Application of combination of emodin and antibiotics in preparation of medicine for preventing and treating salmonella infection

Abstract

The invention discloses application of combination of emodin and antibiotics in preparation of a medicine for preventing and treating salmonella infection, and belongs to the field of biological medicine. According to the application disclosed by the invention, on the premise of not influencing the proliferation and activity of intestinal epithelial cells, the emodin is respectively combined with ciprofloxacin and tetracycline with minimum inhibitory concentration to achieve a remarkable synergistic effect of inhibiting salmonella from invading the intestinal epithelial cells. The invention provides combination of emodin and ciprofloxacin or tetracycline, application of the emodin and ciprofloxacin or tetracycline in preparation of drugs for inhibiting salmonella from invading intestinal epithelial cells and application of the emodin and ciprofloxacin or tetracycline in preparation of drugs for preventing and treating salmonella infection. According to the invention, in the prevention and treatment of salmonella infection, the risk of antibiotic abuse is reduced by using emodin to reduce and replace antibodies.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an application of rhubarb combined with antibiotics in the preparation of a medicine for preventing and treating salmonella infection. Background Art

[0002] Salmonella is a bacterium widely found in the intestines of animals. It can cause food poisoning and various infections, posing a serious threat to public health. In animal husbandry, agriculture, and medicine, the abuse or improper use of antibiotics has led to increased resistance to Salmonella. Specifically, resistant Salmonella can infect humans through direct contact or fecal contamination of the food chain. In clinical treatment, the emergence of resistant Salmonella makes the infection more difficult to treat, increasing treatment costs and risks for patients. A susceptibility test of 17 antibiotics was conducted on 114 strains of Salmonella isolated from Shiyan, Hubei, and it was found that the resistance rate to six commonly used antibiotics, including chloramphenicol, kanamycin, azithromycin, nalidixic acid, ciprofloxacin and tetracycline, reached more than 25% (Lv J, Geng L, Ye W, et al. Antimicrobial resistance and geneticrelatedness of Salmonella serotypes isolated from food, asymptomatic carriers, and clinical cases in Shiyan, China [J]. Plos one, 2024, 19 (5): e0301388.). The emergence of multidrug-resistant and extensively drug-resistant Salmonella limits the available antibiotic options and makes treatment options more complicated.

[0003] At present, the main response measures include: strengthening the supervision of antibiotic use in animal husbandry and limiting the use of non-therapeutic antibiotics. Improving food safety standards and strengthening hygiene control during food processing and distribution. Carrying out drug resistance monitoring and research to better understand the epidemiology and transmission mechanism of drug-resistant Salmonella. In clinical treatment, rationally use antibiotics, avoid unnecessary prescriptions, and develop new antibiotic alternatives.

[0004] "Reducing and replacing antibiotics" is one of the comprehensive response measures to reduce bacterial resistance. "Reducing antibiotics" refers to reducing unnecessary use of antibiotics in human medicine, animal husbandry and agriculture; "Replacing antibiotics" refers to finding or developing alternatives or supplements to traditional antibiotics to reduce dependence on traditional antibiotics, thereby reducing the development of bacterial resistance.

[0005] Chinese veterinary medicine refers to medicines that are made from traditional Chinese herbal medicines and are extracted, processed or compounded into various dosage forms. It is considered to be one of the feasible ways to achieve "reducing and replacing antibiotics" in the animal husbandry industry. Summary of the invention

