A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease and its preparation method

Abstract

The present invention belongs to the field of medicine and disease treatment technology, and specifically relates to a traditional Chinese medicine compound preparation for preventing Haemophilus parasuis infection in pigs and a preparation method thereof. The traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 2-24 parts of Coptis chinensis, 2-24 parts of Phellodendron chinense, and 1-12 parts of Prunus mume. The traditional Chinese medicine compound preparation prepared by the present invention has an immunomodulatory effect, which can affect the body's immune system to promote the production of antibodies and thus mediate an in vivo bactericidal effect. The results of this study show that the traditional Chinese medicine compound Phellodendron chinense has a certain preventive effect on Haemophilus parasuis infection type 13, providing an effective direction and basis for the prevention and control of Haemophilus parasuis infection.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and disease treatment, and particularly relates to a traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease and a preparation method thereof. Background Art

[0002] Haemophilus parasuis (G. parasuis) is a common pathogen that colonizes the upper respiratory tract of pigs and is a common cause of respiratory diseases in the swine industry. It can cause systemic inflammatory diseases in pigs and is susceptible to mixed infections with multiple bacteria, increasing mortality rates and causing significant losses to the global swine industry. Effectively preventing and controlling G. parasuis disease remains a challenge.

[0003] Currently, prevention of Haemophilus parasuis mainly relies on vaccines and antibiotics. However, due to the numerous serotypes of the bacterium, the immune response is limited, making it ineffective in preventing and controlling the disease. In recent years, the widespread use of traditional Chinese medicine (TCM) in the prevention and control of bacterial infections in the livestock industry has also provided insights into its potential for preventing and controlling Haemophilus parasuis. As a natural resource, Chinese herbal medicines contain numerous trace substances beneficial to the animal body, possessing antibacterial, anti-inflammatory, and immune-regulating properties. They also have minimal adverse reactions, lack drug resistance, and are inexpensive, offering unique advantages for both disease prevention and treatment.

[0004] Currently, the Chinese medicinal materials that can be used to prevent Haemophilus parasuis disease include rhubarb, baicalin, epimedium, scutellaria, phellodendron, and Macleaya cordata extract. These single Chinese medicines all have the problem of being applicable to a single symptom and difficult to achieve the best preventive effect. The current prevention of Haemophilus parasuis disease is mainly based on vaccination. Farmers also add an appropriate amount of Qiqing Baidu Granules to pig feed, which can reduce the number of cases to a certain extent, but cannot play a good preventive role. In summary, the existing preventive drugs for Haemophilus parasuis disease are difficult to achieve the best preventive effect. Therefore, there is an urgent need to develop a new Chinese herbal compound preparation that can be used for the prevention of Haemophilus parasuis disease, and to utilize the synergistic effect between drugs to enhance the preventive effect of Haemophilus parasuis disease. Summary of the Invention

[0005] In order to solve the problem that the existing preventive drugs for swine Haemophilus parasuis disease in the prior art are difficult to achieve the best preventive effect, the present invention screens Chinese medicinal raw materials that can inhibit the growth and reproduction of G. parasuis to form a compound Chinese medicinal preparation, and determines the preparation method of the compound Chinese medicinal preparation by HPLC, providing a new idea for the prevention and control of G. parasuis disease.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention aims to explore the preventive efficacy of a compound Chinese herbal preparation against G. parasuis and to evaluate its potential as a new, effective prevention and control measure. G. parasuis is the causative agent of swine Glaser's disease, a condition characterized by arthritis, meningitis, and polyserositis, and has a significant economic impact on global pig health and production. Although current prevention and control of the disease primarily relies on the use of inactivated vaccines, existing vaccines and prevention and control measures have failed to completely prevent the disease due to the serovar diversity of G. parasuis and its resistance to antibiotics. Therefore, the search for new prevention and control strategies is particularly important.

[0008] Based on the above background, the present invention screens Chinese medicinal raw materials that effectively inhibit the growth and reproduction of G. parasuis, uses different ratios to form a compound Chinese medicinal preparation, determines the optimal extraction process of the Chinese medicinal raw materials by HPLC, and uses an experimental animal model to evaluate the preventive effect of the compound Chinese medicinal preparation on G. parasuis disease.

[0009] This invention provides a new scientific basis and practical means for the prevention and control of G. parasuis, and is of great significance for improving animal health and reducing economic losses in the animal husbandry industry. By studying the disease prevention mechanism and application effects of the compound Chinese medicine preparation, this invention also helps promote the modern application of traditional Chinese medicine and provides new ideas for the comprehensive prevention and control of animal diseases. Therefore, this invention not only has a positive impact on the sustainable development of the pig farming industry, but also opens up new areas for the application of traditional Chinese medicine in the field of animal disease prevention and control.

[0010] Based on this, the present invention provides a traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs.

[0011] The traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 2-24 parts of coptis root, 2-24 parts of phellodendron amurense, and 1-12 parts of ebony root.

[0012] Based on the results of single-herb drug sensitivity testing, coptis root, phellodendron chinense, and ebony root showed the strongest antibacterial effects, so these three ingredients were selected for the preparation of the compound Chinese medicine preparation. In the screening of the optimal Chinese medicine compound formulation, a mass ratio of coptis root: phellodendron chinense: ebony root: ebony root showed the best antibacterial effect, so this ratio was selected for the preparation of the compound Chinese medicine preparation.

[0013] Preferably, the traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 4-20 parts of coptis chinensis, 4-20 parts of phellodendron amurense, and 2-10 parts of ebony trees.

[0014] More preferably, the Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 8-16 parts of Coptis chinensis, 8-16 parts of Phellodendron chinense, and 4-8 parts of Prunus mume.

[0015] The present invention also provides a method for preparing the traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease, comprising the following steps:

[0016] Weigh the raw materials according to weight.

[0017] After mixing coptis root, phellodendron chinense, and black plum, a medicinal material mixture is obtained; the medicinal material mixture is soaked in water for 20-30 minutes, then decocted and extracted 2-3 times, and the decoction is collected. Soaking the medicinal materials in water increases the solubility of the medicinal materials, making it easier to extract the active ingredients; decocting 2-3 times can more effectively extract the ingredients of the traditional Chinese medicine, allowing the medicinal materials to exert their effective value.

