A traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome and its preparation method

Abstract

The invention belongs to the technical field of Chinese medicine preparations, and in particular to a kind of Chinese medicine oral liquid for preventing and controlling blue ear disease of pigs and its preparation method. The present invention adopts ethanol percolation extraction after pulverizing rhubarb, coptis root, peppermint, bupleurum and schizonepeta to obtain extract A, scutellaria baicalensis, arctium lappa, dried tangerine peel, forsythia suspensa, cimicifuga heracleifolia, platycodon grandiflorum, liquorice, puffball, figwort, isatis root, indigo naturalis, talcum powder and percolation extraction medicine residues are subjected to water extraction and then reflux extraction of volatile oil, extract B and distillate are obtained, and then extract B is subjected to secondary alcohol precipitation and then N-methyl pyrrolidone and extract A and distillate are added, sodium benzoate and water are added, pH value is adjusted, and final product is obtained. In the Chinese medicine oral liquid for preventing and controlling blue ear disease provided by the present invention, effective ingredient content is high, stability is high and good, can effectively resist PRRSV virus, treat blue ear disease of pigs.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs and a preparation method thereof. Background Art

[0002] Blue ear disease (PRRS) is a highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). It affects pigs of all ages and is characterized by decreased reproductive capacity in sows, high fever, respiratory distress, and acute mortality in piglets. The incidence rate can reach 100%. Once infected, the disease can cause immunosuppression and lead to other secondary diseases, significantly impacting the development and socioeconomic benefits of pig farming.

[0003] Puji Xiaodu San (Puji Xiaodu San) is a traditional Chinese medicine powder included in the Veterinary Pharmacopoeia of the People's Republic of China (Part II, 2020 edition). It boasts the efficacy of clearing heat and detoxifying, dispersing wind and relieving swelling. It is clinically used to prevent and treat heat-toxin-induced upward surges in livestock and poultry. In this formula, Scutellaria baicalensis and Coptis chinensis clear and purge heat and toxins in the upper burner, serving as the main ingredients. Arctium lappa, Forsythia suspensa, Mentha arvense, Schizonepeta tenuifolia, Bupleurum chinense, and Cimicifuga heracleifolia dispel wind and expel pathogenic factors, serving as the assistant ingredients. Puffball, Isatis root, and Indigo naturalis detoxify, reduce swelling, and clear the throat. Scrophularia ningpoensis nourishes yin and reduces internal heat, Tangerine peel regulates qi and disperses stagnation, while Talc and Rhubarb guide heat downward, serving as the adjuvant ingredients. Platycodon grandiflorum invigorates the lungs, expels pathogenic factors, and guides the upward movement of pathogenic factors. Licorice root harmonizes the ingredients, serving as the guiding ingredient. Combined, these ingredients achieve the effects of clearing heat and detoxifying, dispersing wind and relieving swelling. This formula is effective in treating recurrent fever and persistent high fever in livestock and poultry caused by various viruses and bacteria. It is also highly effective in preventing and treating febrile viral diseases such as the currently prevalent "swine fever" and blue ear disease. However, under the intensive farming model, Chinese herbal powders have disadvantages such as uneven mixing and difficult quality control, which cannot meet market demand.

[0004] The preparation method for Puji Disinfection Granules, disclosed in patent CN108125912A, uses different treatments to extract different active ingredients. The extracts are dissolved in ethanol, starch and sucrose are then added to form granules. Since no purification is performed after extraction, impurities precipitate after dissolution in ethanol. The preparation method for Puji Disinfection Oral Liquid, disclosed in patent CN108079116A, uses different extracts from coptis chinensis, scutellaria baicalensis, dried tangerine peel, and forsythia suspensa to form an oral liquid. Because coptis chinensis contains berberine and scutellaria baicalensis contains baicalin, extracting them together can easily lead to chemical reactions and precipitation, thus affecting their content. Furthermore, herbs like mint and schizonepeta tenuifolia contain large amounts of volatile oils, which are lost during decoction, thus affecting their efficacy in dispelling wind and reducing swelling. Published patent CN106361987A describes a traditional Chinese medicine composition for treating blue ear disease in pigs. It boils and concentrates Cuscuta australis, Glycyrrhiza uralensis, Scutellaria baicalensis, and Bambusa guanyinensis with water, removes most impurities, but lacks preservatives, which can affect the stability of the liquid. Published patent CN104069478A describes a traditional Chinese medicine composition for preventing and treating blue ear disease in pigs and its preparation method. It mixes Radix Isatidis, Folium Isatidis, and Radix Puerariae, extracts the mixture by soaking in ethanol and water, and then filters and concentrates the mixture to form a finished product. The extraction and preparation process is safe, but the process simply soaks the medicinal materials in ethanol and water, resulting in a low extraction rate. Furthermore, the process lacks sterilization and preservatives, which can affect the stability of the liquid.

[0005] In summary, the existing technologies generally have technical problems such as low purity, unstable content and quality, lack of volatile oil, and poor efficacy. Summary of the Invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a Chinese medicine oral liquid for preventing and treating blue ear disease in pigs and a preparation method thereof. The Chinese medicine oral liquid for preventing and treating blue ear disease provided by the present invention has a high content of active ingredients, high stability, and can effectively fight against PRRSV virus and treat blue ear disease in pigs.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for preparing a Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs, comprising the following steps:

[0009] S1. Rhubarb, coptis root, mint, bupleurum root, and schizonepeta are mixed, ethanol aqueous solution is added, and percolation extraction is performed. The mixture is concentrated under reduced pressure to obtain extract A and medicinal residue A.

