Application of apiose liquiritin in preparation of medicine for treating or preventing pig main respiratory tract pathogenic bacteria infection

Abstract

The invention belongs to the field of application of traditional Chinese veterinary medicines, and discloses application of apiose liquiritin in preparation of a medicine for treating or preventing pathogenic bacteria infection of main respiratory tracts of pigs. The liquiritin apioside has antibacterial and bactericidal effects on Actinobacillus pleuropneumoniae (APP), Streptococcus suis (SS) and HPS (haemophilus parasuis), the minimum inhibitory concentrations (MIC) of the liquiritin apioside on the APP, the SS and the HPS are respectively 128 mu g / ml, 128 mu g / ml and 256 mu g / ml, the minimum bactericidal concentrations (MBC) of the liquiritin apioside on the SS and the HPS are respectively 128 mu g / ml, 256 mu g / ml and 512 mu g / ml, the toxic and side effects of chemical drugs, antibiotics, vaccines and the like are overcome, and the liquiritin apioside has wide application prospects in the fields of veterinary drugs and breeding industry.

Description

Technical Field

[0001] The invention belongs to the application field of traditional Chinese veterinary medicines, and specifically relates to the application of apigenin in the preparation of medicines for treating or preventing main respiratory pathogenic bacteria infections in pigs. Background Art

[0002] Actinobacillus pleuropneumoniae is the main pathogen of porcine contagious pleuropneumonia. Vaccines are one of the effective measures to prevent porcine contagious pleuropneumonia, but there are many APP serotypes, and it is difficult for existing vaccines to cover all serotypes. Clinically, antibiotics are often used for treatment. The irrational use of drugs has led to a very serious problem of APP resistance. After conducting drug sensitivity tests on 137 APP strains isolated from 2017 to 2022, Romanian scholar Madalina found that the resistance to streptomycin, gentamicin, tilmicosin and erythromycin was 77%, 45%, 39% and 33% respectively. Czech expert Kucerova et al. found that the resistance of APP isolates to tetracycline was 23.9%. Wang Dandan et al. conducted drug sensitivity tests on APP in central Jiangsu and showed that all isolated strains were resistant to streptomycin; Yu Bo et al. conducted resistance phenotype analysis on APP isolated strains from Guizhou Province and found that the strains were resistant to tetracycline and sulfonamides; Xu Minsheng et al. conducted resistance gene and resistance phenotype detection and analysis on 20 APP isolated strains and found that all strains showed multidrug resistance, with a high resistance rate to sulfamethoxazole, lincomycin and tetracycline reaching 100%, and strains with 8 or more resistances accounting for about 50%.

[0003] Streptococcus suis is an important zoonotic pathogen that can infect pigs and cause meningitis, pneumonia, arthritis, and sepsis, causing economic losses to the pig industry (Lun et al 2007, Obradovic et al 2021). It can also infect humans through infected pigs or pork by-products, causing meningitis, arthritis, sepsis, and septic shock, often accompanied by irreversible sequelae (Feng et al 2010, Feng et al 2014). Clinically, the drug resistance problem of Streptococcus suis is also very serious. The resistance of Streptococcus suis to a variety of broad-spectrum antibiotics has shown an increasing trend year by year, especially for tetracyclines, lincosamides, and macrolides (Uruén et al 2022). Between 2013 and 2017, Zhang et al. isolated nearly 7,000 strains of Streptococcus suis from 16 provinces in China for drug resistance experiments. The results showed that the isolated strains showed high resistance to β-lactams, macrolides, aminoglycosides, lincomycins, tetracyclines, quinolones, polymyxins, sulfonamides, etc. (Zhang et al. 2019); between 2017 and 2019, Tan Meifang et al. isolated 112 strains of Streptococcus suis from healthy pig herds in Jiangxi Province. Through resistance testing of 16 antibiotics, it was found that more than 90% of the strains were resistant to clindamycin and tetracycline, and more than 75% of the resistant bacteria were 5 or more (Tan Meifang et al. 2020); in 2019-2020, the Streptococcus suis isolated by Mao Congjian et al. from Sichuan Province were also multidrug-resistant, accounting for 83% (Mao Congjian et al. 2023). Yang Yanbei et al. showed that the rate of drug-resistant gene carriage of Streptococcus suis has been increasing year by year (Yang Yanbei et al. 2023).