[0018] The ratio of water to the medicinal material mixture is 8-12:1. When the ratio is 8-12 times, the extraction efficiency is the best and the content of the extracted effective substances is the highest.

[0019] When decocting, first use high heat at 150-180℃ to boil the Chinese medicine, then adjust to low heat at 80-100℃ to decoct.

[0020] The first, second and third decoction times were 40-45 minutes. The highest content of effective substances was obtained when the decoction time was 40-45 minutes.

[0021] The decoction was filtered and concentrated to obtain a concentrate.

[0022] The concentrated solutions are combined, filtered through a 0.2-1000 μm filter membrane, and then packaged to obtain a traditional Chinese medicine composition.

[0023] After filtering through the filter membrane, impurities and residues in the traditional Chinese medicine decoction can be separated, thereby improving the purity and quality of the traditional Chinese medicine.

[0024] The Chinese medicine composition is prepared into a preparation together with pharmaceutically acceptable auxiliary materials to obtain the Chinese medicine compound preparation for preventing Haemophilus parasuis disease.

[0025] Preferably, the dosage form of the Chinese herbal compound preparation for preventing Haemophilus parasuis disease is a Chinese herbal decoction, or dried into powder or granules.

[0026] Preferably, the auxiliary materials include liquid auxiliary materials and solid materials, such as water, starch, dextrin, sucrose and honey.

[0027] Specifically, 20-50 g of the above-mentioned traditional Chinese medicine composition is dispersed in 200-400 mL of water, and decocted 2-3 times for 25-45 minutes to obtain the traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention provides a traditional Chinese medicine compound preparation for preventing Haemophilus parasuis in pigs and a preparation method thereof, providing a new method for preventing Haemophilus parasuis in pigs. The present invention screens traditional Chinese medicine raw materials that effectively inhibit the growth and reproduction of Haemophilus parasuis and formulates the traditional Chinese medicine compound preparation through different proportions. Furthermore, after using high-performance liquid chromatography to screen the optimal extraction process for the traditional Chinese medicine raw materials, the present invention uses a mouse model infected with Haemophilus parasuis type 13 as a research subject to evaluate the preventive effect of the traditional Chinese medicine compound preparation on Haemophilus parasuis disease, providing a scientific basis for the prevention and control of the disease and the development of the pig farming industry. The research results have important application value.

[0030] The above research results show that: (1) Through the in vitro antibacterial test of Chinese medicine against type 13 G. parasuis, it was found that Rhizoma Coptidis, Phellodendron Amurense and Prunus mume had the strongest antibacterial effect, with the diameters of the antibacterial rings being 25mm, 22mm and 20mm respectively. (2) The best Chinese medicine compound preparation was screened out by preparing compound and different proportions of Rhizoma Coptidis, Phellodendron Amurense and Prunus mume, that is, the mass ratio of Rhizoma Coptidis: Phellodendron Amurense: Prunus mume was 2:2:1; and the in vitro inhibitory effect of the Chinese medicine compound preparation on type 13 G. parasuis was determined by growth inhibition test. (3) Using berberine hydrochloride and citric acid, the main active ingredients in the Chinese medicine compound preparation, as indicators, the optimal factor levels were determined by high performance liquid chromatography, and the optimal extraction process of the Chinese medicine raw materials was screened out as A3B3C2, that is, decocting 3 times, the material-liquid ratio was 12 times, and the decocting time was 45 minutes. (4) In animal experiments, the T lymphocyte subsets CD4 + / CD8 +The ratio showed that the positive group was significantly lower than the blank and negative groups (P<0.01); the compound Chinese medicine Wulianbai decoction group was significantly higher than the positive group (P<0.05); the compound Chinese medicine Wulianbai alcohol extract group was significantly higher than the positive group (P<0.01), indicating that the Chinese medicine compound preparation can effectively enhance the body's immunity and thus resist the pathogenic effects of G. parasuis. (5) The peripheral blood cytokines of mice were detected. The IL-1β, IFN-γ, and IL-10 levels of the positive group mice were significantly higher than those of the blank and negative control groups (P<0.01). The IL-1β, IFN-γ, and IL-10 levels of the compound Chinese medicine Wulianbai decoction group and the compound Chinese medicine Wulianbai alcohol extract group were significantly or extremely significantly lower than those of the positive control group (P<0.05 or P<0.01), indicating that the Chinese medicine compound preparation can effectively inhibit the production of inflammatory factors. (6) The compound Chinese medicine Wulianbai decoction group can provide a 60% protection rate against mice infected with Haemophilus parasuis, and the compound Chinese medicine Wulianbai alcohol extract group can provide a 70% protection rate against mice infected with Haemophilus parasuis. (7) In the liver bacterial load test, the liver bacterial load of mice protected by the compound Chinese medicine Wulianbai decoction group was significantly lower than that of the positive group (P<0.01), indicating that the mice enhanced their ability to kill bacteria after taking the Chinese medicine. (8) The whole blood bactericidal test showed that the Chinese medicine compound preparation has an immunomodulatory effect, which can affect the body's immune system to promote the production of antibodies and thus mediate the in vivo bactericidal effect.

[0031] In summary, the results of the present invention show that the Chinese herbal compound preparation has a certain preventive effect on the infection and pathogenicity of Haemophilus parasuis type 13, and provides an effective direction and basis for the prevention and control of Haemophilus parasuis disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the inhibition ring of the drug sensitivity test of the Chinese medicinal raw materials with different proportions in the optimal Chinese medicinal compound preparation of the present invention.

[0033] Figure 2 is the absorbance value of G. parasuis bacterial solution in the present invention at 13h (10 -3 dilution factor).

[0034] Figure 3 is the absorbance value of G. parasuis bacterial solution in the present invention at 13h (10 -4 dilution factor).

[0035] Figure 4 is the absorbance value of G. parasuis bacterial solution in the present invention at 13h (10 -5 dilution factor).