[0010] S2, take scutellaria baicalensis, arctium bark, tangerine peel, forsythia suspensa, cimicifuga, platycodon, licorice, puffball, figwort, isatis root, indigo naturalis, talc and the medicinal residue A prepared in step S1, add water for heating and refluxing extraction, collect the distillate, and filter the remaining medicinal liquid to obtain filtrate I and medicinal residue B, filtrate I is extract B;

[0011] S3, subjecting the extract B obtained in step S2 to alcohol precipitation treatment with ethanol, separating, and concentrating the supernatant under reduced pressure to obtain extract C;

[0012] S4. Add a stabilizer to the extract A obtained in step S1, stir evenly and heat, then add the extract C obtained in step S3, stir evenly and cool, add the distillate obtained in step S2, stir evenly, add purified water and sodium benzoate, stir to dissolve, adjust the pH value and filter to obtain the product.

[0013] During the research and development process of the present invention, the applicant discovered that the hydrochloride of berberine in the medicinal material Coptis chinensis has a low solubility in water and is easily combined with baicalin in the medicinal material Scutellaria baicalensis to form an insoluble salt, affecting the content of the main components and the clarity of the medicinal solution. In the present invention, Coptis chinensis and Scutellaria baicalensis are extracted separately, which not only significantly improves the extraction rate of berberine and baicalin, but also effectively improves the clarity of the oral solution.

[0014] Medicinal materials such as mint, bupleurum, and schizonepeta contain a large amount of fat-soluble volatile oil components, among which volatile oil is the main substance that exerts the effect of dispelling wind and reducing swelling. The present invention adopts percolation extraction followed by heating and reflux to extract volatile oil, which not only significantly improves the extraction rate of fat-soluble components in medicinal materials, but also significantly improves the efficacy of oral liquid in dispelling wind and reducing swelling.

[0015] Furthermore, in the preparation method of the Chinese herbal oral liquid for preventing and controlling blue ear disease in porcine, in step S1, rhubarb, coptis root, mint, bupleurum root, and schizonepeta are mixed and then crushed; the mass percentage of the ethanol aqueous solution is 60-70%; and the solution is concentrated under reduced pressure to a relative density of 1.06-1.10 g / ml.

[0016] Furthermore, the percolation extraction in step S1 of the preparation method of the Chinese herbal oral liquid for preventing and controlling blue ear disease in pigs is specifically as follows: rhubarb, coptis chinensis, mint, bupleurum, and schizonepeta are mixed, crushed, sieved, moistened with an appropriate amount of ethanol aqueous solution, and then loaded into a percolation device, and then added with 20 times ethanol aqueous solution to soak for 3 hours and then soak for 24 hours, and concentrated under reduced pressure to obtain extract A and residue A.

[0017] Furthermore, the step S2 of the preparation method of the Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs also includes decocting the residue B, filtering, collecting the filtrate II, combining the filtrate I and the filtrate II, and concentrating to obtain the extract B.

[0018] Furthermore, in the preparation method of the Chinese herbal oral liquid for preventing and controlling porcine blue ear disease, the filtration in step S2 is to pass through a 200-mesh sieve, heat under reflux for extraction for 2 hours, and decoct for 2 hours; and concentrate to a relative density of 1.08-1.10 g / ml.

[0019] Furthermore, in the step S3 of the preparation method of the Chinese herbal oral liquid for preventing and controlling blue ear disease in pigs, the ethanol added to the system has an alcohol content of 75%.

[0020] Furthermore, the alcohol precipitation treatment in step S3 of the method for preparing a traditional Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs is specifically as follows: ethanol is added to the extract B obtained in step S2 while stirring until the alcohol content in the system is 75%, and the mixture is allowed to stand at room temperature for 12 hours to obtain supernatant liquid I and precipitate I; 75% by weight ethanol aqueous solution is added to the precipitate I while stirring, and the mixture is allowed to stand at room temperature for 12 hours to obtain supernatant liquid II and precipitate II; supernatant liquid I and supernatant liquid II are combined and concentrated under reduced pressure to a relative density of 1.06-1.10 g / ml to obtain extract C. Puji Xiaodu Powder has a complex formula with many components and many impurities. The secondary alcohol precipitation performed in this application can not only significantly reduce the impurities in the liquid but also increase the content of the main components.

[0021] Furthermore, in the method for preparing a traditional Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs, the stabilizer in step S4 is N-methylpyrrolidone. N-methylpyrrolidone is a polar aprotic solvent. In the formulation of the present invention, it can not only effectively increase the solubility of the main ingredients of the traditional Chinese medicine, but also improve the stability of the traditional Chinese medicine oral liquid.

[0022] Furthermore, in the preparation method of the Chinese herbal oral liquid for preventing and controlling blue ear disease in porcine, the heating temperature in step S4 is 78-82°C, the cooling temperature is 37-45°C; and the pH value is adjusted to 4.5-5.0.

[0023] Furthermore, the weight dosage of each Chinese medicinal component in the preparation method of the Chinese medicinal oral liquid for preventing and controlling porcine blue ear disease is: 1-3 parts of rhubarb, 1-2 parts of scutellaria, 1-2 parts of coptis, 1 part of licorice, 1-2 parts of puffball, 1-2 parts of mint, 1-2 parts of Scrophularia, 3 parts of burdock, 1-2 parts of Cimicifuga, 1-2 parts of bupleurum, 1-2 parts of platycodon, 1-2 parts of tangerine peel, 2 parts of forsythia, 1-2 parts of schizonepeta, 2 parts of isatis root, 1-2 parts of indigo, 5-6 parts of talc, 0.1-0.5 parts of stabilizer, 20-40 parts of purified water, and 0.1-0.5 parts of sodium benzoate.

[0024] The present invention also provides a traditional Chinese medicine oral liquid for preventing and controlling blue ear disease of pigs, which is prepared by utilizing the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling blue ear disease of pigs.

