Composition against Haemophilus parasuis and its application

Abstract

The present invention relates to the field of biomedicine, and in particular to a composition for resisting Haemophilus parasuis and an application thereof. The composition adopts 3-hydroxyflavone as an active ingredient for resisting Haemophilus parasuis and can exhibit a very good bactericidal effect. At the same time, the present invention also discloses a use of the composition and a medicine.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a composition for resisting Haemophilus parasuis and application thereof. Background Art

[0002] Haemophilus parasuis, also known as Glasser's disease, is a systemic inflammatory response in pigs caused by Haemophilus parasuis (HPS), which is mainly manifested as acute exudative cellulitis, polyfibrinous serositis, arthritis and meningitis. The occurrence of Haemophilus parasuis is usually closely related to animal stress and is mostly sporadic. With the change of production mode and the emergence of immunosuppressive viruses, HPS is often mixed with porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus, streptococcus, Escherichia coli, etc., coupled with the long-term abuse of antibiotics, HPS has serious drug resistance, making it one of the common, frequent and serious infectious diseases of the respiratory system in pigs in the current pig farming process. Adding antibiotics to feed is a common method used by the breeding industry to control animal bacterial diseases and promote the growth of livestock and poultry. In clinical practice, ampicillin, fluoroquinolones, cephalosporins, tetracycline, gentamicin, sulfonamides and other drugs are commonly used to prevent and treat HPS.

[0003] Trihydroxyflavone is the main component of the traditional Chinese medicine Scutellaria baicalensis, which is mainly found in the roots of Scutellaria baicalensis, a plant of the Labiatae family, and the seeds and stem bark of Oroxylum indica, a plant of the Bignoniaceae family. In the past decade, many studies and literature have shown that trihydroxyflavone has significant antibacterial effects, as well as antioxidant and anti-radiation effects. It can also regulate certain diseases, making flavonoids increasingly favored by scholars and considered one of the best candidates for alternatives to antibiotics.

[0004] The molecular formula of trihydroxyflavone is C15H10O5, which contains 3 hydroxyl groups. It is commonly reported that it is used in the field of antibacterial. However, the application of 3-hydroxyflavone in the field of antibacterial is uncommon. For example, CN 118490896 A discloses an antibacterial composition, an antibacterial coating, a medical device, and a method for forming an antibacterial coating. 3-hydroxyflavone derivatives are used to release carbon monoxide gas under ultrasonic conditions using derivative groups to achieve effective sterilization of the infected part. It mainly relies on the release of carbon monoxide from the groups on the derivatives to sterilize, and it can only be carried out under ultrasonic conditions, and the use conditions are relatively harsh.

[0005] Therefore, the technical problem solved in this case is: how to develop a new anti-Haemophilus parasuis composition. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a composition for resisting Haemophilus parasuis. The composition uses 3-hydroxyflavone as an active ingredient for resisting Haemophilus parasuis and can show a very good bactericidal effect;

[0007] Meanwhile, the invention also discloses a use of the composition and a medicine.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] A composition for resisting Haemophilus parasuis comprises the compound 3-hydroxyflavone.

[0010] The trihydroxyflavone used in the prior art has three hydroxyl groups, which can form hydrogen bonds or other weak interactions with lipid molecules in the phospholipid bilayer of the cell membrane, thereby changing the fluidity and permeability of the cell membrane, allowing the substances in the bacterial cells to leak out, and achieving the purpose of bactericidal and antibacterial. However, the use conditions of trihydroxyflavone are relatively harsh, such as the need for ultrasound, etc. During the research process of the present invention, it was found that the 3-hydroxyflavone of the present invention has good bactericidal and antibacterial effects on Haemophilus parasuis H45 strain and Haemophilus parasuis H137 strain, and most importantly, it can work under normal conditions. We speculate that it may be because it has only one hydroxyl group, which leads to significant differences in antibacterial effects based on structural differences.

