Medical application of sanguinarine in preparation of salmonella flagellum inhibitor

Abstract

The invention provides medical application of sanguinarine in preparation of a salmonella flagellum inhibitor, and belongs to the technical field of biological medicine. According to the invention, the flagellum of salmonella is used as a potential target for researching and developing the anti-salmonella toxicity drug, and a natural product capable of inhibiting the flagellum of salmonella is screened through a salmonella sliding motion inhibition test and a real-time fluorescent quantitative PCR test; in combination with tests such as a minimum inhibitory concentration determination test, a growth curve, inhibition of salmonella from adhesion to Caco-2 cells and the like, it is verified that sanguinarine can effectively inhibit the salmonella flagellum-mediated swimming ability on the basis of not influencing normal growth of bacteria, so that the pathogenic potential of salmonella is remarkably reduced, the effect of bacteriostasis instead of sterilization is achieved, and the application prospect is broad. A new research thought and a candidate natural compound are provided for prevention and treatment of salmonella infection, and potential practical application value is achieved.

Description

Technical Field

[0001] The invention relates to the medical use of sanguinarine in preparing a salmonella flagella inhibitor, and belongs to the technical field of biomedicine. Background Art

[0002] Salmonella typhimurium is a foodborne pathogen that is widely found in nature, mainly in natural water bodies, sewage and drinking water, soil, animal intestines, and food processing and storage links. Salmonella has strong pathogenic characteristics, and its main transmission route is the digestive tract. It is one of the main pathogens causing bacterial food poisoning worldwide, causing many people to suffer from related diseases every year. The pathogenicity of Salmonella covers a variety of virulence factors, such as diverse secretion systems, membrane structures, genetic information carried by plasmids, and flagella, which together constitute its complex pathogenic mechanism. Flagella play a vital role in the infection mechanism of Salmonella. When Salmonella enters the body through the oral route, they use flagella to successfully colonize in the intestine. In this process, the protein components on the flagella are recognized by Toll-like receptor 5 (TLR-5), which then triggers a pro-inflammatory response and prompts the body to release cytokines that are crucial for activating innate and adaptive immune responses. Despite the activation of innate immunity, flagella possess important virulence characteristics that mediate bacterial attachment and invasion. In addition, flagella-driven motility is also considered an important determinant of Salmonella virulence. It enables bacteria to actively migrate to nutrient-rich areas or target sites of infection, increasing the pathogenicity of pathogens by enhancing adhesion and invasion capabilities. With the help of flagella, Salmonella can penetrate the mucus barrier and successfully invade the epithelial layer. Therefore, the flagella of Salmonella have become a potential target for the development of anti-Salmonella virulence drugs, and in-depth research and discovery of flagellar inhibitors are of great significance for the prevention and control of Salmonella infections. This invention reveals for the first time that sanguinarine has the potential to act as a Salmonella flagella inhibitor, which can effectively combat the pathogenic effects of Salmonella.

[0003] Sanguinarine, also known as pseudo-chelidonium quaternary ammonium base, sanguinarine chloride, and free sanguinarine, is obtained by ultrasonic extraction of sanguinarine or macleaya from the poppy family through industrial ethanol, and is a pale yellow needle-shaped crystal. At present, the source of sanguinarine is extensive, mainly found in plants of the poppy family, corydalis, rutaceae, and ranunculaceae. Among them, the content of sanguinarine in macleaya is relatively high, so it is often used as the main raw material for extracting sanguinarine. At the same time, sanguinarine is also found in many plants such as chelidonium and corydalis, and is one of the main active ingredients in traditional Chinese herbal medicines in my country. Modern medical research results have revealed the various pharmacological effects of sanguinarine, including antibacterial properties, improvement of liver function, analgesic and anti-inflammatory effects, treatment of infectious diseases, and protection of brain nerves, etc., and are widely used in the fields of medicine, animal health, plant protection, and health care product development. In recent years, many documents have widely reported these pharmacological activities of sanguinarine. However, up to now, there is no relevant research on the inhibition of Salmonella flagella by sanguinarine at home and abroad. Summary of the invention

[0004] The invention proposes the medical use of sanguinarine in preparing a salmonella flagella inhibitor, and provides a new research idea and candidate natural compounds for preventing and treating salmonella infection.