[0025] Compared with the existing technology, the Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs and the preparation method thereof provided by the present invention have the following technical advantages:

[0026] (1) The present invention is based on actual production, combines traditional Chinese medicine theory with the characteristics of modern pharmaceutical technology, develops a new product of Chinese medicine oral liquid that can be administered through drinking water, and verifies the preventive and therapeutic effect of this product on blue ear disease through in vivo and in vitro research tests, providing technical support for the convenience of clinical medication;

[0027] (2) The present invention adopts the dynamic leaching percolation method to extract Chinese medicinal materials, which not only has a high solvent utilization rate, but also completely extracts the effective ingredients, and the leachate can be directly collected;

[0028] (3) The Chinese medicine oral liquid for preventing and controlling blue ear disease provided by the present invention contains Chinese medicine volatile oil, which has better therapeutic effect on highly pathogenic blue ear disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Comparison chart for clinical symptom scores of animals;

[0030] Figure 2 This is a comparison chart of animal weight gain;

[0031] Figure 3 The results of the cytotoxicity test;

[0032] Figure 4 Indirect immunofluorescence test: The inhibitory effect of Chinese herbal oral solution on different genotypes of PRRSV after 48 hours;

[0033] Figure 5 Indirect immunofluorescence test: The inhibitory effect of Chinese herbal medicine oral solution on PRRSV after 48 hours;

[0034] Figure 6 The results of the virus adsorption test;

[0035] Figure 7 The results of the virus penetration test;

[0036] Figure 8 The results of the virus replication test;

[0037] Note: Figure 2 Compared with the positive control group, * indicates significant difference, P < 0.05, ** indicates extremely significant difference, P < 0.01. DETAILED DESCRIPTION

[0038] The present invention is further described below by specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art can make various modifications based on the basic ideas of the present invention, but as long as they do not deviate from the basic ideas of the present invention, they are all within the scope of the present invention.

[0039] Example 1: Preparation method of a Chinese medicine oral liquid for preventing and controlling blue ear disease

[0040] A method for preparing a Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs, comprising the following steps:

[0041] S1. Rhubarb, coptis root, mint, bupleurum, and schizonepeta were mixed, crushed, and passed through a 40-mesh sieve. An appropriate amount of 65% by mass ethanol aqueous solution was added to moisten the mixture, and then the mixture was placed in a percolation apparatus. Then, 20 times the mass percentage of 65% by mass ethanol aqueous solution was added to soak the mixture for 3 h and then for 24 h. The mixture was concentrated under reduced pressure to a relative density of 1.08 g / ml to obtain extract A and medicinal residue A.

[0042] S2, take scutellaria baicalensis, burdock fruit, tangerine peel, forsythia, cimicifuga, platycodon, licorice, puffball, scrophulariaceae, isatis root, indigo naturalis, talcum powder and the medicinal residue A prepared in step S1, add 10 times the amount of water and heat reflux extraction for 2h, and collect the distillate, the remaining medicinal liquid is passed through a 200 mesh sieve to obtain filtrate I and medicinal residue B; and add 7 times the amount of water to the medicinal residue B, decoct for 2h, pass through a 200 mesh sieve, and collect filtrate II; combine filtrate I and filtrate II, and concentrate to a relative density of 1.09g / ml to obtain extract B;

[0043] S3. Adding ethanol to the extract B obtained in step S2 while stirring until the alcohol content in the system is 75%, and letting it stand at room temperature for 12 hours to obtain supernatant solution I and precipitate I; adding 75% by mass ethanol aqueous solution to the precipitate I while stirring, and letting it stand at room temperature for 12 hours to obtain supernatant solution II and precipitate II; combining supernatant solution I and supernatant solution II, and concentrating under reduced pressure to a relative density of 1.09 g / ml to obtain extract C;

[0044] S4. Add N-methylpyrrolidone to the extract A obtained in step S1, stir evenly, and heat to 80°C. Then add the extract C obtained in step S3, stir evenly, and cool to 40°C. Add the distillate obtained in step S2, stir evenly, add purified water and sodium benzoate, stir to dissolve, adjust the pH to 4.7, and filter to obtain the product.

[0045] The dosage of each Chinese medicinal component in the preparation method of the Chinese medicinal oral liquid for preventing and controlling porcine blue ear disease is as follows: 2 parts of rhubarb, 1.5 parts of scutellaria, 1.5 parts of coptis, 1 part of liquorice, 1.5 parts of puffball, 1.5 parts of mint, 1.5 parts of scrophularia, 3 parts of burdock fruit, 1.5 parts of cimicifuga, 1.5 parts of bupleurum, 1.5 parts of platycodon, 1.5 parts of tangerine peel, 2 parts of forsythia, 1.5 parts of schizonepeta, 2 parts of isatis root, 1.5 parts of indigo, 5.5 parts of talc, 0.5 part of N-methylpyrrolidone, 33 parts of purified water, and 0.1 part of sodium benzoate.

[0046] Example 2: Preparation method of a Chinese medicine oral liquid for preventing and controlling blue ear disease

[0047] A method for preparing a Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs, comprising the following steps:

[0048] S1. Rhubarb, coptis root, mint, bupleurum, and schizonepeta were mixed, crushed, and passed through a 40-mesh sieve. An appropriate amount of 60% by mass ethanol aqueous solution was added to moisten the mixture, and then the mixture was placed in a percolation apparatus. Then, 20 times the mass percentage of 60% by mass ethanol aqueous solution was added to soak the mixture for 3 h and then for 24 h. The mixture was concentrated under reduced pressure to a relative density of 1.06 g / ml to obtain extract A and medicinal residue A.