[0011] Preferably, the structural formula of the 3-hydroxyflavone is Formula I:

[0012] ;

[0013] Formula I

[0014] Preferably, the 3-hydroxyflavone is a pharmaceutically acceptable salt, derivative, hydrate, solvate or polymorph thereof.

[0015] In addition, the present invention provides a drug for resisting Haemophilus parasuis, comprising the composition as described above.

[0016] Preferably, the medicine is an injection, solution, powder, tablet, emulsion or pill.

[0017] Meanwhile, the present invention also provides the use of the above-mentioned composition in a drug for treating Haemophilus parasuis disease. Preferably, the Haemophilus parasuis disease is a bacterial disease of pigs caused by Haemophilus parasuis.

[0018] Finally, the present invention also provides use of the above-mentioned composition in a preparation for preventing infection by Haemophilus parasuis.

[0019] Preferably, the preparation for preventing Haemophilus parasuis infection is applicable to pigs.

[0020] The beneficial effects of the present invention are:

[0021] The 3-hydroxyflavone of the present invention is different from the trihydroxyflavone used in the prior art. We speculate that it may be because it has only one hydroxyl group. Based on the structural difference, it leads to a significant difference in antibacterial effect. It has good bactericidal and antibacterial effects on Haemophilus parasuis H45 strain and Haemophilus parasuis H137 strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the growth curve of strain H45 under different 3-hydroxyflavone concentrations;

[0023] Figure 2 This is the growth curve of H137 strain under different 3-hydroxyflavone concentrations. DETAILED DESCRIPTION

[0024] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0025] Product information:

[0026] 3-Hydroxyflavone: Beijing Bailingwei Technology Co., Ltd.

[0027] Haemophilus parasuis strain H45 and Haemophilus parasuis strain H137 were isolated from a pig farm in Guangdong Province, China and maintained by the Animal Health Institute of Guangdong Academy of Agricultural Sciences;

[0028] 1. Bacterial culture

[0029] The strains were inoculated into TSB medium containing 10 wt % calf serum and 30 mg / L NAD, respectively, and cultured at 37°C, 220 rpm overnight, and then the bacteria were collected by centrifugation.

[0030] 2. Determination of bacterial resistance

[0031] After the isolated strain was inoculated into the nutrient broth and cultured at 37 °C for 24 h, the concentration of the bacterial solution was adjusted to 1.5 × 108 cells / mL using a McFarland turbidimeter tube. According to the conventional bacterial inoculation method, 100 μL of the bacterial solution was evenly spread on the TSA medium, and a standard drug sensitivity paper (6 mm in diameter) was pasted and placed at 4 °C for 30 min. Then, it was moved to a 37 °C constant temperature incubator for 24 h, and the diameter of the inhibition zone was observed and measured. Finally, the results were determined according to the American Clinical Laboratory Standards Institute (CLSI) standards. The results are shown in Table 1.

[0032] Table 1 Results of drug sensitivity test of Haemophilus parasuis strains to commonly used clinical drugs

[0033]

[0034] It can be seen from this that Haemophilus parasuis H137 is a typical strain of drug-resistant Haemophilus parasuis.

[0035] 3. Bacterial MIC determination

[0036] The minimum inhibitory concentration was measured by microdilution susceptibility test. The specific operation is as follows:

[0037] Add TSB liquid culture medium to each well of the 96-well plate (200 μL in the first well, 100 μL in the second to 12 wells), use the microbroth dilution method to add the prepared test solution to wells 1 to 10, and do not add the solution to wells 11 and 12 as blank controls (the concentration of the prepared solution is 100 μg / mL, use a pipette to draw 100 μL of the solution and add it to the first well, pipette to mix, and then draw out 100 μL and add it to the second well. In the same way, after mixing the first well, draw 100 μL and add it to the next well until the 10th well. After mixing in the 10th well, draw 100 μL and discard it so that the volume of each well is 100 μL). Then, 100 μL of Haemophilus parasuis bacterial solution with a concentration of 1X106 cfu / mL was added to wells 1 to 10 and 12, and no solution was added to well 11. At this time, the concentration of bacteria in each well was 5 X 105 cfu / mL. Three replicates were set for each drug, and a control group was made to avoid drug color interference. The method was the same as the previous groups, except that physiological saline was used instead of bacterial solution to ensure the same volume. After adding each well, the volume was 200 μL, and then the 96-well plate was placed in a constant temperature incubator and cultured at 37 °C for 24 h. The lowest drug concentration well with no bacterial growth is the minimum inhibitory concentration (MIC) of the drug against Haemophilus parasuis. The results are shown in Table 2.

[0038] 4. Bacterial MBC determination

[0039] According to the MIC of different strains of Haemophilus parasuis that have been measured, the culture medium of each well with no bacterial growth was aspirated in turn, and dropped onto TSA (containing 10% FBS, 30 mg / L NAD) bacterial culture plates. The plates were cultured at 37°C overnight. The minimum dilution of the drug with less than 5 colonies on the plate was the minimum bactericidal concentration (MBC). The results are shown in Table 2.

[0040] Table 2 MIC and MBC values ​​of 3-hydroxyflavone against Haemophilus parasuis

[0041] strain MIC (μg / mL) MBC (μg / mL) H45 3.125 6.25 H137 3.125 6.25

[0042] 5. Effect of drugs on bacterial growth curve

[0043] The bacterial suspension of Haemophilus parasuis was inoculated into TSB medium, and 3-hydroxyflavone solution with a final concentration of 1 / 2MIC, MIC, and 2MIC was added to each strain. No 3-hydroxyflavone was added to the negative control. The medium was placed in a 37°C incubator for shaking culture, and samples were taken at 0, 1, 2, 4, 5, 6, 8, 10, 12, 14, and 24 hours. The D600 nm value was determined using an ultraviolet spectrophotometer. This was repeated 3 times, and the growth curves of bacteria at different drug concentrations were plotted. The results are shown in Figure 2. Figure 1 , Figure 2 shown.

[0044] Results Analysis

[0045] The present invention found through drug sensitivity test that the virulent strain H45 of Haemophilus parasuis is sensitive to 18 common antibiotics, except for ampicillin, tetracycline and bacitracin, and erythromycin. The clinically isolated strain H137 of Haemophilus parasuis is resistant to all 18 antibiotics, which shows that it is a highly resistant strain (Table 1).

[0046] The compounds of the present invention have obvious inhibitory effects on the virulent strains and clinical highly resistant strains of Haemophilus parasuis. The MIC values ​​of 3-hydroxyflavone to the virulent strain H45 of Haemophilus parasuis and the clinically resistant strain H137 are both 3.125 μg / mL; the MBC values ​​of 3-hydroxyflavone to the virulent strain H45 of Haemophilus parasuis and the clinically resistant strain H137 are both 6.25 μg / mL.

[0047] The MBC / MIC ratios of 3-hydroxyflavone against different isolates showed different antibacterial and bactericidal effects. The MBC / MIC values ​​of Cistanche naphthoquinone against the two strains were both 1, indicating that 3-hydroxyflavone was a bactericide against Haemophilus parasuis and had a strong bactericidal effect against Haemophilus parasuis (Table 2).

[0048] Effect of 3-hydroxyflavone on the growth curve of Haemophilus parasuis Figure 1 , Figure 2 It can be seen that compared with the negative control group, different concentrations of 3-hydroxyflavone have an effect on the growth curves of different strains, and the bacterial growth of each strain is significantly inhibited at 1 / 2MIC, 1MIC and 2MIC 3-hydroxyflavone concentrations.

[0049] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. Use of 3-hydroxyflavone in the preparation of drugs against Haemophilus parasuis.

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