Application of combination of magnolol and polymyxin in preparation of salmonella infection resisting medicine

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of magnolol as a polymyxin synergist in inhibition of salmonella. The application finds that the combination of magnolol and polymyxin has a good synergistic effect on salmonella in vitro and in vivo experiments. Compared with the traditional method of killing salmonella through combination of antibiotics, the method has the advantages that the magnolol and the polymyxin which are plant-derived synergistically kill bacteria, the generation of bacterial drug resistance is not easy to induce, and the magnolol has the characteristics of wide source, low price and good treatment effect; the method has good research and application significance for excavating antibiotic synergistic substitution and solving bacterial drug resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and discloses the application of magnolol as a polymyxin synergist in inhibiting salmonella. Background Art

[0002] Salmonella is a Gram-negative bacterium that causes foodborne illness when humans or farmed animals consume food contaminated with Salmonella, which can cause local gastroenteritis to systemic diseases. The drug resistance of Salmonella has become a threat to global public health. With the increasing rate of Salmonella resistance in clinical tests and the limited supply of new antibiotics, polymyxin has become the last line of defense for antibiotic treatment of multidrug-resistant Salmonella.

[0003] Since the 1950s, polymyxins have been used as antibiotics to combat clinical infections caused by Gram-negative bacteria in humans and veterinary settings. However, due to the clinical toxicity of polymyxins, their use rate decreased significantly in the 1980s. However, as the harm of drug-resistant bacteria intensified, polymyxins were used again as a last resort for the treatment of multidrug-resistant bacteria. Since 2016, when the plasmid-mediated mobile polymyxin resistance gene (mcr-1) was reported, polymyxin resistance has become an emerging public health threat worldwide. Polymyxins have been discontinued in the veterinary field in my country and have effectively reduced the detection rate of polymyxin-resistant Escherichia coli. However, in clinical treatment, polymyxins are still very important antibiotics.

[0004] Extracting natural compounds that have synergistic effects with polymyxin from natural compound libraries can effectively restore the sensitivity of polymyxin-resistant Enterobacteriaceae to polymyxin, which is also one of the strategies to effectively explore alternative methods for antibiotics and solve the problem of bacterial resistance. The problem of polymyxin resistance based on Enterobacteriaceae is quite important and prominent, so screening effective polymyxin enhancers to treat polymyxin-resistant Enterobacteriaceae infections has become a top priority and deserves further research and development. At present, there are no reports of magnolol as a synergist to enhance the sensitivity of Salmonella to polymyxin. Summary of the invention

[0005] One of the purposes of the present invention is to use magnolol as a polymyxin synergist in inhibiting Salmonella. At the same time, the present application has found that in an in vitro environment, a better inhibitory effect can be achieved in combination with a specific culture medium. The present invention uses the survival rate of Salmonella as a target for screening polymyxin synergists, and magnolol (MAG) is screened to significantly reduce the survival rate of Salmonella. In an in vivo experiment, by constructing a mouse Salmonella intestinal infection model, MAG combined with polymyxin intraperitoneal injection can reduce the amount of Salmonella in feces, intestines, kidneys, liver and spleen and significantly improve the survival rate of animals.

[0006] The above technical objectives are achieved through the following solutions:

[0007] Application of magnolol combined with polymyxin (CS) in the preparation of drugs for resisting Salmonella infection.

[0008] Magnolia nobile (MAG) is a hydroxylated biphenyl isolated from the root and stem bark of Magnolia officinalis. MAG has a wide range of biological activities, such as muscle relaxant, antioxidant, anti-atherosclerotic, anti-inflammatory and antimicrobial effects. MAG has been reported to have a range of pharmacological effects, including antioxidant, anti-inflammatory, antimicrobial, antithrombotic or antiplatelet, anti-stress, anxiolytic, anti-Alzheimer's, anti-stroke, hypoglycemic, smooth muscle relaxant, weight control, anti-dyspepsia / prokinetic, anti-epileptic and hepatoprotective effects.

[0009] Preferably, in the in vitro experiment, the Salmonella is first cultured and incubated to the logarithmic growth phase, and then incubated with LPM broth. After the incubation is completed, it is diluted, and then magnolol and colistin are added in combination.