[0049] S2, take scutellaria baicalensis, arctium lappa, tangerine peel, forsythia suspensa, cimicifuga, platycodon, licorice, puffball, scrophulariaceae, isatis root, indigo naturalis, talcum powder and the medicinal residue A prepared in step S1, add 10 times the amount of water and heat reflux extraction for 2h, and collect the distillate, the remaining medicinal liquid is passed through a 200 mesh sieve to obtain filtrate I and medicinal residue B; and add 7 times the amount of water to the medicinal residue B, decoct for 2h, pass through a 200 mesh sieve, and collect filtrate II; combine filtrate I and filtrate II, concentrate to a relative density of 1.08g / ml, and obtain extract B;

[0050] S3. Add ethanol to the extract B obtained in step S2 while stirring until the alcohol content in the system is 75%, and let it stand at room temperature for 12 hours to obtain supernatant solution I and precipitate I; add 70% by mass ethanol aqueous solution to the precipitate I while stirring, and let it stand at room temperature for 12 hours to obtain supernatant solution II and precipitate II; combine supernatant solution I and supernatant solution II, and concentrate under reduced pressure to a relative density of 1.06 g / ml to obtain extract C;

[0051] S4. Add N-methylpyrrolidone to the extract A obtained in step S1, stir evenly, and heat to 80°C. Then add the extract C obtained in step S3, stir evenly, and cool to 40°C. Add the distillate obtained in step S2, stir evenly, add purified water and sodium benzoate, stir to dissolve, adjust the pH to 4.5, and filter to obtain the product.

[0052] The dosage of each Chinese medicinal component in the preparation method of the Chinese medicinal oral liquid for preventing and controlling porcine blue ear disease is as follows: 3 parts of rhubarb, 2 parts of scutellaria, 2 parts of coptis, 1 part of liquorice, 2 parts of puffball, 2 parts of mint, 2 parts of scrophularia, 3 parts of burdock fruit, 2 parts of cimicifuga, 2 parts of bupleurum, 2 parts of platycodon, 2 parts of tangerine peel, 2 parts of forsythia, 2 parts of schizonepeta, 2 parts of isatis root, 2 parts of indigo, 6 parts of talc, 0.3 parts of N-methylpyrrolidone, 40 parts of purified water, and 0.4 parts of sodium benzoate.

[0053] Example 3: Preparation method of a Chinese medicine oral liquid for preventing and controlling blue ear disease

[0054] A method for preparing a Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs, comprising the following steps:

[0055] S1. Rhubarb, coptis root, mint, bupleurum, and schizonepeta were mixed, crushed, and passed through a 40-mesh sieve. An appropriate amount of 70% by mass ethanol aqueous solution was added to moisten the mixture, and then the mixture was placed in a percolation apparatus. Then, 20 times the mass percentage of 70% by mass ethanol aqueous solution was added to soak the mixture for 3 h and then for 24 h. The mixture was concentrated under reduced pressure to a relative density of 1.10 g / ml to obtain extract A and medicinal residue A.

[0056] S2, take scutellaria baicalensis, arctium lappa, tangerine peel, forsythia suspensa, cimicifuga, platycodon, licorice, puffball, scrophulariaceae, isatis root, indigo naturalis, talcum powder and the medicinal residue A prepared in step S1, add 10 times the amount of water and carry out heating reflux extraction for 2h, and collect the distillate, and the remaining medicinal liquid is passed through a 200 mesh sieve to obtain filtrate I and medicinal residue B; and add 7 times the amount of water to the medicinal residue B, decoct for 2h, pass through a 200 mesh sieve, and collect filtrate II; combine filtrate I and filtrate II, and concentrate to a relative density of 1.10g / ml to obtain extract B;

[0057] S3. Add ethanol to the extract B obtained in step S2 while stirring until the alcohol content in the system is 75%, and let it stand at room temperature for 12 hours to obtain supernatant solution I and precipitate I; add 60% by mass ethanol aqueous solution to the precipitate I while stirring, and let it stand at room temperature for 12 hours to obtain supernatant solution II and precipitate II; combine supernatant solution I and supernatant solution II, and concentrate under reduced pressure to a relative density of 1.10 g / ml to obtain extract C;

[0058] S4. Add N-methylpyrrolidone to the extract A obtained in step S1, stir evenly, and heat to 80° C., then add the extract C obtained in step S3, stir evenly, and cool to 40° C., add the distillate obtained in step S2, stir evenly, add purified water and sodium benzoate, stir to dissolve, adjust the pH to 5.0, and filter to obtain the product.

[0059] The dosage of each Chinese medicinal component in the preparation method of the Chinese medicinal oral liquid for preventing and controlling porcine blue ear disease is as follows: 1 part of rhubarb, 1 part of scutellaria, 1 part of coptis, 1 part of liquorice, 1 part of puffball, 1 part of mint, 1 part of scrophularia, 3 parts of burdock fruit, 1 part of cimicifuga, 1 part of bupleurum, 1 part of platycodon, 1 part of tangerine peel, 2 parts of forsythia, 1 part of schizonepeta, 2 parts of isatis root, 1 part of indigo, 5 parts of talc, 0.1 part of N-methylpyrrolidone, 20 parts of purified water, and 0.5 part of sodium benzoate.

[0060] Comparative Example 1

[0061] This comparative example is similar to Example 2, and the difference between this comparative example and Example 2 is that: in step S1 of this comparative example, water extraction is adopted, and the specific method is: rhubarb, coptis chinensis, mint, bupleurum, and schizonepeta are mixed, crushed, passed through a 40-mesh sieve, added with 10 times the amount of deionized water, decocted for 12 hours, passed through a 200-mesh sieve, and the filtrate is concentrated under reduced pressure to a relative density of 1.06 g / ml to obtain extract A and medicinal residue A.