[0036] Figure 5 is a chromatogram of a standard substance used to detect the content of effective substances in a sample in the present invention; wherein Figure 5A is the chromatogram of berberine hydrochloride standard; Figure 5 B is the chromatogram of citric acid standard.

[0037] Figure 6 The berberine hydrochloride standard curve of the present invention

[0038] Figure 7 This is the citric acid standard curve of the present invention.

[0039] Figure 8 is the sample chromatogram at the optimal factor level in the present invention; wherein, Figure 8 A is the chromatogram of berberine hydrochloride in the sample; Figure 8 B is the chromatogram of citric acid in the sample.

[0040] Figure 9 is the weight change of mice in the present invention.

[0041] Figure 10 The CD4 + / CD8 + The ratio changes.

[0042] Figure 11 This is the result of the whole blood sterilization test in the present invention. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0044] Example 1

[0045] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 12g of coptis root, 12g of phellodendron chinense, and 6g of ebony root.

[0046] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0047] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps:

[0048] S1. Weigh the Chinese medicinal raw materials: coptis root, phellodendron amurense, and ebony plum according to weight; wash the decoction pot and the above Chinese medicinal raw materials with clean water, and then mix the coptis root, phellodendron amurense, and ebony plum to obtain a medicinal material mixture.

[0049] S2. Soak the medicinal material mixture in water for 30 minutes, add water to the medicinal material mixture and boil and extract three times, and collect the decoction.

[0050] The material-liquid ratio of water added to the medicinal material mixture is 12:1.

[0051] When decocting, first boil the herbs at 180°C, then reduce to 100°C. The water level should be 3cm above the surface of the herbs.

[0052] The first decoction lasts for 45 minutes. After the decoction is complete, the Chinese medicine is poured out through a strainer. Then, the mixture of herbs in the pot is decocted a second time. The second decoction lasts for 45 minutes. After the decoction is complete, the Chinese medicine is poured out through a strainer. Then, the mixture of herbs in the pot is decocted a third time. The third decoction lasts for 45 minutes. After the decoction is complete, the Chinese medicine is poured out through a strainer.

[0053] The Chinese medicinal decoctions prepared by the first decoction, the second decoction and the third decoction are mixed to obtain a decoction liquid.

[0054] The decoction was filtered through a filter, and the filtrate was collected and concentrated using a rotary evaporator to obtain a concentrate;

[0055] The concentrated solution was filtered through a 0.45 μm filter membrane and then packaged to obtain a traditional Chinese medicine compound preparation.

[0056] 30 g of the above-mentioned traditional Chinese medicine composition was dispersed in 360 mL of water, and decocted three times for 45 minutes to obtain the traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease.

[0057] Example 2

[0058] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis infection in pigs is composed of the following raw materials: 2g of coptis root, 2g of phellodendron amurense, and 1g of ebony root.

[0059] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0060] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0061] Example 3

[0062] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 24g of coptis root, 24g of phellodendron amurense, and 12g of ebony root.

[0063] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0064] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0065] Example 4

[0066] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 13g of coptis root, 13g of phellodendron chinense, and 6g of ebony root.

[0067] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0068] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0069] Example 5

[0070] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis infection in pigs is composed of the following raw materials: 4g of coptis root, 4g of phellodendron chinense, and 2g of ebony root.

[0071] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0072] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0073] Example 6

[0074] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 20g of coptis root, 20g of phellodendron amurense, and 10g of ebony root.

[0075] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0076] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0077] Example 7

[0078] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis infection in pigs is composed of the following raw materials: 13g of coptis root, 12g of phellodendron chinense, and 6g of ebony root.

[0079] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0080] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0081] Example 8

[0082] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis infection in pigs is composed of the following raw materials: 8g of coptis root, 8g of phellodendron chinense, and 4g of ebony root.

[0083] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0084] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0085] Example 9

[0086] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 16g of coptis root, 16g of phellodendron chinense, and 8g of ebony root.

[0087] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0088] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0089] Example 10

[0090] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 20g of coptis root, 20g of phellodendron amurense, and 10g of ebony root.

[0091] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0092] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps:

[0093] S1. Weigh the Chinese medicinal raw materials: coptis root, phellodendron amurense, and ebony plum according to weight; wash the decoction pot and the above Chinese medicinal raw materials with clean water, and then mix the coptis root, phellodendron amurense, and ebony plum to obtain a medicinal material mixture.

[0094] S2. Soak the medicinal material mixture in water for 30 minutes, add water to the medicinal material mixture and boil and extract three times, and collect the decoction.

[0095] The material-liquid ratio of water added to the medicinal material mixture is 8:1.

[0096] 11. When decocting, first boil the herbs at 180°C, then reduce heat to -100°C. The water level should be 3cm above the surface of the herbs.

[0097] The decoction time is 30 minutes. After decoction, pour out the medicine soup through a filter to obtain the decoction liquid.

[0098] The decoction was filtered through a filter, and the filtrate was collected and concentrated using a rotary evaporator to obtain a concentrated solution.

[0099] The concentrated solution was filtered through a 0.45 μm filter membrane and then packaged to obtain a traditional Chinese medicine compound preparation.

[0100] 50 g of the above-mentioned traditional Chinese medicine composition was dispersed in 400 mL of water, and decocted three times for 30 minutes to obtain the traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease.

[0101] Example 11

[0102] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 20g of coptis root, 20g of phellodendron amurense, and 10g of ebony root.

[0103] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0104] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps:

[0105] S1. Weigh the Chinese medicinal raw materials: coptis root, phellodendron amurense, and ebony plum according to weight; wash the decoction pot and the above Chinese medicinal raw materials with clean water, and then mix the coptis root, phellodendron amurense, and ebony plum to obtain a medicinal material mixture.

[0106] S2. Soak the medicinal material mixture in water for 30 minutes, add water to the medicinal material mixture and boil and extract three times, and collect the decoction.

[0107] The material-liquid ratio of water added to the medicinal material mixture is 10:1.