[0005] The invention verifies that sanguinarine can inhibit flagella-mediated sliding of bacteria, thereby reducing the pathogenicity of Salmonella, through tests such as a Salmonella sliding motion inhibition test, a minimum inhibitory concentration determination test, and a Salmonella adhesion cell inhibition test. The Salmonella used in the test is SL1344.

[0006] The molecular formula of sanguinarine of the present invention is [C 20 H 14 NO4] + , with a molecular weight of 332.33. The molecular structure of sanguinarine is as follows:

[0007]

[0008] The positive effects of the present invention are:

[0009] A new medical use of sanguinarine in preparing a salmonella flagella inhibitor is provided, and it is disclosed that sanguinarine can inhibit flagella-mediated bacterial sliding, thereby reducing the pathogenicity of salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : Sanguinarine inhibits the sliding movement of Salmonella SL1344 on semi-solid LB plates;

[0011] Figure 2 :The measurement results of sanguinarine on the sliding movement diameter of Salmonella SL1344;

[0012] Figure 3: Sanguinarine does not inhibit the growth of Salmonella SL1344 within the effective concentration range;

[0013] Figure 4 : Sanguinarine inhibits the transcription of the flagellar gene of Salmonella SL1344 within the effective concentration range;

[0014] Figure 5 : Sanguinarine has no toxic effect on Cacao-2 cells within the effective concentration range;

[0015] Figure 6 :Sanguinarine inhibits the adhesion of Salmonella SL1344 to Caco-2 cells. DETAILED DESCRIPTION

[0016] The present invention is further described by way of the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily implemented by a person of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0017] Example 1

[0018] Sanguinarine can be used as a Salmonella flagella inhibitor in any pharmaceutically acceptable carrier.

[0019] Example 2

[0020] Sanguinarine can be used as a salmonella flagella inhibitor for preparing medicines for treating infectious diseases.

[0021] Example 3

[0022] Sanguinarine is used as a Salmonella flagella inhibitor to treat infectious diseases caused by bacteria.

[0023] Test Example 1

[0024] Salmonella gliding motility inhibition assay

[0025] The overnight cultured Salmonella SL1344 was centrifuged at 8000 g for 2 min to collect the cells, which were then washed with autoclaved PBS and resuspended in PBS (the inoculum size was about 2 × 10 6 CFU) was dripped vertically onto the LB soft agar solid plate treated with sanguinarine, and then placed in a 37°C constant temperature incubator for 10-12 hours. The size of the colony moving around was observed and measured. The larger the sliding circle, the stronger the sliding ability; conversely, the weaker the sliding ability.

[0026] The results showed that compared with the control group (without sanguinarine treatment), different concentrations of sanguinarine (16-64 μg / mL) could significantly inhibit the sliding of Salmonella on 0.3% LB soft agar solid plates (see Appendix Figure 1 -Attached Figure 2 ).

[0027] Test Example 2

[0028] Determination of the minimum inhibition concentration (MIC) of sanguinarine against Salmonella SL1344

[0029] The determination of MIC of sanguinarine against Salmonella was carried out according to the test standard of the National Committee for Clinical Laboratory Standards (NCCLS). The SL1344 strain of Salmonella typhimurium was cultured at 37°C and 180 rpm until the logarithmic growth phase and the bacterial culture was diluted to OD 600nm = 0.1, add the diluted bacterial suspension (the final concentration of bacterial suspension is 5×10 5 CFUs / ml), with three replicates for each concentration, and the cells were cultured at 37°C. The lowest concentration at which no bacteria could be seen growing was taken as the MIC value of sanguinarine.

[0030] The results showed that the MIC value of sanguinarine against Salmonella SL1344 was greater than 64μg / ml.

[0031] Test Example 3

[0032] Growth curve of Salmonella SL1344

[0033] Salmonella SL1344 was inoculated into 2 mL LB medium and cultured overnight. The next day, the culture was expanded to a 100 mL conical flask at a ratio of 1:100 and continued to be cultured until OD 600nm =0.3, the bacterial solution was divided equally into 5 conical flasks, and sanguinarine was added to each flask to make the final concentrations of 16, 32, and 64 μg / mL, respectively. A DMSO control group was set up at 37°C and 180 rpm to continue culturing. The OD of the bacterial culture was measured every 1 h. 600nm The values ​​were recorded until the bacteria grew to the plateau phase and a growth curve was drawn.