[0062] Comparative Example 2

[0063] This comparative example is similar to Example 2, but differs from Example 2 in that: in step S3 of this comparative example, a single alcohol precipitation is adopted, and the specific method is as follows: ethanol is added to the extract B obtained in step S2 under stirring until the alcohol content in the system is 75%, and the mixture is allowed to stand at room temperature for 12 hours to obtain supernatant solution I and precipitate I; deionized water is added to the precipitate I under stirring, and the mixture is allowed to stand at room temperature for 12 hours to obtain supernatant solution II and precipitate II; the supernatant solution I and the supernatant solution II are combined, and the mixture is concentrated under reduced pressure to a relative density of 1.06 g / ml to obtain extract C.

[0064] Comparative Example 3

[0065] This comparative example is similar to Example 2, and the difference between this comparative example and Example 2 is that an equal amount of sodium lauryl sulfate is used instead of N-methylpyrrolidone in step S4 of this comparative example.

[0066] Comparative Example 4

[0067] This comparative example is similar to Example 2, and the difference between this comparative example and Example 2 is that in step S4 of this comparative example, an equal amount of deionized water is used instead of the distillate prepared in step S2.

[0068] Experimental Example 1: Comparative Study on Single Extraction and Mixed Extraction of Coptis chinensis and Scutellaria baicalensis

[0069] Test method:

[0070] Chromatographic conditions: Column: Agilent HC-C18 (2) (4.6 mm × 250 mm, 5 μm); Mobile phase: methanol-0.1% phosphoric acid solution (50:50), flow rate: 1.0 mL min -1 ; Detection wavelength: 230nm; Column temperature: 30℃.

[0071] Preparation of reference solution: Accurately weigh 2.55 mg of baicalin reference substance and 3.09 mg of berberine hydrochloride reference substance, place them in 10 ml volumetric flasks respectively, dilute to the mark with methanol, and set aside.

[0072] Preparation of test solution: Coptidis rhizome and Scutellaria baicalensis were crushed into coarse powder (about 40 mesh sieve), 2g of Coptidis rhizome and 2g of Scutellaria baicalensis were mixed as a mixed sample for extraction, 2g of Coptidis rhizome was used as a Coptidis rhizome sample for separate extraction, and 2g of Scutellaria baicalensis was used as a Scutellaria baicalensis sample for separate extraction. 10 times the amount of 50% ethanol was added to the powder respectively, and extracted twice, each time for 1 hour. After each extraction, centrifuge at 4000r / min for 5 minutes, take the supernatant, accurately measure 1ml to a 10ml volumetric flask, dilute to the mark with methanol, filter, and set aside. Determine under the above chromatographic conditions and calculate the contents of baicalin and berberine hydrochloride in the test sample by the external standard method.

[0073] The test results are shown in Table 1.

[0074] Table 1 Comparison of single and mixed extraction results of Coptis chinensis and Scutellaria baicalensis

[0075]

[0076] As can be seen from Table 1, when Coptidis rhizome and Scutellaria baicalensis are extracted together, the contents of berberine hydrochloride and baicalin are greatly reduced, which shows that the separate extraction of Coptidis rhizome and Scutellaria baicalensis in the present invention effectively increases the contents of baicalin and berberine hydrochloride in the Chinese medicine oral liquid.

[0077] Test Example 2: Detection of Berberine Hydrochloride and Baicalin Content

[0078] Test samples: oral solutions prepared in Examples 1-3, Comparative Examples 1-2, and Comparative Example 4;

[0079] Test method:

[0080] Chromatographic conditions: Zorbax Eclipse SB C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.2% phosphoric acid (B) (triethylamine added to adjust pH = 2.5), gradient elution (0-5 min, 12% A; 5-10 min, 12%→20% A; 10-20 min, 20%→27% A; 20-25 min, 27%→31% A; 25-30 min, 31%→35% A); flow rate: 1.0 mL / min; detection wavelength: 275 nm; column temperature: 25°C; injection volume: 10 μL. Samples were injected and measured under the above chromatographic conditions.

[0081] Preparation of reference substance: Accurately weigh 10.21 mg of the reference substance baicalin, dissolve it in methanol, and dilute to 10 mL; dissolve 1.21 mg of berberine hydrochloride in methanol, and dilute to 10 mL. Shake well, take 1 mL and dilute to 10 mL with methanol to obtain reference substance stock solutions with concentrations of baicalin and berberine hydrochloride of 0.1021 and 0.0121 mg / mL, respectively.

[0082] Preparation of test solution: Take 1 ml of each of three batches of Puji Disinfection Oral Liquid samples, accurately measure into a 100 ml volumetric flask, dissolve in an appropriate amount of methanol, then dilute to the scale, shake well, and filter. Determine according to the above chromatographic conditions and calculate the content of baicalin and berberine hydrochloride in the test sample by the external standard method.

[0083] The test results are shown in Table 2.

[0084] Table 2 Test results of berberine hydrochloride and baicalin

[0085]

[0086] As can be seen from Table 2, the content of berberine hydrochloride in the Chinese medicine oral liquid for preventing and controlling blue ear disease provided by the present invention is 1.32-1.50 mg / mL, and the content of baicalin is 4.96-5.82 mg / mL. Among them, the contents of berberine hydrochloride and baicalin in the oral liquid prepared in Example 2 are both the highest, which is the best embodiment of the present invention.

[0087] Compared with Example 2, in Comparative Example 1, water extraction was used in step S1, but the content of berberine hydrochloride in the obtained oral liquid was greatly reduced, which indicates that percolation extraction can improve the extraction rate of berberine hydrochloride in Coptis chinensis; in Comparative Example 2, one-step alcohol precipitation was used in step S3, but the content of baicalin in the obtained oral liquid was greatly reduced, which indicates that the use of secondary alcohol precipitation in step S3 of the present invention can effectively increase the content of baicalin; in Comparative Example 4, the distillate prepared in step S2 was not added, but the content of baicalin in the obtained oral liquid was greatly reduced, which indicates that the present invention uses percolation extraction followed by heating and reflux to extract volatile oil, which can significantly improve the extraction rate of fat-soluble components in medicinal materials.