[0006] The purpose of the present invention is to overcome the existing problem of drug resistance of Salmonella and provide an application of rhubarb combined with antibiotics in the preparation of a medicine for preventing and treating Salmonella infection.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention found that the minimum inhibitory concentrations of 6 commonly used antibiotics, chloramphenicol, kanamycin, azithromycin, nalidixic acid, ciprofloxacin and tetracycline, on the standard strain of Salmonella SL1344 were 2μg / mL, 4μg / mL, 8μg / mL, 1μg / mL, 0.016μg / mL and 2μg / mL respectively. Rheum sulphate does not affect the growth of SL1344 Salmonella at a concentration of 1000μM and lower, that is, Rheum sulphate has no antibacterial effect on Salmonella at a concentration of 1000μM and lower. Compared with the use of various antibiotics alone, Rheum sulphate is used in combination with 6 commonly used antibiotics, chloramphenicol, kanamycin, azithromycin, nalidixic acid, ciprofloxacin and tetracycline, respectively, and there is no significant difference in the diameter of the antibacterial ring, that is, the combination of Rheum sulphate and these antibiotics does not affect the antibacterial effect of antibiotics on Salmonella. 50μM Rheum sulphate does not affect the proliferation of intestinal epithelial cells SW480. Without affecting the proliferation of intestinal epithelial cells SW480, 50μM emodin combined with 0.016μg / mL ciprofloxacin and 2μg / mL tetracycline showed a significant synergistic inhibitory effect on the invasion of SL1344 into intestinal epithelial cells SW480.

[0009] Based on the above findings, the present invention provides the use of emodin and antibiotics in the preparation of a drug for inhibiting the invasion of Salmonella into intestinal epithelial cells. The antibiotic is ciprofloxacin or tetracycline, preferably tetracycline.

[0010] The present invention also provides the use of a composition comprising emodin and an antibiotic in the preparation of a drug for inhibiting salmonella from invading intestinal epithelial cells. The antibiotic is ciprofloxacin or tetracycline, preferably tetracycline.

[0011] The present invention also provides the use of rheum officinale and antibiotics in the preparation of medicines for preventing and treating salmonella infection. The antibiotics are ciprofloxacin or tetracycline, preferably tetracycline.

[0012] The present invention also provides the use of a composition comprising rhein and an antibiotic in the preparation of a drug for preventing and treating salmonella infection. The antibiotic is ciprofloxacin or tetracycline, preferably tetracycline.

[0013] The present invention also provides a drug for inhibiting salmonella from invading intestinal epithelial cells or preventing salmonella infection, which comprises rhamnosin and antibiotics, and may also comprise auxiliary materials acceptable in pharmaceutical preparation. The antibiotic is ciprofloxacin or tetracycline, preferably tetracycline.

[0014] The present invention has the following advantages and effects compared with the prior art: The present invention finds that the combination of emodin and ciprofloxacin or tetracycline has a synergistic effect in inhibiting the invasion of salmonella into intestinal epithelial cells. The present invention provides the use of emodin in combination with ciprofloxacin or tetracycline in the preparation of a drug for inhibiting the invasion of salmonella into intestinal epithelial cells or preventing and treating salmonella infection. The present invention reduces the risk of antibiotic abuse by using emodin to reduce antibiotics and replace antibiotics in the prevention and treatment of salmonella infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results are as follows: the minimum inhibitory concentration of six commonly used antibiotics, including chloramphenicol (C), kanamycin (K), azithromycin (AZM), nalidixic acid (NA), ciprofloxacin (CIP) and tetracycline (TE), was determined against the standard strain of Salmonella SL1344.

[0016] Figure 2 These are the test results of the inhibition of Salmonella by different concentrations of rheum-emodin and the determination results of the growth curve of Salmonella.

[0017] Figure 3 The antibiotic susceptibility paper method (KB method, Kirby-Bauer test) is used to test the diameter of the inhibition zone of rheumatoid artemisia combined with different antibiotics.

[0018] Figure 4 The effect of different concentrations of rheumatoid arthritis on the proliferation and activity of SW480 intestinal epithelial cells.