[0004] Haemophilus parasuis is the main pathogen of Haemophilus parasuis disease. There are currently 15 known serotypes, and the most common serotypes reported in clinical reports in my country are serotypes 4 and 5 (Zhang et al, 2012b; Ding Wenwen et al, 2019). HPS is a conditional pathogen that often causes disease when the body's immunity is reduced or under stress. It can cause symptoms such as fibrinous serositis, arthritis and meningitis in pigs, bringing great harm to the world's pig industry (Brockmeier et al, 2014, Liu et al, 2016). The drug resistance problem of HPS is also very serious in clinical practice. Danish researchers conducted drug sensitivity tests on 52 strains of HPS, and the results showed that 10 strains had reduced sensitivity to trimethoprim + sulfamethoxazole (Aarestrup et al, 2004). Zhou found that the HPS strains isolated from China had a high resistance rate to enrofloxacin and trimethoprim-sulfamethoxazole, and the strains resistant to more than three antimicrobial drugs reached 23.6% (Zhou et al, 2010). Wang Feihong et al. conducted a susceptibility test of 32 HPS isolates with 20 commonly used clinical antimicrobial drugs using the drug sensitivity paper disk method, and found that the resistance rate to kanamycin, neomycin, streptomycin and sulfonamide drugs was as high as 100% (Wang Feihong et al, 2022).

[0005] The emergence of multi-drug resistant strains not only increases the difficulty of disease prevention and control, but also generates problems such as antibiotic residues, which are potential hazards to the environment and human health. Therefore, how to produce high-quality meat products, increase the efforts of "ecological and efficient breeding", and solve the safety problems of animal products caused by antibiotic residues have become urgent problems that need to be solved.

[0006] Liquiritin apioside (LA) (chemical formula: C 26 H 30 O 13; Molecular weight: 550.51) is a flavonoid compound isolated and purified from licorice, with a molecular weight of 550.51, and has multiple biological activities such as anti-inflammatory, antioxidant, neuroprotective and anti-aging. Gong Xue et al. reported that apiolactone can activate the immune system to increase the number of NK cells and drive phagocytosis. At the same time, apiolactone also has the effect of inhibiting the growth of tumor cells. Jiang Ruichan et al.'s research results showed that apiolactone at a concentration of 6.25umol / L can significantly increase the number of Ki67-positive cells, reduce the number of β-galactosidase-positive cells, intracellular ROS levels and the proportion of G1 phase cells, weaken the adipogenic differentiation ability of hADSCs, promote the osteogenic differentiation ability of cells, and achieve anti-aging effects. KIMA et al. found that apiolactone can reduce MMP activity by inhibiting the migration of human fibrosarcoma HT1080 cells, inhibit the activation of NF-κB, and then inhibit tumor growth. Gong Xue et al. reported that apiolactone can activate the immune system to increase the number of NK cells and drive phagocytosis.

[0007] However, there are no reports on the use of licorice root glycoside as an active ingredient in drugs that inhibit major respiratory pathogens in pigs. Summary of the invention

[0008] The purpose of the present invention is to provide the use of apigenin in the preparation of drugs for treating or preventing major respiratory pathogens in pigs. The apigenin has good antibacterial effects on porcine Actinobacillus pleuropneumoniae, Streptococcus suis, and Haemophilus parasuis, and has good application potential in the prevention and treatment of porcine infectious pleuropneumoniae, porcine streptococcal disease, and Haemophilus parasuis disease. To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] The invention discloses an application of liquiritin in preparing a medicine for treating or preventing pig respiratory pathogen infection, comprising preparing the medicine for treating or preventing pig respiratory pathogen infection with liquiritin as the only effective ingredient or one of the effective ingredients, wherein the pig respiratory pathogen is porcine Actinobacillus pleuropneumoniae type 5 or type 1, Streptococcus suis type 2 and / or Haemophilus parasuis type 5.