[0010] Preferably, the Salmonella is the Salmonella standard strain ATCC14028 and the polymyxin-resistant Salmonella.

[0011] Preferably, the concentration of the bacterial solution after incubation is 1×10 6 ~5×10 6 CFU / mL.

[0012] Preferably, the pH value of LPM broth is 5.5-6.0.

[0013] Preferably, the concentration of magnolol is 50-400 μM, and the concentration of polymyxin is 2-16 mg / L.

[0014] A combined culture method comprises first culturing and incubating Salmonella to a logarithmic growth phase, then incubating with LPM broth, diluting after the incubation, and then adding magnolol and colistin for combined use. Preferably, the Salmonella is a standard strain of Salmonella ATCC14028 and a polymyxin-resistant Salmonella.

[0015] Preferably, the concentration of the bacterial solution after incubation is 1×10 6 ~5×10 6 CFU / mL.

[0016] Preferably, the pH value of LPM broth is 5.5-6.0.

[0017] Preferably, the concentration of magnolol is 50-400 μM, and the concentration of polymyxin is 2-16 mg / L.

[0018] In vivo experiments, by constructing a Salmonella intestinal infection model in mice, MAG combined with polymyxin intraperitoneal injection can reduce the amount of Salmonella in feces, intestines and spleen and significantly improve the survival rate of animals. The preferred dosage form of the drug is injection, tablet, pill, capsule, suspension or emulsion.

[0019] Preferably, the dosage of polymyxin in the drug is 5 mg / kg / day, and the dosage of magnolol is 5 mg / kg / day.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present application found that magnolol combined with polymyxin has a good synergistic effect on Salmonella in both in vitro and in vivo experiments. Compared with the combined killing of Salmonella by traditional antibiotics and antibiotics, the synergistic bactericidal effect of magnolol and polymyxin from plant sources is not easy to induce the production of bacterial resistance. In addition, magnolol has the characteristics of wide sources, low price and good therapeutic effect, which has good research and application significance for exploring synergistic substitution of antibiotics and solving bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Showing the checkerboard heat map and in vitro killing curve of MAG combined with polymyxin against Salmonella;

[0023] Figure 2 The checkerboard heat map showing the effect of MAG combined with polymyxin on Salmonella after LB broth culture based on Example 1.

[0024] Figure 3 The checkerboard heat map showing the effect of MAG combined with polymyxin on Salmonella after MH broth culture based on Example 1.

[0025] Figure 4 The chessboard FICI bar graph shows the effect of MAG combined with polymyxin on Salmonella after culture in LPM, LB and MH broths based on Example 1.

[0026] Figure 5 It showed that the combination of MAG and polymyxin significantly reduced the bacterial load in different tissues and organs of Salmonella-infected mice and significantly improved the survival rate of mice. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0029] The polymyxin used in the following examples is polymyxin E. The magnolol described in the present invention has a CAS registration number of 528-43-8 and a molecular formula of C 18 H 18 O 2 , molecular mass 266.33.

[0030] The structural formula of magnolol is as follows:

[0031]

[0032] Example 1 Magnolia officinalis enhances the sensitivity of Salmonella to polymyxin in vitro

[0033] 1. Cool the autoclaved LPM broth with a pH of 5.8 and set aside. The standard strain of Salmonella ATCC 14028 and the polymyxin-resistant Salmonella (17ES) are both laboratory-preserved and commercially available.

[0034] 2. Preparation before the test:

[0035] (1) Prepare an appropriate amount of MAG, using dimethyl sulfoxide as the solvent, so that the final drug concentration is 200 mM, mix well, filter with a filter membrane, and set aside. Prepare a stock solution of polymyxin with a concentration of 5120 mg / L according to CLSI.

[0036] (2) The standard strain of Salmonella ATCC14028 and the polymyxin-resistant Salmonella (17ES) were inoculated onto LB agar plates and cultured to an appropriate size.

