[0148] Group CD4⁺ / CD8⁺ ratio <![CDATA[IgG antibody (OD 450 )]]> ALT (U / L) Discontinuation period (days) Average daily feed intake (kg) Example 3 2.8±0.3 1.85±0.15 38±5 3 2.8±0.2 Comparative Example 1 1.2±0.2 0.72±0.10 42±6 7 2.1±0.3 Comparative Example 2 1.6±0.3 1.05±0.12 45±4 5 2.3±0.2 Comparative Example 3 1.4±0.2 0.93±0.11 50±7 6 2.0±0.4 Comparative Example 4 0.9±0.1 0.61±0.09 58±8 10 1.8±0.3 Comparative Example 5 1.3±0.2 0.68±0.08 47±6 8 2.2±0.3 Comparative Example 6 1.1±0.2 0.65±0.07 62±9 9 1.9±0.2 Comparative Example 7 1.0±0.1 0.59±0.06 60±7 9 2.0±0.3 Comparative Example 8 1.3±0.2 0.71±0.08 55±6 7 2.1±0.2 Comparative Example 9 0.8±0.1 0.51±0.08 65±10 14 1.5±0.4 healthy pigs 1.5-2.0 0.1-0.3 10-50 - 3.0±0.5

[0149] The results above show that the formulation prepared by this invention has a short withdrawal period, improves breeding efficiency, and has good palatability.

[0150] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0151] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of 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 combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A preparation for preventing and treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever, characterized in that, It is composed of the following raw materials in parts by weight: 10-60 parts of modified polysaccharide inclusion complex, 40-80 parts of nano-Astragalus-Houttuynia cordata complex, 10-30 parts of medium-chain fatty acid glycerides, 8-40 parts of organic acid sustained-release microcapsules, 5-25 parts of fermented glycyrrhizic acid ammonium, 5-20 parts of selenized Isatis indigotica polysaccharide, 10-20 parts of modified quercetin, 5-10 parts of modified mannitol, and 20-100 parts of pharmaceutical carrier; The modified polydextrin inclusion complex is prepared by mixing polydextrin and β-cyclodextrin at a molar ratio of 1:4-8 and treating with ultrasound at 40-60℃ and 200-500W for 20-40 minutes. The preparation method of the nano-Astragalus-Houttuynia cordata complex is as follows: Astragalus and Houttuynia cordata are mixed at a ratio of 2-5:1, cellulase is added and enzymatically hydrolyzed at 35-45℃ for 1-3 hours; after high-pressure homogenization, liquid nitrogen freezing is performed, and the particles are ground to a particle size of 0.1-2μm. The organic acid sustained-release microcapsules are prepared by dissolving citric acid and fumaric acid in water at a ratio of 1-3:1 and mixing them with 2-5 wt% sodium alginate; then adding the mixture to a coagulation solution containing 1-3 wt% calcium chloride and 0.5-2 wt% chitosan and drying to obtain microcapsules. The preparation method of the fermented ammonium glycyrrhizate is as follows: glycyrrhizic acid and Lactobacillus plantarum are anaerobic fermented at 35-39℃ for 36-60h, and the pH is controlled at 5.5-6.5, so that the microbial fermentation transforms the immune active components; the preparation method of the selenized Isatis indigotica polysaccharide is as follows: Isatis indigotica polysaccharide and sodium selenite are complexed at 60-80℃ for 2-4h at a mass ratio of 10:1-5:

1. The modified quercetin is prepared by: compounding quercetin with soybean lecithin at a mass ratio of 1:2-5, evaporating the solvent to form a thin film, adding phosphate buffer for hydration, and ultrasonically treating to obtain a nanocomposite. The modified mannitol is prepared as follows: mannitol is dissolved in 5-8 times its mass of isopropanol-water mixed solvent, and 2.0-2.5 times its molar amount of NaOH is added. The mixture is stirred at 60-65℃ for 30 min. Chloroacetic acid is added in batches, controlling the molar ratio of chloroacetic acid to mannitol to be 0.5-0.7:

1. The temperature is raised to 70-75℃ and the reaction is carried out for 2-3 h. The pH is adjusted to 6.5-7.5 with glacial acetic acid, and 3 times its volume of ethanol is added to precipitate the product. Byproducts are removed by ultrafiltration. The product is spray-dried to obtain a white powder.

2. The preparation for preventing and treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever as described in claim 1, characterized in that, It is composed of the following raw materials in parts by weight: 20-50 parts of modified polysaccharide inclusion complex, 50-70 parts of nano-Astragalus-Houttuynia cordata complex, 15-25 parts of medium-chain fatty acid glycerides, 18-30 parts of organic acid sustained-release microcapsules, 10-20 parts of fermented glycyrrhizic acid ammonium, 10-15 parts of selenized Isatis indigotica polysaccharide, 12-18 parts of modified quercetin, 6-9 parts of modified mannitol, and 40-80 parts of pharmaceutical carrier.

3. The preparation for preventing and treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever as described in claim 2, characterized in that, It is composed of the following raw materials in parts by weight: 35 parts modified polysaccharide inclusion complex, 60 parts nano-Astragalus-Houttuynia cordata complex, 20 parts medium-chain fatty acid glycerides, 24 parts organic acid sustained-release microcapsules, 15 parts fermented glycyrrhizic acid ammonium, 12.5 parts selenized Isatis indigotica polysaccharide, 15 parts modified quercetin, 7.5 parts modified mannitol, and 60 parts pharmaceutical carrier.

4. The method for preparing the formulation for preventing and treating porcine reproductive and respiratory syndrome (PRRS) and African swine fever as described in any one of claims 1-3, characterized in that, Includes the following steps: Prepare all ingredients according to the formula; Mix all raw materials under an inert atmosphere; The mixture is sieved, dispensed, sealed, and stored away from light.

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