[0088] Test Example 3: Oral Liquid Stability Test

[0089] Test samples: oral liquids prepared in Examples 1-3 and Comparative Example 3.

[0090] Test method: After sterilization and disinfection, the test samples were packaged in sealed brown containers and placed at 40°C for 6 months. Samples were taken at 0, 1, 2, 3, 4, 5, and 6 months, and the properties, relative density, pH, microbial limits, and baicalin content were analyzed.

[0091] The test results are shown in Table 3.

[0092] Table 3 Oral solution stability test results

[0093]

[0094] * indicates a brown to brown-black liquid with a slightly bitter and astringent taste, a small amount of flocculent sediment, which will disperse with a light shake; # indicates a brown to brown-black liquid with a slightly bitter and astringent taste, a large amount of sediment, which will not disperse with a light shake; - indicates that the microbial detection is in compliance with regulations; + indicates that the microbial detection exceeds the standard.

[0095] As can be seen from Table 3, after being placed for 6 months, the Chinese herbal oral liquid for preventing and controlling porcine blue ear disease provided by the present invention has an appearance of brown to brown-black liquid, tastes slightly bitter and astringent, has a small amount of flocculent precipitate, which disperses with a light shake, and has no obvious precipitation. The relative density, pH value, and microbial limit all show no significant changes and meet the requirements.

[0096] Comparative Example 3 Compared with Example 2, an equal amount of sodium lauryl sulfate was used instead of N-methylpyrrolidone. However, the properties of the oral liquid prepared changed in the third month, the relative density became lower and lower, the pH value varied widely, and as the storage time increased, the microbial test exceeded the standard, and the baicalin content became lower and lower. This indicates that N-methylpyrrolidone in the present invention is a polar aprotic solvent, which can not only effectively improve the solubility of the main ingredients of traditional Chinese medicine in the formula of the present invention, but also improve the stability of the oral liquid of traditional Chinese medicine.

[0097] Test Example 4: Evaluation of the efficacy of the drug against blue ear disease

[0098] Test samples: oral liquid prepared in Example 2 (volatile oil group), Comparative Example 4 (oral liquid group), and commercially available Puji Xiaodu Powder (powder group) (Puji Xiaodu Powder was purchased from the Animal Health Products Factory of Guangdong Wenshi Dahuanong Biotechnology Co., Ltd., and its medicinal material formula can refer to the Puji Xiaodu Powder in Part II of the 2020 edition of the Chinese Veterinary Pharmacopoeia. Its preparation method is to crush, sieve, and mix the 17 medicinal materials in the formula);

[0099] Experimental Methods: 60 pigs were purchased and randomly divided into five groups of 12 each. Following the challenge, the pigs were administered medication according to the dosing schedule described in Table 4. All pigs were housed under identical experimental conditions. During the trial, clinical symptoms of the animals in each group were observed, recorded, and scored for 28 days after challenge. Animals were weighed and weight gain was recorded one day before and at the end of the trial.

[0100] Table 4. Dosage methods for pigs

[0101]

[0102] After the experiment, the clinical symptom scores of the animals in each group were shown in Figure 1 The weight gain of animals in each group was Figure 2 .

[0103] Depend on Figure 1It can be seen that the clinical symptom scores of the pigs in Example 2, Comparative Example 4 and Puji Xiaodu San groups after medication were significantly reduced compared with those in the positive control group. Among them, the clinical symptom scores of the pigs in Example 2 group decreased faster than those in Comparative Example 4 and Puji Xiaodu San groups.

[0104] Depend on Figure 2 It can be seen that after the experiment, compared with the positive control group, the weight gain of pigs in Example 2 group, Comparative Example 4 group and Puji Xiaodusan group was significantly increased (P>0.01), among which the weight gain of pigs in Example 2 group was closest to that of the negative control group.

[0105] Depend on Figure 1 、 Figure 2 The results show that the volatile oil contained in the Chinese medicine oral liquid for preventing and controlling blue ear disease of pigs of the present invention has a better therapeutic effect on highly pathogenic blue ear disease of pigs.

[0106] Test Example 5: In vitro anti-PRRSV test

[0107] 1. Experimental plan

[0108] 1.1 Test materials

[0109] DMEM culture medium, fetal bovine serum, PBS, CCK8 detection kit, reverse transcription kit, PCR kit, protease inhibitors, BCA protein concentration determination kit, SDS-PAGE gel preparation kit, SDS-PAGE electrophoresis buffer, transfer fluid, blocking fluid, antibody diluent, PRRSV N protein monoclonal antibody, horseradish peroxidase-labeled goat anti-rabbit IgG, and the traditional Chinese medicine oral liquid for preventing and controlling blue ear disease prepared in Example 2.

[0110] 1.2 Experimental cells and viruses

[0111] African green monkey fetal kidney Marc-145 cells, PRRSV strains: CH-1a, VR-2332, JXA1, TF.

[0112] 1.3 Test methods

[0113] 1.3.1 Recovery of Marc-145 cells

[0114] Remove a 1ml tube of Marc-145 cells from the liquid nitrogen tank and thaw in a 37°C water bath. Once thawed, place the tube in a clean biosafety cabinet. Add 6ml of 10% FBS DMEM and 1ml of Marc-145 cells to a 25T cell flask. Mix thoroughly by pipetting. After observation under a microscope, place the tube in a 37°C, 5% CO2 incubator. Subculture the cells when they reach 80%-90% saturation.