[0108] When decocting, first boil the herbs at 180°C, then reduce heat to 100°C and simmer. The water level should be 3cm above the surface of the herbs.

[0109] The first decoction time is 25 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter. Then, the medicinal material mixture in the pot is decocted for the second time. The second decoction time is 25 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter.

[0110] The Chinese medicinal decoctions prepared by the first decoction and the second decoction are mixed to obtain a decoction liquid.

[0111] The decoction was filtered through a filter, and the filtrate was collected and concentrated using a rotary evaporator to obtain a concentrated solution.

[0112] The concentrated solution was filtered through a 0.45 μm filter membrane and then packaged to obtain a traditional Chinese medicine compound preparation.

[0113] 50 g of the above-mentioned traditional Chinese medicine composition (compound traditional Chinese medicine urianbai decoction) was dispersed in 500 mL of water, and decocted twice for 25 minutes to obtain the traditional Chinese medicine compound preparation for preventing swine Haemophilus parasuis disease.

[0114] Comparative Example 1

[0115] A method for preparing a Chinese medicine preparation comprises the following steps:

[0116] S1. Weigh 20 g of Coptis chinensis; clean the decoction pot and the Coptis chinensis with clean water, then soak the cleaned Coptis chinensis in water for 30 minutes, then add water to the decoction for extraction three times, and collect the decoction liquid.

[0117] Among them, the material-liquid ratio of water added to Coptis chinensis is 10:1.

[0118] When decocting, first boil the Chinese medicine at 180℃, then reduce the heat to 100℃ and simmer. The water level should be 3cm above the surface of the coptis root.

[0119] The first decoction time is 45 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter. Then, the medicinal material mixture in the pot is decocted for the second time. The second decoction time is 45 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter.

[0120] The Chinese medicinal decoctions prepared by the first decoction and the second decoction are mixed to obtain a decoction liquid.

[0121] The decoction was filtered through a filter, and the filtrate was collected and concentrated using a rotary evaporator to obtain a concentrated solution.

[0122] The concentrated solution was filtered through a 0.45 μm filter membrane and then packaged to obtain the Chinese medicine Coptis chinensis water decoction.

[0123] The above 20 g of the Chinese medicinal herb Coptis chinensis is dispersed in 200 mL of water, and decocted twice for 45 minutes to obtain the Chinese medicinal herb Coptis chinensis water decoction for preventing Haemophilus parasuis disease in pigs.

[0124] Comparative Example 2

[0125] A method for preparing a Chinese medicine preparation comprises the following steps:

[0126] S1. Weigh 20g of Phellodendron amurense; clean the decoction pot and Phellodendron amurense with clean water, then soak the cleaned Phellodendron amurense in water for 30 minutes, add water to it and boil and extract three times, and collect the decoction.

[0127] Among them, the material-liquid ratio of water added to Phellodendron chinense is 10:1.

[0128] When decocting, first boil the Chinese medicine at 180℃, then reduce the heat to 100℃ and simmer. The water level should be 3cm above the surface of the cork.

[0129] The first decoction time is 45 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter. Then, the medicinal material mixture in the pot is decocted for the second time. The second decoction time is 45 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter.

[0130] The Chinese medicinal decoctions prepared by the first decoction and the second decoction are mixed to obtain a decoction liquid.

[0131] The decoction was filtered through a filter, and the filtrate was collected and concentrated using a rotary evaporator to obtain a concentrated solution.

[0132] The concentrated solution was filtered through a 0.45 μm filter membrane and then packaged to obtain the Chinese medicine Phellodendron amurense decoction.

[0133] The above 20 g of the Chinese medicine Phellodendron chinense was dispersed in 200 mL of water, and the mixture was decocted twice for 45 minutes to obtain the Chinese medicine Phellodendron chinense water decoction for preventing Haemophilus parasuis disease in pigs.

[0134] Comparative Example 3

[0135] A method for preparing a Chinese medicine preparation comprises the following steps:

[0136] S1. Weigh 20g of black plum; wash the decoction pot and the black plum with clean water, then soak the washed black plum in water for 30 minutes, add water and boil and extract three times, and collect the decoction.

[0137] Among them, the material-liquid ratio of water added to black plum is 10:1.

[0138] When decocting, first boil the Chinese medicine at 180℃, then reduce the heat to 100℃ and simmer. The water level should be 3cm above the surface of the black plum.

[0139] The first decoction time is 45 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter. Then, the medicinal material mixture in the pot is decocted for the second time. The second decoction time is 45 minutes. After the decoction is completed, the Chinese medicine is poured out through a filter.

[0140] The Chinese medicinal decoctions prepared by the first decoction and the second decoction are mixed to obtain a decoction liquid.

[0141] The decoction was filtered through a filter, and the filtrate was collected and concentrated using a rotary evaporator to obtain a concentrated solution.

[0142] The concentrated solution was filtered through a 0.45 μm filter membrane and then packaged to obtain the Chinese medicine Wumei decoction.

[0143] The above-mentioned 20g of the traditional Chinese medicine Wumei was dispersed in 200mL of water, and the mixture was decocted twice for 45 minutes to obtain the traditional Chinese medicine Wumei water decoction for preventing Haemophilus parasuis disease.

[0144] Comparative Example 4

[0145] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 10g of coptis root, 20g of phellodendron amurense, and 10g of ebony root.

[0146] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0147] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease includes the following steps: the same as those in Example 6.

[0148] Comparative Example 5

[0149] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 10g of coptis root, 20g of phellodendron amurense, and 20g of ebony root.

[0150] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0151] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps: the same as those in Example 1.

[0152] Comparative Example 6

[0153] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 20g of coptis root, 10g of phellodendron chinense, and 10g of ebony root.

[0154] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0155] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease includes the following steps: the same as those in Example 6.

[0156] Comparative Example 7

[0157] A traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease in pigs is composed of the following raw materials: 20g of coptis root, 20g of phellodendron amurense, and 10g of ebony root.

[0158] Among them, Coptis chinensis, Phellodendron amurense and Prunus mume are sourced from Tianyitang Guoda Pharmacy in Shenyang.

