Application of oridonin as polymyxin antibiotic synergist and application of oridonin in preparation of anti-salmonella drugs

Abstract

The invention belongs to the technical field of bacterial infection and drug treatment, and discloses an ability of oridonin in combined synergism of anti-salmonella effect of polymyxin. The invention verifies that oridonin and polymyxin are combined to resist salmonella in a macrophage intracellular environment simulation culture medium that oridonin has a remarkable synergistic capability on the salmonella inhibition capability of polymyxin; a growth curve experiment and a combined sterilization curve experiment verify that oridonin enhances the salmonella infection resistance of polymyxin. A novel combined method and a research and development strategy are provided for clinically treating salmonella infection by polymyxin. Compared with a traditional action mechanism that antibiotics directly kill salmonella, oridonin inhibits salmonella infection by effectively enhancing the sterilization effect of polymyxin, bacterial infection can be effectively prevented and treated while the application concentration of polymyxin can be reduced, and salmonella is not prone to being induced to generate related drug resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial infection and drug treatment, and discloses oridonin A as a polymyxin antibiotic synergist and use thereof in preparing an anti-Salmonella drug. Background Art

[0002] Salmonella is an important zoonotic foodborne pathogen that can be transmitted between humans and animals, causing serious harm to the development of various types of breeding industries and human health. Salmonella typhimurium is a pantropic salmonella with a wide range of hosts. It is currently one of the pathogens with the highest isolation rate in various countries. It can cause gastrointestinal diseases in various livestock, dogs, cats and experimental animals, and can also cause gastroenteritis or sepsis in humans. In today's society, antibiotics are seriously abused, Salmonella resistance is increasing year by year, and multidrug resistance is also common, which has a major negative impact on animal husbandry production and even public health safety. The continuous strengthening of bacterial resistance has caused a serious decline in the antibacterial effect of traditional antibiotics, and it has become an urgent need to find new antibacterial treatment routes.

[0003] Polymyxin E is an important drug for treating bacterial infections and is of great significance in fighting against Salmonella infections. Salmonella invades the whole body by invading host macrophages and is desensitized to polymyxin E. This characteristic greatly challenges the ability of polymyxin E to fight against Salmonella infections.

[0004] Oridonin is a natural product of kaurene diterpenoids extracted from Oridonium rubescens. It has effective anti-tumor, anti-inflammatory, heat-clearing and detoxifying effects. According to existing research, Oridonin is believed to have a certain relationship with various kinases in mammalian cells. At present, the various effects of Oridonin on bacteria have not been deeply studied and explored. Summary of the invention

[0005] The primary purpose of the present invention is to provide a polymyxin antibiotic synergist.

[0006] Another object of the present invention is to provide a combination of a polymyxin antibiotic potentiator and an antibiotic.

[0007] Another object of the present invention is to provide the use of the above-mentioned combination substance in the preparation of anti-Salmonella infection drugs.

[0008] The present invention takes the enhancement of the anti-Salmonella ability of polymyxin E as the drug action target, and through the combined drug verification experiment on Salmonella, it is found that Rubescensine A can significantly enhance the anti-Salmonella ability of polymyxin E in LPM culture medium (macrophage intracellular environment simulation culture medium), and can fully enhance the sub-inhibitory concentration of polymyxin E to eliminate Salmonella without affecting the growth of bacteria.

[0009] The above technical objectives are achieved through the following solutions:

[0010] The invention discloses an antibacterial composition, which consists of Rubescensine A and polymyxin.

[0011] Furthermore, the polymyxin is polymyxin E.

[0012] The present invention also protects the use of the antibacterial composition in the preparation of anti-Salmonella drugs.

[0013] Furthermore, the combination of Rubescensine A and polymyxin can reduce the concentration of polymyxin required for anti-Salmonella.

[0014] Furthermore, the application also includes application in drugs for preventing and / or treating infectious diseases caused by Salmonella.

[0015] Furthermore, the infectious disease includes human and animal infection caused by Salmonella.

[0016] Furthermore, the human and animal infections include one or more of typhoid, paratyphoid, gastroenteritis or pullorum.

[0017] In addition, the drug also includes a pharmaceutically acceptable carrier.

[0018] Preferably, the drug is in the form of injection, tablet, pill, capsule, suspension or emulsion.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention finds that Rubescensine A itself has no obvious antibacterial effect on Salmonella, but after combined use, it can synergistically enhance the antibacterial effect of polymyxin E on Salmonella. Compared with the direct application of polymyxin E to kill Salmonella, the combination of Rubescensine A and polymyxin E can greatly reduce the concentration of polymyxin E used, and it is not easy to induce Salmonella to produce relevant drug resistance while effectively killing Salmonella. Rubescensine A itself can effectively resist mammalian tumors, has anti-inflammatory and heat-clearing effects, and has safe ingredients, providing new application ideas and research and development strategies for the clinical application of polymyxin E to fight Salmonella infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To show that Rubescensine A itself does not affect the normal growth of Salmonella typhimurium (the horizontal axis represents time, and the vertical axis represents the measured value);

[0022] Figure 2 To reflect that Rubescensine A can enhance the ability of polymyxin E against Salmonella typhimurium (the horizontal axis represents the change in polymyxin E concentration, and the vertical axis represents the change in Rubescensine A concentration);

[0023] Figure 3To demonstrate that Rubescensine A enhances the ability of polymyxin E against Salmonella typhimurium and is also effective against drug-resistant bacteria (the horizontal axis represents the change in polymyxin E concentration, and the vertical axis represents the change in Rubescensine A concentration);

[0024] Figure 4 This is a bactericidal curve of the combination of Rubescensine A and polymyxin E against Salmonella typhimurium (the horizontal axis represents time, and the vertical axis represents the bacterial count). DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] The CAS registration number of the oridonin of the present invention is 28957-04-2, and the molecular formula is C 20 H 28 O6, molecular weight is 364.44.