[0115] 1.3.2 Passaging and culture of Marc-145 cells

[0116] Observe under a microscope. When the cells have grown to a dense state, passage the Marc-145 cells. Add 1ml of trypsin to the 25T cell flask to digest the Marc-145 cells. Wash them twice, leaving 0.5ml for the third digestion. Place the cells in a 37°C, 5% CO2 incubator for 1 minute to digest. When the cells are suspended as a monolayer, passage them. Observe with the naked eye that the attached cells have fallen off and become a quicksand. 1 / 3 of the cells are passaged. Add 3ml of 10% FBS DMEM to terminate the digestion and pipette the cells evenly. Add 17ml of 10% FBS DMEM to the 75T cell flask. Transfer 3ml of the 25T cell flask to the 75T cell flask to ensure a 1 / 3 ratio of passage. Observe under a microscope and place the cells in a 37°C, 5% CO2 incubator.

[0117] 1.3.3 Cell seeding and culture

[0118] Take a vial of Marc-145 cells grown to a confluent state and adjust the cell concentration to 7.5 x 104 cells / ml using 10% FBS medium. Seed the cells into various cell culture plates according to the specific experimental requirements: 100 μl per well of a 96-well plate; 1 ml per well of a 24-well plate; and 2 ml per well of a 6-well plate. Incubate in a 37°C, 5% CO2 incubator for 24 hours before conducting subsequent experiments.

[0119] 1.3.4 TCID50 determination of PRRSV strains

[0120] Marc-145 cells were seeded in 96-well plates and allowed to adhere for 24 hours. Then, 100 μL of a 10-fold serial dilution of PRRSV (CH-1a, VR-2332, JXA1, and TF) was added to each well, with seven dilutions and eight replicate wells. A normal cell control was also established. The cells were cultured in a 37°C, 5% CO2 incubator. 100 μL of 2% FBS-containing DMEM was added to each well and incubated for 72 hours at 37°C, 5% CO2. Cytopathic effects were observed daily, and the TCID50 of each strain was calculated using the Reed-Muench method.

[0121] 1.3.5 Cytotoxicity of oral solution to Marc-145 cells

[0122] Marc-145 cells were seeded in 96-well plates and cultured for 24 hours. Then, 100 μl of a two-fold serial dilution of Puji Disinfection Oral Solution in DMEM was added to each well in duplicate. Cell controls and blank controls were also included. After incubation for 48 hours at 37°C in a 5% CO2 incubator, cytotoxicity was determined using a CCK8 assay, and the maximum safe concentration of the drug for the cells was calculated.

[0123] 1.3.6 Indirect immunofluorescence test

[0124] Marc-145 cells were seeded in a 48-well plate and the cell concentration was adjusted to 7.5 x 104 cells / ml using 10% FBS medium. After 24 hours, the cells were inoculated with 100 TCID50 of PRRSV (CH-1a strain, VR-2332 strain, JXA1 strain, TF strain) at 250 μL / well. After incubation at 37°C and 5% CO2 for 2 hours, the oral solution prepared in Example 2 was serially diluted 2-fold with 2% FBS DMEM medium and added to the cell plate at an equal volume of 1:1 with the virus solution. After incubation at 37°C and 5% CO2 for 48 hours, an indirect immunofluorescence assay was performed.

[0125] 1.3.7 Effect of oral solution on PRRSV

[0126] The maximum safe concentration for Marc-145 cells was used as the highest drug concentration for oral administration. Five concentrations were prepared by serial two-fold dilution using DMEM medium. The two-fold dilutions were mixed with equal volumes of 200 TCID50 PRRSV dilutions (drug and virus concentrations were halved after mixing) and incubated for 2 hours in a 37°C, 5% CO2 incubator (vortexed every 30 minutes). 500 μL of the solution was then added to a 24-well plate containing Marc-145 cells that had been adherently cultured for 24 hours, in triplicate. A blank control, a normal cell control, a ribavirin control, and a virus control were also established. The cells were incubated for 2 hours at 37°C, 5% CO2. The virus and drug mixture was discarded, the cells were washed twice with PBS, and 500 μL of 2% FBS medium was added to each well. The cells were incubated at 37°C, 5% CO2, for 48 hours. Cytopathic effect (CPE) was observed daily. Samples were collected and processed for Q-PCR and Western blot.

[0127] 1.3.8 Effect of oral solution on PRRSV

[0128] The maximum safe concentration for Marc-145 cells was used as the highest concentration of Puji Disinfection Oral Liquid. Five concentration gradients were prepared using DMEM culture medium. 500 μL of the solution was added to each well of a 24-well plate containing Marc-145 cells that had been adherently cultured for 24 hours. A blank control, normal cells, ribavirin, and virus controls were also set up. The cells were incubated at 37°C, 5% CO2 for 2 hours. The solution was discarded, and the cells were washed twice with PBS. Except for the cell control, 100 TCID50 of PRRSV virus dilution was added to each well and incubated at 37°C, 5% CO2 for another 2 hours (gently shaking every 30 minutes to ensure uniform distribution of virus particles). The virus solution was discarded, the cells were washed twice with PBS, and 500 μL of 2% FBS medium was added to each well. The cells were incubated at 37°C, 5% CO2 for 48 hours. Cytopathic effect (CPE) was observed daily. Samples were collected and processed for Q-PCR and Western blot.

[0129] 1.3.9 Inhibitory effect of oral solution on PRRSV

[0130] Marc-145 cells were cultured adherently for 24 hours in a 24-well plate. Except for the cell control, 500 μL of 100 TCID50 PRRSV virus dilution was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 2 hours (gently shaking every 30 minutes to ensure uniform distribution of virus particles). The virus solution was discarded, and the cells were washed twice with PBS. A 500 μL oral solution dilution (prepared with serial dilutions of DMEM culture medium, using the maximum safe concentration as the highest drug concentration) was added to each well in triplicate. A blank control, a cell control group, a ribavirin control group, and a virus control group were also set up. The cells were incubated at 37°C in a 5% CO2 incubator. After 2 hours, the culture medium was discarded, the cells were washed twice with PBS, and 500 μL of maintenance medium was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. Cytopathic effect (CPE) was observed daily. Samples were collected and processed for Q-PCR and Western blot.