[0037] 3. MIC and FICI of MAG and polymyxin against Salmonella

[0038] (1) The tested Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) were inoculated into a test tube containing 4 mL of MH broth and incubated in a shaker at 37°C and 180 rpm until the logarithmic growth phase;

[0039] (2) Use LPM broth with a pH of 5.8 to dilute the incubated bacteria to 100 times, about 10 6 CFU / mL, reserve;

[0040] (3) Take a sterile 96-well plate, add 180 μL of LPM broth medium to well 1, and add 100 μL of LPM broth medium to wells 2-11 respectively;

[0041] (4) Add 20 μL of drug to the first column, pipette and mix well, then pipette 100 μL into the second well, and so on, pipette 100 μL into the tenth well and discard;

[0042] (5) Add 100 μL of diluted bacterial solution to wells 1 to 11 and 200 μL of LPM broth to well 12;

[0043] (6) Repeat steps (3) to (5) three times in parallel;

[0044] (7) Place the inoculated 96-well plate in a 37°C incubator and incubate for 16-18 hours before reading the results;

[0045] (8) Based on the MIC results, the checkerboard method was performed. The MAG concentration range was 6.25-400 μM, and the effective concentration range of polymyxin antibiotics was 2-128 mg / L.

[0046] The MIC results are shown in Table 1. In LPM medium, the MIC values ​​of MAG against Salmonella standard bacteria and Salmonella resistant to polymyxin are >10mM. When MAG is used in combination with polymyxin, the sensitivity of resistant bacteria and standard bacteria to polymyxin can be significantly reduced. The results of the chessboard method are shown in Figure 1 As shown, the results of the chessboard method showed that FICI was less than 0.5, indicating that MAG and polymyxin had a good synergistic effect in the treatment of Salmonella.

[0047] Table 1

[0048]

[0049] In addition, referring to the above 3, MIC and FICI of MAG and polymyxin against Salmonella, steps (2) to (8) were compared with the incubation effects of LB and MH medium. Figures 2 to 4 As shown, it was shown that the synergistic effect of MAG combined with polymyxin against Salmonella was the best after LPM broth culture.

[0050] 4. In vitro bactericidal curves of MAG and polymyxin against Salmonella

[0051] (1) The tested Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) were inoculated into a test tube containing 4 mL of MH broth and incubated in a shaker at 37°C and 180 rpm until the logarithmic growth phase;

[0052] (2) Use LPM broth with a pH of 5.8 to dilute the incubated bacteria 10-fold, about 10 7 CFU / mL, reserve;

[0053] (3) Add 100% MAG or polymyxin with an antibacterial concentration alone or in combination to a centrifuge tube of LPM broth at a pH of 5.8, then add 0.4 mL of the diluted bacterial solution obtained in step (2) and vortex for 5 seconds to mix. Set up a blank control group as a quality control. Except for not adding drugs, the other conditions of the control group are the same as those of the experimental group. The bacterial solution concentration at this time is about 5×10 6 CFU / mL, placed in a 37℃ shaker at 180rpm for culture, the bacterial solution system is 4mL;

[0054] (4) Take 100 μL of the bacterial solution at 0, 3, 6, and 9 h of culture time, add it to a 2 mL centrifuge tube containing 900 μL of 0.85% saline, and perform a 10-fold gradient dilution. After dilution, take 25 μL and drop it on MH agar medium. Incubate it in a 37°C incubator for 18 h and count it. The experimental results are statistically analyzed after three biological replicates. Count the number of colonies at each time point and draw a sterilization curve.

[0055] result: Figure 1 The in vitro bactericidal curve showed that the bacterial load of MAG and polymyxin decreased by more than 100 times after 9 hours of combined use compared with single drug, with significant synergy, demonstrating that the combination of MAG and polymyxin has an excellent effect in killing Salmonella in vitro.

[0056] Example 2 MAG enhances the killing effect of polymyxin on Salmonella in vivo

[0057] 1. Experimental materials: 80 7-week-old C57BL / 6J female mice, mouse gavage needles, and sterile syringes were purchased from Southern Medical University in Guangdong Province.

[0058] 2. Preparation before the experiment: Prepare 5mg / kg polymyxin, 50mg / kg MAG stock solution, and 100000mg / L streptomycin. Inoculate the Salmonella ATCC14028 strain onto the LB agar plate and culture to an appropriate size. There were 9 mice in each group, and there were 4 groups in total (control group, polymyxin treatment group, MAG treatment group, and combined treatment group).