[0010] The invention discloses an application of liquiritin in non-therapeutic inhibition or killing of porcine respiratory pathogens, wherein the porcine respiratory pathogens are porcine Actinobacillus pleuropneumoniae type 5 or type 1, Streptococcus suis type 2 and / or Haemophilus parasuis type 5.

[0011] Compared with the prior art, the present invention has the following advantages and effects:

[0012] The present invention first discovered that apigenin has the effect of inhibiting and killing the main respiratory pathogens of pigs. The present invention confirmed that apigenin has good antibacterial effect on porcine Actinobacillus pleuropneumoniae, Streptococcus suis and Haemophilus parasuis, providing experimental basis for the development of drugs and feed additives for the efficient prevention and treatment of porcine infectious pleuropneumoniae, porcine streptococcal disease and Haemophilus parasuis disease, and also providing new ideas and methods for the clinical prevention and treatment of major respiratory diseases of pigs by Chinese medicine monomer compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the growth curve of the effect of liquiritin on the growth of porcine Actinobacillus pleuropneumoniae serotype 5.

[0014] Figure 2 This is the growth curve of the effect of liquiritigenin on the growth of Streptococcus suis type 2.

[0015] Figure 3 This is the growth curve of the effect of liquiritin on the growth of Haemophilus parasuis serotype 5. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0017] Unless otherwise specified, all reagents and consumables used in the following examples were purchased from the market.

[0018] Embodiment 1:

[0019] Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Liquiritin against Actinobacillus pleuropneumoniae, Streptococcus suis and Haemophilus parasuis

[0020] 1. Materials

[0021] TSA, TSB, Mueller-Hinton (MH) broth and other culture media were purchased from BD Company, NAD was purchased from Biosharp Co., Ltd., and calf serum was purchased from Sijiqing Materials Co., Ltd. for bacterial culture, drug sensitivity testing, etc.

[0022] 2. Test methods

[0023] Refer to the broth dilution method.

[0024] Preparation of MIC plate: under sterile conditions, add 100 μL of MH medium to wells 1 to 11; then add 100 μL of apigenin solution to well 1, mix well, and add 100 μL to well 2, and so on, until well 10, then aspirate and discard 100 μL of the solution from well 10; add 200 μL of MH medium to well 12.

[0025] Preparation of inoculum: porcine Actinobacillus pleuropneumoniae K17 (serotype 5, "Nested PCR Detection of Porcine Infectious Actinobacillus pleuropneumoniae"), porcine Actinobacillus pleuropneumoniae S4074 (serotype 1), clinical strain Streptococcus suis (SS2HG006, serotype 2), Streptococcus suis SC19 (serotype 2), Haemophilus parasuis SH0165 (serotype 5), clinical isolate Haemophilus parasuis XY201501 (serotype 5), Escherichia coli, and Enterobacter faecium (see Table 1 for details) were streaked and inoculated into the corresponding solid culture medium and cultured at 37°C overnight. Subsequently, single colonies were picked and placed in the corresponding liquid culture medium, and cultured at 37°C with shaking for 10-12h; then transferred to a new liquid culture medium at a dilution of 1:1000, and cultured at 37°C until the bacterial mid-logarithmic period, centrifuged at 5000r / min for 5min to collect the bacteria, and resuspended and diluted to 1×10 8 CFU / mL. After the bacterial suspension was diluted 1:100 with MH liquid medium, 100 μL of bacterial suspension was added to the 1st to 11th wells of the three sterile 96-well polystyrene plates to make the final bacterial concentration in each well about 5×10 5 CFU / mL, cultured in a 37°C incubator for 12 h, and the MIC value was determined.

[0026] Mix the bacterial solution in the wells for determining the MIC value, aspirate 100 μL onto the prepared culture plate, cover it, and gently shake the culture plate to cover the plate with the bacterial solution. Place it in a 37°C incubator for overnight culture. Observe whether there is bacterial growth to determine the MBC value.

[0027] Result determination: When the bacteria in the negative control well grow well, the lowest drug concentration at which there is no obvious bacterial growth and a clear state when observed with the naked eye is the MIC value of the bacteria to be tested; the drug concentration at which there are no bacterial colonies on the plate is the MBC value of the bacteria to be tested.