[0131] 2. Test results

[0132] The results of cytotoxicity test were as follows Figure 3 As shown in FIG, the Chinese medicine oral solution prepared in Example 2 had no toxicity to Marc-145 cells when diluted 160 times. Figure 4 and Figure 5 As shown in Figure 2, the Chinese medicine oral solution reduced the viral content of different genotypes of PRRSV and Marc-145 cells in a dose-dependent manner. The results of the virus adsorption test are shown in Figure 2. Figure 6 As shown in Figure 2, Chinese medicine oral liquid can inhibit the adsorption of PRRSV TF strain on Marc-145 cells. Figure 7As shown in Figure 2, the Chinese medicine oral solution can inhibit the penetration of PRRSV TF strain into Marc-145 cells. Figure 8 As shown, the Chinese medicine oral liquid can inhibit the replication of PRRSV TF strain in Marc-145 cells.

[0133] It can be seen from the above test results that the Chinese herbal oral liquid for preventing and controlling porcine blue ear disease provided by the present invention inhibits the proliferation of different genetic subtypes of PRRSV in Marc1-145 cells in a dose-dependent manner, has no toxic effect on Marc1-145 cells when diluted 160 times, and has inhibitory effects on the adsorption, penetration and replication stages of the virus.

[0134] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for preparing a Chinese medicine oral liquid for preventing and controlling blue ear disease in pigs, characterized in that: The following steps are involved: S1. Rhubarb, coptis root, mint, bupleurum root, and schizonepeta are mixed, 60-70% ethanol aqueous solution is added, and the mixture is subjected to percolation extraction, and concentrated under reduced pressure to obtain extract A and medicinal residue A; S2, take scutellaria baicalensis, arctium bark, tangerine peel, forsythia suspensa, cimicifuga, platycodon, licorice, puffball, figwort, isatis root, indigo naturalis, talc and the medicinal residue A prepared in step S1, add water for heating and refluxing extraction, collect the distillate, and filter the remaining medicinal liquid to obtain filtrate I and medicinal residue B, filtrate I is extract B; S3, subjecting the extract B obtained in step S2 to alcohol precipitation with an ethanol aqueous solution, separating the extract, and concentrating the supernatant under reduced pressure to obtain extract C; S4, add a stabilizer to the extract A obtained in step S1, stir evenly and then heat, then add the extract C obtained in step S3, stir evenly and then cool, add the distillate obtained in step S2, stir evenly, add purified water and sodium benzoate, stir to dissolve, adjust the pH value and filter to obtain; the alcohol precipitation treatment in step S3 is specifically as follows: add ethanol to the extract B obtained in step S2 under stirring until the alcohol content in the system is 75%, let it stand at room temperature for 12 hours to obtain supernatant solution I and precipitate I; add 75% by mass ethanol aqueous solution to the precipitate I under stirring, let it stand at room temperature for 12 hours to obtain supernatant solution II and precipitate II; combine supernatant solution I and supernatant solution II to obtain extract C; the stabilizer in step S4 is N-methylpyrrolidone; adjust the pH value to 4.5-5.0; Among them, the weight dosage of each component is as follows: 1-3 parts of rhubarb, 1-2 parts of scutellaria, 1-2 parts of coptis, 1 part of licorice, 1-2 parts of puffball, 1-2 parts of mint, 1-2 parts of scrophularia, 3 parts of burdock, 1-2 parts of cimicifuga, 1-2 parts of bupleurum, 1-2 parts of platycodon, 1-2 parts of tangerine peel, 2 parts of forsythia, 1-2 parts of schizonepeta, 2 parts of isatis root, 1-2 parts of indigo, 5-6 parts of talc, 0.1-0.5 parts of stabilizer, 20-40 parts of purified water and 0.1-0.5 parts of sodium benzoate.

2. The method for preparing the Chinese medicine oral liquid for preventing and controlling blue ear disease according to claim 1, wherein In step S1, rhubarb, coptis root, mint, bupleurum root, and schizonepeta are mixed and then crushed; and concentrated under reduced pressure to a relative density of 1.06-1.10 g / mL.

3. The method for preparing the Chinese medicine oral liquid for preventing and controlling blue ear disease according to claim 1, wherein The percolation extraction in step S1 is specifically as follows: rhubarb, coptis root, mint, bupleurum, and schizonepeta are mixed, crushed, sieved, moistened with an appropriate amount of ethanol aqueous solution, and then loaded into a percolation device, and then soaked in 20 times ethanol aqueous solution for 3 hours and then for 24 hours, and concentrated under reduced pressure to obtain extract A and medicinal residue A.

4. The method for preparing the Chinese medicine oral liquid for preventing and controlling blue ear disease according to claim 1, wherein In step S2, the filtration is performed through a 200-mesh sieve, and the mixture is heated under reflux for extraction for 2 hours.

5. The method for preparing the Chinese medicine oral liquid for preventing and controlling blue ear disease according to claim 1, wherein The step S2 further includes decocting the medicinal residue B for 2 hours, filtering, collecting the filtrate II, combining the filtrate I and the filtrate II, and concentrating to a relative density of 1.08-1.10 g / mL to obtain the extract B.

6. A Chinese herbal oral liquid for preventing and controlling blue ear disease of pigs prepared by the preparation method of the Chinese herbal oral liquid for preventing and controlling blue ear disease of pigs according to any one of claims 1 to 5.

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