Medical application of apigenin in preparation of salmonella flagellum inhibitor

Abstract

The invention belongs to the technical field of anti-infection drugs, and particularly relates to medical application of apigenin in preparation of a salmonella flagellum inhibitor. A migration inhibition test (0.3% agar), minimum inhibitory concentration (MIC) determination, a Caco-2 cell adhesion test and a molecular docking test prove that the apigenin can specifically inhibit salmonella flagellum-mediated migration ability (inhibition rate gt; 49%) and host cell adhesion capability (reduced by more than or equal to 58%), and does not affect bacterial growth (pgt; 0.05), the apigenin can be used for targeting flagellin FljB, FliC, FlgN, FlgA and FlgG (binding energy per lt; -5kcal / mol) can block flagellar movement, and has an application prospect in prevention and control of livestock salmonella related diseases.

Description

Technical Field

[0001] The present invention relates to the use of apigenin as an inhibitor of Salmonella flagellar function in the preparation of anti-virulence drugs, belonging to the technical field of anti-infective drugs. Background Art

[0002] Salmonella typhimurium is a Gram-negative bacillus that widely exists in nature, especially in the intestines of animals. It causes global foodborne diseases by contaminating food and water sources, and the clinical manifestations are fever, diarrhea and systemic infections. Statistics from the World Health Organization show that Salmonella causes more than 100 million infections every year, and the proportion of its multi-drug resistant (MDR) strains continues to rise, seriously threatening public health safety. Traditional antibiotics control infections through direct bactericidal effects, but long-term use has led to two major dilemmas: ① the selective pressure of drug resistance; ② the destruction of the host symbiotic flora, exacerbating the imbalance of the intestinal microecosystem. In response to the above problems, the anti-virulence strategy has become a hot international research topic - by specifically inhibiting pathogen virulence factors (such as adhesion, invasion, toxin release) rather than directly killing bacteria, it can not only relieve clinical symptoms but also reduce the evolutionary pressure of drug resistance. The flagellar system of Salmonella is the core of its virulence. Therefore, targeting flagellar function can double-block the motility-pathogenic axis of Salmonella and is an ideal target for anti-virulence treatment. In recent years, natural compounds have become an important source for the development of anti-virulence drugs due to their low drug resistance induction and multi-target regulation potential.

[0003] Apigenin, also known as apigetrin or apiin, is a flavonoid compound. It exists in a variety of fruits and vegetables, and the main sources include parsley, chamomile, celery, echinacea, artichoke and oregano. Apigenin has various pharmacological effects such as anti-cancer, antioxidant, anti-inflammatory, blood pressure lowering and antiviral. However, there is no relevant research on apigenin inhibiting Salmonella flagella at home and abroad so far. The apigenin (purity ≥ 98%) used in the experiments was from Beijing Solarbio Science & Technology Co., Ltd. Summary of the Invention

[0004] The present invention proposes the use of apigenin as an inhibitor of Salmonella flagellar function in the preparation of anti-virulence drugs, providing a new research idea and candidate natural compound for the prevention and treatment of Salmonella infections.

[0005] Using Salmonella as the research model bacterium, the present invention verified through experiments such as Salmonella swimming inhibition test, minimum inhibitory concentration determination test, growth curve test, Salmonella adhesion to Caco-2 cells test and molecular docking that apigenin can significantly inhibit the swimming of Salmonella flagella on semi-solid medium without affecting bacterial growth, reduce the adhesion rate of Salmonella to Caco-2 cells, thereby reducing the pathogenicity of Salmonella. The Salmonella used in the experiments was SL1344.

[0006] The apigenin described in the present invention has the molecular formula C 15 H 10 O 5 , and its molecular weight is 270.24. The molecular structure of apigenin is as follows:

[0007]

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

[0009] It provides a new medical use of apigenin in the preparation of Salmonella flagella inhibitors, and discloses that apigenin can inhibit flagella-mediated bacterial motility, thereby reducing the pathogenicity of Salmonella. Description of the Drawings

[0010] Figure 1 : Apigenin inhibits the motility of Salmonella SL1344 on semi-solid LB plates;

[0011] Figure 2 : Measurement results of the motility diameter of Salmonella SL1344 by apigenin;

[0012] Note: Compared with the control group, significant differences are indicated as *p<0.05, **p<0.01;