[0028] 3. Results

[0029] As can be seen from Table 1, apigenin has antibacterial and bactericidal effects on porcine Actinobacillus pleuropneumoniae K17 (serotype 5, "Nested PCR Detection of Porcine Infectious Actinobacillus pleuropneumoniae"), porcine Actinobacillus pleuropneumoniae S4074 (serotype 1), clinical strain Streptococcus suis (SS2HG006, serotype 2), Streptococcus suis SC19 (serotype 2), Haemophilus parasuis SH0165 (serotype 5), and clinical isolate Haemophilus parasuis XY201501 (serotype 5), but has no inhibitory effect on Enterococcus faecalis.

[0030] Table 1 MIC and MBC of apigenin against bacteria

[0031]

[0032] Embodiment 2:

[0033] Effects of apigenin on the growth of Actinobacillus pleuropneumoniae K17, Streptococcus suis type 2 SS2HG006, and Haemophilus parasuis XY201501

[0034] 1. Materials

[0035] TSA, TSB, Mueller-Hinton (MH) broth and other culture media were purchased from BD Company, NAD was purchased from Biosharp Co., Ltd., and calf serum was purchased from Sijiqing Materials Co., Ltd. for bacterial culture, drug sensitivity testing, etc.

[0036] 2. Test methods

[0037] Refer to the broth dilution method.

[0038] Preparation of MIC plate: Under sterile conditions, 50 μL of MIC solution of different concentrations after multiple dilution was added to the first to eleventh wells of a sterile 96-well polystyrene plate; well 12 was not added with drug as a growth control.

[0039] Preparation of inoculum: Actinobacillus pleuropneumoniae K17, Streptococcus suis type 2 SS2HG006, and Haemophilus parasuis XY201501 were streaked on TSA plates (containing 5% calf serum and 10ug / mL NAD at a final concentration) and cultured at 37°C for 12h. Then, a single colony was picked and placed in TSB medium (containing 5% calf serum and 10ug / mL NAD at a final concentration) and cultured at 37°C overnight with shaking; the cultured bacterial solution was transferred to a new TSB medium (containing 5% calf serum and 10ug / mL NAD at a final concentration) at a ratio of 1:1000, cultured at 37°C until the bacterial mid-logarithmic phase, centrifuged at 5000r / min for 5min to collect the bacteria, resuspended the bacteria in the corresponding liquid culture medium, and adjusted to a concentration of 1×10 8CFU / mL. Dilute the bacterial suspension 1:5 with TSB medium (final concentration containing 5% calf serum and 10ug / mL NAD), and add 50μL of bacterial suspension to wells 1 to 11, so that the final bacterial concentration in each well is about 5×10 5 CFU / mL, cultured in a 37°C incubator.

[0040] Growth curve drawing: After inoculation of bacterial solution, the plate was placed in a 37℃ incubator for incubation. Every 1 hour, 100μL of bacterial solution was taken and coated on TSA plates (final concentration containing 5% calf serum and 10ug / mL NAD) to count the viable bacteria. Three plates were coated each time and the final average value was taken. The curve was drawn with time as the horizontal axis and the number of colonies as the vertical axis. The results are shown in Figure 2. Figure 1 , Figure 2 , Figure 3 As shown, when the concentration of apigenin is 1MIC, it can significantly inhibit the growth of porcine Actinobacillus pleuropneumoniae, Streptococcus suis, and Haemophilus parasuis.

Claims

1. Use of liquiritigenin in the preparation of a drug for treating or preventing porcine respiratory pathogen infection, wherein the porcine respiratory pathogen is porcine Actinobacillus pleuropneumoniae type 5 or type 1, Streptococcus suis type 2 and / or Haemophilus parasuis type 5.

2. Application of liquiritigenin in non-therapeutic killing or inhibition of porcine respiratory pathogens, wherein the porcine respiratory pathogens are porcine Actinobacillus pleuropneumoniae type 5 or type 1, Streptococcus suis type 2 and / or Haemophilus parasuis type 5.

3. The use according to claim 1, wherein the only active ingredient of the drug is liquiritigenin.

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