[0059] 3. Establishment of a Salmonella infection mouse model

[0060] (1) Each mouse was orally administered 20 mg of streptomycin to treat its intestinal flora, and was deprived of water and food 4 h before treatment.

[0061] (2) One day after streptomycin treatment, 100 μL of the drug was administered intragastrically. 8 The tested ATCC14028 strain was inoculated into a test tube containing 4 mL of LB broth and incubated in a shaker at 37°C at 180 rpm until the logarithmic growth phase. The centrifuge tube was taken out (about 108 Bacterial count).

[0062] 4. Detection of colony counts in target organs after mouse infection

[0063] (1) One day after infection, mice were given MAG (50 mg / kg, intraperitoneal injection, once a day) and polymyxin (5 mg / kg, intraperitoneal injection, once a day) as monotherapy or in combination.

[0064] (2) After continuous treatment with the above-mentioned drug doses for 3 days, the mice in the treatment group and the quality control group were killed by cervical dislocation, and various organs were collected and colony counts were performed.

[0065] Results: Compared with the best single-drug treatment group, the combined treatment of MAG and polymyxin significantly reduced the number of target organs (spleen, intestines, and feces) of mice, and the bacterial count decreased by more than 100 times compared with the single-drug treatment (see Figure 5 ).

[0066] 5. Survival rate test of mice after infection

[0067] (1) One day after infection, mice were given MAG (50 mg / kg, intraperitoneal injection, once a day) and polymyxin (5 mg / kg, intraperitoneal injection, once a day) as monotherapy or in combination.

[0068] (2) After 7 days of continuous treatment with the drug dose in (1), record and plot the survival curves of mice in the treatment group and the quality control group.

[0069] in, Figure 5 The figure a shows that in the Salmonella infection model in mice, the bacterial load in the feces of animals treated with MAG and polymyxin combined was significantly reduced compared with the bacterial load after treatment with MAG and polymyxin alone. Figure 5 b shows that the bacterial load in the colon of animals treated with MAG and polymyxin combined was significantly reduced compared with that after treatment with MAG and polymyxin alone. Figure 5 c shows that the bacterial load in the spleen of animals treated with MAG and polymyxin combined was significantly reduced compared with that after treatment with MAG and polymyxin alone; Figure 5 d shows that the survival rate of animals treated with the combination of MAG and polymyxin was significantly increased compared with the survival rate of animals treated with MAG and polymyxin alone.

[0070] Conclusion: After 7 days of treatment, the survival rate of the control group was 11%, the survival rate of the MAG treatment group was 33%, the survival rate of the polymyxin treatment group was 44%, and the survival rate of MAG combined with polymyxin was 89%; the protection rate of MAG combined with polymyxin was significantly higher than that of the polymyxin treatment group (see Figure 5 ). This suggests that the combined use of MAG and polymyxin in the treatment of Salmonella infection in vivo also has a good synergistic effect.

[0071] In summary, this study targeted the survival rate of Salmonella and screened out that the natural product MAG combined with polymyxin had a good in vitro killing effect on Salmonella. Finally, a Salmonella-infected mouse model was successfully established by oral infection with Salmonella, further confirming that the combination of MAG and polymyxin can reduce the colonization of different tissues and organs and reduce the mortality of Salmonella-infected mice. These studies will provide ideas and basis for the development of new drugs for polymyxin enhancers.

[0072] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. The application of magnolol combined with polymyxin in the preparation of drugs against Salmonella infection.

2. The use according to claim 1, characterized in that: The Salmonella is the standard strain of Salmonella ATCC14028 and / or the polymyxin-resistant Salmonella.

3. The use according to claim 1, characterized in that: The polymyxin is polymyxin E.

4. The use according to claim 1, characterized in that: The concentration of bacterial solution was 1×10 6 ~5×10 6 CFU / mL.

5. The use according to claim 3 or 4, wherein the concentration of magnolol is 50-400 μM, and the concentration of polymyxin is 2-16 mg / L.

6. The use according to claim 1, characterized in that: The dosage form of the drug is injection, tablet, pill, capsule, suspension or emulsion.

7. The use according to claim 6, characterized in that: The dosage of polymyxin in the medicine is 5 mg / kg / day, and the dosage of magnolol is 5 mg / kg / day.

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