[0013] Figure 3 : Apigenin does not inhibit the growth of Salmonella SL1344 within the sub-inhibitory concentration range;

[0014] Figure 4 : Molecular docking results of apigenin with Salmonella flagellin;

[0015] Figure 5 : Apigenin has no toxic effect on Caco-2 cells within the sub-inhibitory concentration range;

[0016] Figure 6 : Apigenin inhibits the adhesion of Salmonella SL1344 to Caco-2 cells;

[0017] Note: Compared with the control group, significant differences are indicated as *p<0.05, **p<0.01. Detailed Embodiments

[0018] The present invention will be further described by 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 that is easily achieved by those of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0019] Example 1

[0020] Apigenin can be used as a Salmonella flagella inhibitor in any pharmaceutically acceptable carrier.

[0021] Example 2

[0022] Apigenin can be used as a Salmonella flagella inhibitor for the preparation of drugs for preventing related infectious diseases.

[0023] Example 3

[0024] Apigenin can be used as a Salmonella flagella inhibitor for the preparation of drugs for treating related infectious diseases.

[0025] Test Example 1

[0026] Salmonella motility inhibition test

[0027] Take 10 mL of sterile LB medium (containing 0.3% agar and glucose), add apigenin to reach the specified concentration, and pour it into a sterile petri dish. Take the overnight cultured SL1344 bacterial solution, centrifuge (10000 rpm, 5 min) to collect the bacteria, wash the bacteria three times with PBS and resuspend the bacteria to make its OD 600nm = 0.1, pipette an appropriate amount of the bacterial solution to the center of the solid plate, and place it in an incubator at 37°C for 12 h. Evaluate the motility ability according to the diameter of the motility zone of each group of colonies. The larger the motility zone, the stronger the motility ability; conversely, the weaker the motility ability.

[0028] The results showed that: compared with the control group (not treated with apigenin), treatment with different concentrations of apigenin (0.5 - 2 mg / mL) could significantly inhibit the motility of Salmonella on the LB soft agar solid plate, and the inhibition rate > 49% (p < 0.01, n = 3, see attachment Figure 1 ).

[0029] Test Example 2

[0030] Determination of the minimum inhibitory concentration (MIC) value of apigenin against Salmonella SL1344

[0031] According to the standard dilution method for determining the minimum inhibitory concentration value published by the American Society for Clinical and Laboratory Standards, the MIC value of apigenin against Salmonella SL1344 was determined. Add apigenin to a sterile 96-well plate to make its final concentration 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and simultaneously inoculate the overnight cultured Salmonella SL1344 (adjust the bacterial amount to 5×10 5(CFU / mL). After shaking and mixing evenly, it was placed in an incubator at 37°C for 20 h - 24 h to observe the growth of bacteria. The concentration of the compound that visibly inhibited the growth of bacteria was determined as the MIC value of apigenin against Salmonella.

[0032] Conclusion: The MIC value of apigenin against Salmonella SL1344 ≥ 4 mg / mL.

[0033] Test Example 3

[0034] Growth curve of Salmonella SL1344

[0035] Salmonella SL1344 was inoculated into 2 mL of LB medium and cultured overnight. The next day, it was expanded 100-fold to 100 mL of LB medium and continued to be cultured until OD 600nm = 0.3. The bacterial solution was equally divided into 5 conical flasks, and apigenin was added to each flask to make the final concentration 0.5, 1, 2 mg / mL respectively. At the same time, a DMSO control group was set up. It was continuously cultured at 37°C and 180 rpm. The OD 600nm value of the bacterial culture was measured at different time points and recorded until the bacteria grew to the plateau phase, and the growth curve was plotted.

[0036] Conclusion: Within the sub-inhibitory concentration range (0.5 - 2 mg / mL), apigenin did not affect the growth of Salmonella SL1344 (see attachment Figure 3 ).

[0037] Test Example 4

[0038] Molecular docking of apigenin and flagellin of Salmonella SL1344

[0039] The 2D structure of the apigenin small molecule was downloaded from the PubChem database (http / / zinc.docking.org / ) and converted into a mol2 file. The 3D structures of the Salmonella flagellar target proteins FljB, FliC, FlgN, FlgA, and FlgG were downloaded from the PDB database (https: / / www.rcsb.org). The ligands and water molecules were deleted using Pymol software. The small molecule and the target protein were imported into AutoDock software for molecular docking.