[0159] The preparation method of the above-mentioned traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease comprises the following steps:

[0160] S1. Weigh the Chinese medicinal raw materials: coptis root, phellodendron amurense, and ebony plum according to weight; wash the decoction pot and the above Chinese medicinal raw materials with clean water, and then mix the coptis root, phellodendron amurense, and ebony plum to obtain a medicinal material mixture.

[0161] S2. Extract the medicinal material mixture twice with 75% ethanol at a solid-liquid ratio of 10, and collect the extract.

[0162] The decoction was filtered through a filter and concentrated using a rotary evaporator to obtain a concentrate;

[0163] The concentrated solutions after the extraction were combined, filtered through a 0.22 μm filter membrane and then packaged to obtain a Chinese medicine composition (compound Chinese medicine wulianbo ethanol extract).

[0164] 50 g of the above-mentioned traditional Chinese medicine composition was dispersed in 500 mL of 75% ethanol, and extracted twice using a heating reflux device to obtain the traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease.

[0165] In order to illustrate the effect of a traditional Chinese medicine compound preparation for preventing Haemophilus parasuis disease provided by the present invention, the present invention selects Example 6, Example 10, Example 11 and Comparative Examples 1 to 6 as examples (the water-decocted black plum, coptis root, and phellodendron amurense are in a ratio of 1:2:2, respectively) as the compound traditional Chinese medicine wulianbai water decoction group and (the alcohol-extracted black plum, coptis root, and phellodendron amurense are in a ratio of 1:2:2, respectively) as the compound traditional Chinese medicine wulianbai alcohol extract group), and conducts the following study:

[0166] 1. Drug sensitivity testing

[0167] (1) Preparation of Chinese medicine sensitive tablets:

[0168] Spread a 6 mm diameter filter paper on a plate with tweezers, and slowly add the Chinese medicine composition prepared in Example 10 and the Chinese medicine solution prepared in Comparative Examples 4 to 6 to the filter paper using a pipette to ensure uniform drug concentration on the paper. After the paper is fully absorbed for 20 minutes, take out the filter paper, wrap it with sterile gauze, and place it in a 37 ° C incubator to dry. After drying, use tweezers to pick up the filter paper in a clean bench and place it in a 5 mL cillin bottle. Cover it with a stopper and wrap it tightly with sealing film. Store it at -20 ° C.

[0169] The concentration of the Chinese medicine composition prepared in Example 10 was 0.1 g / mL. The concentration of the Chinese medicine solutions prepared in Comparative Examples 4 to 6 was 1 g / mL.

[0170] The reason for selecting the above concentration is that the drug-sensitive tablets prepared with the traditional Chinese medicine composition prepared at 1 g / mL have a strong antibacterial effect, resulting in no colony growth on the culture medium and the inability to compare the diameters of the inhibition rings. Therefore, the replicated traditional Chinese medicine composition was diluted to 0.1 g / mL for the preparation of drug-sensitive tablets.

[0171] (2) Detection

[0172] In Example 10 and Comparative Examples 4 to 6, the Chinese medicine compositions prepared by mixing different proportions of three Chinese medicinal raw materials, Phellodendron chinense, Coptis chinensis, and Prunus mume, were applied to TSA culture medium inoculated with type 13 G. parasuis at 37°C for 12 hours and then the diameter of the inhibition zone was observed. The drug sensitivity results are shown in Table 1 and Figure 1 shown.

[0173] Table 1 Results of drug sensitivity test with different ratios

[0174]

[0175] The results showed that in the Chinese medicine susceptibility test, the largest inhibition ring diameter was 24 mm in the ratio of Phellodendron chinense: Coptis chinensis: Prunus mume = 2:2:1.

[0176] 2. Growth inhibition test

[0177] G. parasuis type 13 was cultured on TSA medium at 37°C, and the G. parasuis type 13 grown to the logarithmic phase on the TSA medium was washed with PBS solution to obtain a bacterial solution, which was diluted 10-fold to 10 -3 , 10 -4 , 10 -5 Three concentrations were tested.

[0178] 100 μL of the Chinese medicine composition prepared in Example 1 and the Chinese medicine solution prepared in Comparative Examples 1 to 3, as well as 200 μL of bacterial solution, were added to the Chinese medicine wells. Only 300 μL of bacterial solution was added to the control wells. 300 μL of TSB medium was added to the culture medium wells. The microplate reader was preheated to 37°C in advance. After adding the sample, the cover was covered and cultured for 13 hours (G. parasuis reached the logarithmic phase at this time). According to the OD at 13 hours, the 600 The results were used to determine whether the drug inhibited the growth of G. parasuis.

[0179] The concentration of PBS solution is 0.01 mol / L, the pH is 7.4, and the source is Beijing Solebow Technology Co., Ltd.

[0180] TSA culture medium was obtained from Beijing Solebow Technology Co., Ltd.

[0181] Type 13 G. parasuis was isolated and frozen by our research group.

[0182] The formula or source of TSB medium is Beijing Solebow Technology Co., Ltd.

[0183] Chinese medicine Ulva ulmifolia group: 100 μL of drug solution and 200 μL of G. parasuis bacterial solution.

[0184] Positive group: 300 μL G. parasuis bacterial solution.

[0185] See the results Figures 2 to 4 According to the change of absorbance at 13h, it was found that the Chinese medicine composition prepared in Example 10 had an inhibitory effect on G. parasuis type 13. The absorbance value of the Chinese medicine Ulva uliensis group was significantly lower than that of the positive group (P<0.01).

[0186] The above results show that different concentrations of the Chinese herbal composition can inhibit the growth of type 13 G. parasuis.