[0034] The results showed that within the effective concentration range (16-64 μg / mL), sanguinarine had no effect on the growth of Salmonella SL1344 (see Appendix Figure 3 ).

[0035] Test Example 4

[0036] Effects of sanguinarine on the flagellar gene of Salmonella SL1344

[0037] Salmonella SL1344 was inoculated into 2 mL LB medium and cultured overnight. The next day, it was expanded into fresh LB medium at a ratio of 1:20, and sanguinarine was added to make the final concentrations 16 and 64 μg / mL, respectively. The culture was continued at 37°C and 180 rpm for 4 h. The bacteria were collected by centrifugation at 12000 rpm for 5 min, and the RNA of the bacteria was extracted by Trizol method. 1.5-2.0 μg of cDNA was synthesized according to the instructions of the one-step cDNA synthesis kit. The flagellar genes FlhC, FlgM, FliD, and FliC were quantified in real time according to the instructions of the real-time fluorescence quantitative PCR kit.

[0038] The results showed that sanguinarine had a significant inhibitory effect on the synthesis of flagellar genes of Salmonella SL1344 within the effective concentration range (16, 64 μg / ml) (see Appendix Figure 4 ).

[0039] Test Example 5

[0040] Cytotoxicity of sanguinarine on Caco-2 cells

[0041] The digested Caco-2 cells were inoculated into 96-well culture plates and the concentration was adjusted to 2×10 4 cells / well, add 200 μL of cell suspension to each well, and continue to culture in a 37°C incubator containing 5% carbon dioxide overnight. Discard the culture medium in each well, add sanguinarine to each well to prepare a solution with a final concentration of 16, 32, and 64 μg / mL, and set up an equal volume of DMSO control. After incubation in a 37°C incubator for 4 hours, add 10% CCK-8 solution to each well, incubate at 37°C for 30 minutes, and measure the absorbance of the sample at 450nm on an enzyme reader.

[0042] The results showed that sanguinarine had no significant cytotoxic effect on Caco-2 cells within the effective concentration range (16-64 μg / ml) (see Appendix Figure 5 ).

[0043] Test Example 6

[0044] Sanguinarine inhibits the adhesion of Salmonella SL1344 to Caco-2 cells

[0045] The digested Caco-2 cells were inoculated into 24-well culture plates and the concentration was adjusted to 4 × 10 4cells / well, and continue to culture overnight in a 37°C incubator containing 5% carbon dioxide. Salmonella SL1344 was inoculated into 2 mL LB medium and cultured overnight. The next day, it was expanded into fresh LB medium at a ratio of 1:20, and sanguinarine was added to make the final concentrations of 16, 32, and 64 μg / mL, respectively, and an equal volume of DMSO was set as a control. Culture was continued at 37°C and 180 rpm for 4 hours. The concentration of the bacterial solution was adjusted to 4.0×10 6 CFUs / mL, for future use.

[0046] Remove the culture medium in the 24-well plate and add the prepared bacterial solution, 1 mL / well. After 1 hour of infection, add 1 mL of 0.2% TritonX-100 to each well and pipette repeatedly. Transfer the lysate to a centrifuge tube, dilute it in multiples, and spread it on streptomycin-resistant LB solid culture medium and culture it at 37°C overnight. Count the next day.

[0047] The results showed that sanguinarine (16-64 μg / mL) treatment could significantly inhibit the adhesion of Salmonella SL1344 to Caco-2 cells (see Appendix Figure 6 ).

Claims

1. The medical use of sanguinarine in the preparation of Salmonella flagella inhibitors.

2. The medical use according to claim 1, characterized in that The medical use refers to the biological function of sanguinarine in inhibiting bacterial sliding mediated by salmonella flagella.

3. The medical use according to claim 1, characterized in that The Salmonella flagella inhibitor effectively inhibits the motility mediated by the flagella of the Salmonella, thereby reducing the pathogenicity of the Salmonella.

4. The medical use according to claim 1, characterized in that The salmonella flagella inhibitor sanguinarine does not affect the growth of salmonella within an effective concentration range, but can significantly inhibit the adhesion of salmonella to host cells.

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