[0040] The results showed that: The lowest binding energy between the active ingredient and the target was calculated using Autodock software. The specific results are shown in attachment Figure 4 and Table 1. Apigenin had a strong binding ability with the key proteins of Salmonella flagella, and the binding energy was less than -5 kcal / mol. The results indicated that there might be a regulatory effect between apigenin and Salmonella flagellin.

[0041] Table 1 Molecular docking scoring results

[0042]

[0043] Note: FljB and FliC: These two proteins are the main components of Salmonella flagella. They are involved in the formation of the flagellar filaments, which are the motility organs of bacteria and consist of helical filaments and rotary motors for generating thrust. FlgN, FlgA, and FlgG are flagellar export chaperone proteins involved in flagellar filament synthesis.

[0044] Test Example 5

[0045] Cytotoxicity of Apigenin on Caco-2 Cells

[0046] The digested Caco-2 cells were inoculated into 96-well culture plates, and the concentration was adjusted to 2×10 4 cells / well with DMEM medium without antibiotics. 200 μL of cell suspension was added to each well, and the cells were continued to be cultured overnight in a 5% carbon dioxide incubator at 37°C. The medium in each well was discarded, and solutions with final concentrations of 0.5, 1, and 2 mg / mL of apigenin were added to each well, and an equal volume of DMSO was set as a control. After static incubation in a 37°C incubator for 4 h, 10% CCK-8 solution was added to each well. After incubation at 37°C for 30 min, the absorbance value of the sample at 450 nm was measured on an enzyme-linked immunosorbent assay (ELISA) reader.

[0047] The results showed that apigenin had no obvious cytotoxic effect on Caco-2 cells within the sub-inhibitory concentration range (0.5 - 2 mg / mL) (see Appendix Figure 5 ).

[0048] Test Example 6

[0049] Apigenin Inhibits the Adhesion of Salmonella SL1344 to Caco-2 Cells

[0050] Caco-2 cells were seeded into 24-well culture plates (2×10 4 cells / well) and cultured in a 37°C constant temperature cell incubator for 12 h. The medium was replaced with DMEM (containing 10% fetal bovine serum) containing SL1344 (MOI = 80) to infect the cells. After adding apigenin to reach the specified concentration, the cells were placed in a 37°C constant temperature incubator and incubated for 1.5 h. The cells were washed three times with PBS, 1 mL of sterile distilled water was added to lyse the cells for 5 min, followed by dilution and plating. The plates were placed in a 37°C constant temperature incubator and cultured for 12 h, and then the colonies were counted and statistically analyzed.

[0051] Conclusion: Treatment with apigenin (0.5 - 2 mg / mL) can significantly inhibit the adhesion of Salmonella SL1344 to Caco-2 cells (reduction ≥ 58%, p < 0.01, n = 3, see Appendix Figure 6)。

Claims

1. The medical use of apigenin in the preparation of Salmonella flagella inhibitors, characterized in that: Apigenin is used to prepare an anti-toxic drug for inhibiting the flagella-mediated swimming ability of Salmonella. The drug contains a sub-inhibitory concentration of apigenin, which is 0.5-2 mg / mL, and inhibits the movement of Salmonella and the adhesion to host cells by targeting the flagellar proteins FljB (6RGV), FliC (91WQ), FlgN (5B3D), FlgA (3TEE), and FlgG (6JF2).

2. The use according to claim 1, characterized in that: The apigenin inhibits the swimming ability of Salmonella mediated by flagella, and the inhibition rate is >49%.

3. The use according to claim 1, characterized in that: The apigenin inhibits the adhesion efficiency of Salmonella to Caco-2 cells, reducing the efficiency by ≥58%.

4. An anti-toxic drug for inhibiting the flagella-mediated swimming ability of Salmonella, characterized in that: The drug contains a sub-inhibitory concentration of apigenin, which is 0.5-2 mg / mL, and inhibits the movement of Salmonella and the adhesion to host cells by targeting flagellin FljB (6RGV), FliC (91WQ), FlgN (5B3D), FlgA (3TEE), and FlgG (6JF2).

5. The drug according to claim 4, characterized in that: Used to prevent or treat Salmonella infection and does not inhibit the growth of Salmonella.

Images