[0187] 3. Water extraction process research

[0188] Example 6, Example 10 and Example 11 are respectively methods for preparing Chinese medicine compositions under different decoction times (A), material-liquid ratios (B) and decoction times (C), in order to compare the effects of the above different conditions. The present invention uses the Chinese medicine compositions prepared in Example 6, Example 10 and Example 11 as research objects, and uses high performance liquid chromatography to detect the content of berberine hydrochloride and citric acid therein (berberine hydrochloride is the main effective antibacterial component in Coptis chinensis and Phellodendron chinense; citric acid is the main effective antibacterial component in Plum, so these two effective substances are selected for content detection). The specific method is as follows:

[0189] (1) Preparation of chromatographic standards: Accurately measure 2 mg of each of berberine hydrochloride and citric acid standards into a 10 mL volumetric flask, add 2 mL of chromatographic grade methanol, and prepare 1 mg / mL berberine hydrochloride standard solution and 1 mg / mL citric acid standard solution, respectively.

[0190] (2) Preparation of chromatographic test samples: The Chinese medicine compositions prepared in Example 6, Example 10, and Example 11 were diluted with methanol to a concentration of 25 mg / mL, and 2 mL of each solution was placed in a 50 mL volumetric flask. Chromatographic grade methanol was added to the scale, and the mixture was weighed and ultrasonicated for 25 min. Methanol was added to make up the weight, and the mixture was filtered through a 0.22 μm filter membrane. The filtrate was taken as a 1 mg / mL test sample solution.

[0191] Among them, the prepared test solution includes the test solution prepared from the traditional Chinese medicine composition prepared in Example 6, Example 10 and Example 11.

[0192] (3) Chromatographic conditions:

[0193] The chromatographic conditions for berberine hydrochloride were as follows: 3C18-EB column, mobile phase: 0.02 mol / L potassium dihydrogen phosphate (pH 2.6): acetonitrile = 75:25, flow rate: 1 mL / min, detection wavelength: 345 nm, column temperature: 30°C.

[0194] The chromatographic conditions for citric acid were as follows: C18 column, mobile phase consisting of 0.02 mol / L potassium dihydrogen phosphate (pH 2.6) and acetonitrile for gradient elution, flow rate of 0.8 mL / min, detection wavelength of 210 nm, and column temperature of 30°C.

[0195] The present invention has carried out methodological verification on the above-mentioned high performance liquid chromatography method, and the specific process is as follows:

[0196] (1) Specificity investigation: 10 μL of the above-prepared chromatographic standard, chromatographic test sample and negative control (methanol solution) were respectively drawn into the injector and tested under the above-mentioned chromatographic conditions.

[0197] The results showed that the berberine peak and citric acid peak appeared in the same retention time for both the chromatographic standard and the chromatographic test sample, while no chromatographic peak appeared in the negative control solution at the same time. According to the peak area, the effective substance content in 100 μg berberine hydrochloride and citric acid standard was 99.7 μg / mL and 98.34 μg / mL, respectively, indicating that the method had good specificity. Figure 5 shown.

[0198] (2) Precision investigation: 10 μL of the above-prepared chromatographic standard was drawn into the sampler and injected six times continuously. The peak area was read and the content of the effective substance was calculated. The RSD was 1.22%, indicating that the HPLC instrument had good precision.

[0199] (3) Repeatability study: 10 μL of the prepared chromatographic sample was drawn into the sampler and injected 6 times. The peak area was read and the content of the active substance was calculated. The RSD was 1.35%, indicating good repeatability.

[0200] (4) Linear relationship investigation: The two prepared chromatographic standard solutions (1 mg / mL) were diluted with methanol to a series of concentrations of 500, 250, 100, and 50 μg / mL for HPLC detection. The standard curve was drawn with the concentration of the chromatographic standard as the abscissa and the peak area as the ordinate. The linear equation of berberine was y = 27.079x-197.73, R2 = 0.9999; the linear equation of citric acid was y = 0.2298x + 11.669, R 2 =0.9997, such as Figures 6 and 7 shown.

[0201] The above results show that berberine and citric acid have a good linear relationship and can accurately determine the contents of berberine hydrochloride and citric acid in the traditional Chinese medicine compound Urenba preparation.

[0202] In summary, the above methodological validation results all indicate that the instrument has good precision, and the mobile phase, flow rate, column temperature and other conditions set by HPLC can be used as a method for detecting berberine hydrochloride and citric acid in the traditional Chinese medicine composition prepared by the present invention.

[0203] The results of water extraction process screening are:

[0204] (1) A1B1C1, i.e., decocted once, with a material-liquid ratio of 8 times and a decocting time of 30 min, the berberine content was 4.81 μg / mL; the citric acid content was 37.94 μg / mL.

[0205] (2) A1B2C2, i.e., decocted once, with a material-liquid ratio of 10 times and a decocting time of 45 min, the berberine content was 13.35 μg / mL; the citric acid content was 42.24 μg / mL.

[0206] (3) A1B3C3, i.e., decocted once, with a material-liquid ratio of 12 times and a decocting time of 60 min, the berberine content was 12.72 μg / mL; the citric acid content was 30.45 μg / mL.

[0207] (4) A2B1C2, i.e., decocted twice, with a material-liquid ratio of 8 times and a decocting time of 45 min, the berberine content was 8.98 μg / mL; the citric acid content was 55.89 μg / mL.

[0208] (5) A2B2C3, i.e., decocted twice, with a material-liquid ratio of 10 times and a decocting time of 60 min, the berberine content was 14.55 μg / mL; the citric acid content was 59.81 μg / mL.

[0209] (6) A2B3C1, i.e., decocted twice, with a material-liquid ratio of 12 times and a decocting time of 30 min, the berberine content was 15.22 μg / mL; the citric acid content was 61.29 μg / mL.

[0210] (7) A3B1C3, i.e., decocted 3 times, with a material-liquid ratio of 8 times and a decocting time of 45 min, the berberine content was 8.51 μg / mL; the citric acid content was 45.48 μg / mL.

[0211] (8) A3B2C1, that is, decocted three times, with a material-liquid ratio of 10 times and a decocting time of 30 min, the berberine content was 12.19 μg / mL; the citric acid content was 58.27 μg / mL.

[0212] (9) A3B3C2, i.e., decocted three times, with a material-liquid ratio of 12 times and a decocting time of 45 min, the berberine content was 19.31 μg / mL; the citric acid content was 74.19 μg / mL.