[0028] The chemical structural formula of Rubescensine A is as follows:

[0029]

[0030] The polymyxins used in the following examples are all polymyxin E.

[0031] Example 1 Verification of the effect of Rubescensine A on the normal growth of Salmonella

[0032] In this study, the minimum inhibitory concentration (first start) of Rubescensine A against Salmonella (ATCC 14028) was first determined by MIC experiment, and then the effect of Rubescensine A on the growth of Salmonella standard strains was evaluated at sub-inhibitory concentrations based on the obtained MIC value. The experiment used 96-well plates, and Salmonella was inoculated into 4mL LB broth medium and cultured statically overnight at 37°C. In the 96-well plate, Rubescensine A was diluted in 6 groups with a final concentration of 32μg / mL to 1μg / mL using LPM as the culture medium. One group was not added with drugs or bacteria as a negative control, and one group was not added with drugs or bacteria as a blank control. The overnight culture solution was added at a ratio of 1:1000, and three replicates were made for each group. The samples were cultured statically at 37°C, and the OD of the samples was measured and recorded every 1h. 600 .

[0033] The results are as follows Figure 1 As shown, the conclusion is: 1μg / mL to 32μg / mL of Rubescensine A does not inhibit the normal growth of Salmonella. It can be concluded that Rubescensine A itself has no direct ability to inhibit Salmonella.

[0034] Example 2 Rubescensine A enhances the anti-Salmonella ability of polymyxin E

[0035] Single colonies of Salmonella (ATCC 14028) and clinically resistant Salmonella (S.typhimurium (17ES)) were picked and inoculated into LB broth medium for 4 hours (37°C, 180rpm / min), and then diluted to LPM medium at 1:100. Take a 96-well plate, add 50μL of LPM broth in the 8×8 range, add 50μL of polymyxin E diluted with LPM to the first column, and dilute 7 columns in multiples; add 50μL of oridonin A diluted in multiples to the first 7 rows; finally, add 100μL of diluted bacterial solution to each well. The highest concentration of polymyxin E is 64μg / mL, and the highest concentration of oridonin A is 256μg / mL. Incubate at 37°C for 16-18 hours, and measure the OD 600 .

[0036] Conclusion: According to the test data, the FIC values ​​of standard strains and resistant strains are far less than 0.5. Oridonin and polymyxin E have a combined effect against Salmonella, and the combined effect is significant (see Figure 2 and Figure 3 ).

[0037] Example 3. Bactericidal curve of Rubescensine A combined with polymyxin E against Salmonella

[0038] Salmonella (ATCC 14028) was inoculated into 4 mL of LB broth and cultured overnight at 37°C. 1 mL of the bacterial solution was washed with PBS buffer and the OD was adjusted. 600 To 0.25-0.28. Use 15mL centrifuge tubes and 5mL LPM broth system, and divide them into control group, oridonin group, polymyxin E group, and combination group, and make three replicates for each group. Add drugs according to 16μg / mL of oridonin and 16μg / mL of polymyxin E, and do not add drugs to the control group; add the adjusted bacterial volume of bacterial solution at 1:100. Cultivate on a shaker (37℃, 180rpm / min) for about. Take out 100μL of multiple dilutions from each tube at 0, 1, 3, 6, 9, 12, and 24h of cultivation, and then drop the plate. The agar plate is incubated in a 37℃ incubator for 10-12h and then counted.

[0039] Conclusion: The bactericidal curve experiment of the combination of the two drugs showed that the amount of Salmonella in the environment could be greatly reduced when polymyxin E was used at a sub-inhibitory concentration. A certain concentration of Rubescensine A could effectively reduce the concentration of polymyxin E required to eliminate Salmonella, and could kill bacteria more efficiently (see Figure 3 ).

[0040] In summary, the present invention proposes a synergist, Rubescensine A, which effectively enhances the anti-Salmonella activity of polymyxin E. The synergistic effect is significant after combined use, which provides a new medication idea for effectively preventing and treating Salmonella and other Gram-negative bacterial infections.

[0041] 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. An antibacterial composition, characterized in that It is composed of Rubescensine A and polymyxin.

2. The antibacterial composition according to claim 1, characterized in that The polymyxin is polymyxin E.

3. Use of the antibacterial composition according to claim 1 or 2 in the preparation of anti-Salmonella drugs.

4. The use according to claim 3, characterized in that: The combination of Rubescensine A and polymyxin can reduce the concentration of polymyxin required for anti-Salmonella.

5. The use according to claim 4, characterized in that: Oridonin can effectively improve the anti-Salmonella ability of sub-inhibitory concentration of polymyxin, and the concentration of Oridonin is 4-32 μg / mL.

6. The use according to claim 3, characterized in that: The application also includes application in medicines for preventing and / or treating infectious diseases caused by Salmonella.

7. The use according to claim 6, characterized in that: The infectious diseases include infections in humans and animals caused by Salmonella.

8. The use according to claim 7, characterized in that: The human and animal infections include one or more of typhoid, paratyphoid, gastroenteritis or pullorum.

9. The use according to claim 3, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that: The dosage form of the drug is injection, tablet, pill, capsule, suspension or emulsion.

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