[0213] From the above experimental results, we can see that this experiment used the traditional Chinese medicine decoction form to extract drugs, and conducted drug sensitivity tests on eight Chinese medicines to screen out Coptis chinensis, Phellodendron chinense, and Prunus mume as having the best in vitro antibacterial effects.

[0214] (2) The effective Chinese medicines were screened out by preparing compound prescriptions and different ratios, and the best Chinese medicine formula was selected: Coptis chinensis: Phellodendron chinense: Prunus mume = 2:2:1, and the effectiveness was further verified by growth inhibition test.

[0215] (3) Using berberine hydrochloride and citric acid, the main active ingredients in the traditional Chinese medicine compound wulianbai preparation, as indicators, the optimal factor levels were determined by high performance liquid chromatography, and the optimal extraction process was screened out as A3B3C2, that is, decocting 3 times, the material-liquid ratio was 12 times, and the decocting time was 45 minutes. Figure 8 shown.

[0216] 4. Study on the preventive effect of G. parasuis type 13 infection on mice

[0217] 4.1 Animal grouping and treatment

[0218] Female Kunming mice were housed and observed for 3 days. After confirming normal condition, they were randomly divided into five groups: a blank control group, a negative control group, a positive control group, a Chinese herbal decoction group, and a Chinese herbal alcohol extract group. Mice were gavaged every other day and weighed before each gavage. Specific groupings and treatments are shown in Table 2. Forty-eight hours after the seventh gavage, mice were intraperitoneally injected with G. parasuis serotype 13. Clinical manifestations and disease progression were observed after the challenge.

[0219] Among them, the female mice in Kunming came from Shenyang Changsheng Biological Company.

[0220] Attack strain: G. parasuis type 13.

[0221] Chinese medicinal ingredients: Wumei: Coptis chinensis: Phellodendron amurense = 1:2:2.

[0222] Table 2 Grouping of experimental mice

[0223]

[0224] In the early stage of feeding, the mice in the above different groups had normal food and water intake, and their weight changes showed an upward trend. After the infection, the mice showed rough fur, weakened response to external stimuli, gradually increased eye secretions, decreased food and water intake, and clinical symptoms such as clumping, drowsiness, and tremors. Figure 9 shown.

[0225] Depend on Figure 9As can be seen, the body weight of mice in the G. parasuis 13 challenge group showed a significant or extremely significant decrease compared to the blank control group (P < 0.05 or P < 0.01). This result indicates that G. parasuis 13 causes damage to the mouse body. The weight loss in the compound Chinese herbal medicine Wulianbai decoction and compound Chinese herbal medicine Wulianbai ethanol extract groups after challenge was significantly lower than that in the positive control group (P < 0.05). The clinical symptoms of mice in the compound Chinese herbal medicine Wulianbai decoction and compound Chinese herbal medicine Wulianbai ethanol extract groups after challenge were generally milder than those in the positive control group, and the recovery time was faster, indicating that the compound Chinese herbal medicine Wulianbai decoction and compound Chinese herbal medicine Wulianbai ethanol extract groups can alleviate the pathological damage caused by G. parasuis and accelerate the recovery of mice.

[0226] 4.2 Extraction of lymphocytes from mouse peripheral blood

[0227] The eyeball blood of the mice in the above different groups was collected in sodium heparin anticoagulant tubes; 500 μL of anticoagulant blood was drawn into the sample diluent and slowly pipetted to mix; the liquid was slowly added along the wall of the tube into a centrifuge tube containing 2 mL of lymphocyte separation solution, placed in a horizontal centrifuge, and centrifuged at 1500 rpm for 20 min; after centrifugation, the lymph fluid in the milky white annular layer was slowly aspirated with a pipette, washed twice with PBS, and resuspended in 100 μL of PBS after centrifugation to obtain a lymphocyte suspension.

[0228] The concentration of PBS solution is 0.01 mol / l, the pH is 7.4, and the source is Beijing Solebow Technology Co., Ltd.

[0229] The source of anticoagulated blood was mouse eyeball blood collected in sodium heparin anticoagulation tubes.

[0230] 4.3 Detection of T lymphocyte subsets in mouse peripheral blood by flow cytometry

[0231] To the lymphocyte suspension, 5 μL of FITC anti-mouse CD3, PE anti-mouse CD4, and APC anti-mouse CD8 were added in sequence, the mixture was mixed by pipetting, and the mixture was incubated at 4°C in the dark for 20 min. After incubation, the mixture was centrifuged at 1500 r / min for 10 min, the supernatant was discarded, and the cells were washed three times with PBS. After fixation with 400 μL of 4% paraformaldehyde, the cells were stored at 4°C in the dark until detection. 10,000 cells were taken for each sample.

[0232] CD4 + / CD8 + The ratio is a dynamic value, and the immune capacity of the body can be evaluated according to the ratio. The present invention extracts mouse peripheral blood lymphocytes, stains them with antibodies and fixes them with 4% formaldehyde, and then uses flow cytometry to detect T lymphocyte subsets and calculate the lymphocyte CD4 + / CD8+ The ratio, the result is Figure 10 shown.

[0233] The results showed that the positive control group CD4 + / CD8 + The ratio was significantly lower than that of the blank and negative groups (P<0.01), indicating that the model was successfully established. The mice in the positive control group were in an immunosuppressive state. The CD4 + / CD8 + The ratio was significantly lower than that of the blank control group (P<0.05), and extremely significantly higher than that of the positive control group (P<0.01). + / CD8 + The ratio was significantly lower than that of the blank control group and the negative control group (P<0.01), and significantly higher than that of the positive control group (P<0.05), indicating that both the compound Chinese medicine Urenba ethanol extract group and the compound Chinese medicine Urenba water decoction group played a role in enhancing the body's immune level.

[0234] 4.4 Protection rate against G. parasuis-challenged mice

[0235] Mice were randomly divided into five groups: a blank control group, a negative control group, a positive control group, a Chinese herbal decoction group, and a Chinese herbal alcohol extract group. The blank group was maintained in a normal diet, while mice in the other four groups were treated by oral gavage every other day. Specific grouping and treatment methods are shown in Table 3. Forty-eight hours after the seventh oral gavage, mice were intraperitoneally challenged with the poison. Their condition was observed throughout the day, and morbidity and mortality were recorded to determine the protective effect of the Chinese herbal composition on the mice.

[0236] Type 13 challenge dose selection: 4×10 9 CFU / mouse (minimum lethal dose).

[0237] Table 3 Grouping of experimental mice

[0238]

[0239] After oral administration of the Chinese medicine, mice were challenged with the virus, and the mortality of the mice was observed. The results are shown in Table 3. All mice in the positive control group died within 24 hours after the challenge. The survival rate of mice in the Chinese medicine decoction group and the Chinese medicine alcohol extract group after the challenge was 60%, respectively. This indicates that both the Chinese medicine compound wulianbai alcohol extract group and the Chinese medicine compound wulianbai water decoction group had a protective effect against mice challenged with G. parasuis serotype 13.

[0240] Table 3 Protection rate of Chinese medicine against mice challenged with G. parasuis type 13

[0241]

[0242] 4.5 Detection of liver bacterial load

[0243] Mice sacrificed from the different groups were dissected, and the livers were removed with sterile forceps and placed in a mortar. Sterile PBS was added at a rate of 1 mL / g and ground. The ground solution was diluted 10-fold to 10⁻⁶ and 10⁻⁷. The plates were then spread onto TSA plates using an L-shaped spreader rod and incubated at 37°C for 12 hours before colony counts were performed. The results are shown in Table 4.

[0244] Table 4 Liver bacterial load detection results

[0245]

[0246] The results showed that the bacterial load in the liver of the compound Chinese medicine Ulva ulmoides water decoction group and the compound Chinese medicine Ulva ulmoides alcohol extract group was significantly lower than that in the positive control group (P<0.01).

[0247] 4.6 Whole blood bactericidal test

[0248] G. parasuis type 13 strains were cultured in TSB liquid medium in a 37°C incubator, and the bacterial solution was collected by centrifugation at 3000 rpm for 3 min. The solution was resuspended and washed twice with sterilized PBS solution, and the bacterial solution concentration was diluted to 1.0 × 10 9 CFU / mL, dilute the serum to be tested 2-fold with sterile PBS, take 10L of type 13 G. parasuis and 190L of the serum to be tested and add them to a 1.5mL sterile centrifuge tube, mix thoroughly with a pipette, and incubate in a 37℃ constant temperature incubator for 30min, then add 100L of whole blood from the blank group mice, mix thoroughly and incubate at 37℃ with shaking for 1h, dilute the liquid in the centrifuge tube to 10-8 and evenly spread it on a TSA plate, incubate upright in a 37℃ incubator for 30min and then invert and incubate for 14h, and count the colonies on the plate the next day.

[0249] Bactericidal rate = (CFU of blank group - CFU of Chinese medicine group) / CFU of blank group × 100%

[0250] The calculated results showed that the bactericidal rate of the negative control group was 6.1%, while the growth of G. pararsuis was severely inhibited in the Chinese herbal decoction group and the Chinese herbal alcohol extract group, with bactericidal rates of 60.4% and 70.6% respectively. The differences between the Chinese herbal decoction group and the Chinese herbal alcohol extract group and the negative control group were extremely significant (P<0.01). Figure 11 shown.

[0251] The above results show that the traditional Chinese medicine composition provided by the present invention not only has a good antibacterial effect in vitro, but also can exert a bactericidal and inflammatory response-relieving effect in vivo by regulating the body's immune system. It has a certain preventive effect on the infection and pathogenicity of G. parasuis type 13, providing an effective direction and basis for the prevention and control of the disease.

[0252] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0253] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0254] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of a Chinese medicinal compound preparation in the preparation of a medicament for preventing Haemophilus parasuis infection in pigs, characterized in that: The traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 2-24 parts of coptis root, 2-24 parts of phellodendron amurense, and 1-12 parts of ebony root; The porcine Haemophilus parasuis disease is caused by infection with Haemophilus parasuis type 13.

2. The use according to claim 1, characterized in that The traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 4-20 parts of coptis root, 4-20 parts of phellodendron amurense, and 2-10 parts of ebony root.

3. The use according to claim 1, characterized in that The traditional Chinese medicine compound preparation is prepared from the following raw materials in parts by weight: 8-16 parts of coptis root, 8-16 parts of phellodendron amurense, and 4-8 parts of ebony root.

4. The method for preparing the Chinese medicinal compound preparation according to any one of claims 1 to 3, characterized in that: The following steps are involved: Weigh the raw material medicine according to weight; Mixing coptis root, phellodendron chinense, and plum blossoms to obtain a medicinal material mixture; adding water to the medicinal material mixture and boiling and extracting for 2 to 3 times, and collecting the decoction; The decoction is filtered and concentrated to obtain a concentrated solution; The concentrated solutions are combined, filtered through a 0.2-1000 μm filter membrane, and then packaged to obtain a Chinese medicine composition; The Chinese medicine composition is prepared into a preparation together with pharmaceutically acceptable auxiliary materials to obtain the Chinese medicine compound preparation for preventing Haemophilus parasuis disease.

5. The preparation method according to claim 4, characterized in that The liquid-to-material ratio of water added to the medicinal material mixture is 8-12:

1.

6. The preparation method according to claim 4, characterized in that Before decoction extraction, the herbal mixture was soaked in water for 20-30 min.

7. The preparation method according to claim 4, characterized in that When decocting, first use high heat at 150-180℃ to boil the Chinese medicine, then adjust to low heat at 80-100℃ to decoct.

8. The preparation method according to claim 4, characterized in that The first, second and third decoction times are 40-45 minutes.

9. The preparation method according to claim 4, characterized in that The dosage form of the Chinese medicine compound preparation is Chinese medicine decoction, powder or granule.

10. The preparation method according to claim 4, characterized in that The auxiliary materials include liquid auxiliary materials and solid auxiliary materials.

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