[0019] Figure 5 The effect of rhamnosus combined with antibiotics on the invasion of Salmonella into SW480 human intestinal epithelial cells. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0021] Example 1 Test of the minimum inhibitory concentration of common antibiotics against Salmonella

[0022] According to the National Committee for Clinical Laboratory Standards (NCCLS) of the United States, the minimum inhibitory concentration (MIC) value of Salmonella to antibiotics was determined by the microbroth dilution method. The specific steps are as follows:

[0023] (1) Preparation of antibiotic solution: Six antibiotics, chloramphenicol (C), kanamycin (K), azithromycin (AZM), nalidixic acid (NA), ciprofloxacin (CIP) and tetracycline (TE), were prepared into stock solutions for later use. Except for chloramphenicol and nalidixic acid, which needed to be diluted with dimethyl sulfoxide (DMSO), the stock solutions of other antibiotics were diluted twice with sterile water to obtain different dilution concentration gradients.

[0024] (2) The SL1344 strain of Salmonella typhimurium was activated using the three-zone streaking method and cultured in a 37°C constant temperature incubator overnight. The next day, a single clone was selected and cultured in LB liquid medium for 12 hours. The concentration of the SL1344 strain of Salmonella typhimurium was adjusted to 10 5 ~10 6 CFU / mL. 90μL bacterial solution and 10μL diluted antibiotic solution were added to each well of the experimental group, and sterile water containing 90% bacterial solution was added to the control group. Three parallel experiments were performed each time, and then the 96-well plate was placed at 37°C for 16-18h. Finally, the 96-well plate was placed in an ELISA instrument to measure the absorbance value at OD600nm. The MIC value was read, which is the minimum antibiotic concentration that inhibits bacterial growth.

[0025] result( Figure 1 ) showed that the minimum inhibitory concentrations of six commonly used antibiotics, including chloramphenicol, kanamycin, azithromycin, nalidixic acid, ciprofloxacin and tetracycline, against the standard strain of Salmonella SL1344 were 2μg / mL, 4μg / mL, 8μg / mL, 1μg / mL, 0.016μg / mL and 2μg / mL, respectively.

[0026] Example 2 Test of the Effect of Rheum officinale on the Growth of Salmonella

[0027] (1) Using dimethyl sulfoxide (DMSO) as solvent, prepare 100 mM emodin stock solution and store it at -20°C for later use. Using the two-fold dilution method, dilute 100 mM emodin to 0.1 mM in turn. The preparation method of Salmonella typhimurium SL1344 bacterial solution is the same as that in Example 1.

[0028] The effect of adding different concentrations of emodin on the growth of Salmonella was tested by the microbroth dilution method. The specific method is: set up an experimental group, that is, add 3μL of different diluted concentrations of emodin to every 300μL of bacterial solution, mix well, and add to 3 parallel wells of a 96-well plate, 101μL / well, as a repeat. Set up a control group, that is, add 3μL DMSO to 300μL of bacterial solution, mix well, and add to 3 parallel wells, 101μL / well, as a repeat. After culturing the ELISA plate at 37°C for 16 hours, take out the 96-well plate, place it in an ELISA instrument, and read the OD600nm absorbance value. The results are shown in the table below. Figure 2 A.

[0029] (2) Growth curve determination: Use a sterile test tube to hold 10 mL of 10 5 ~10 6 CFU / mL of Salmonella enterica serovar SL1344 was added with 100 μL of rhamnosine stock solution to make the final concentration of rhamnosine 100 μM. The culture was carried out in a shaker at 37°C and 210 r / min. 500 μL of the sample was taken out every 1 hour to detect the OD600nm absorbance value and record the results. Figure 2 B.

[0030] The above results show that the addition of emodin (not higher than 1000 μM) does not affect the growth of Salmonella SL1344, that is, emodin has no inhibitory effect on Salmonella at a concentration of no more than 1000 μM.

[0031] Example 3 Test of the antibacterial effect of rhein combined with antibiotics on Salmonella

[0032] (1) The preparation method of emodin mother solution is the same as that in Example 2.

[0033] (2) Preparation of drug sensitivity test medium: Weigh 20 g of MH agar (Mueller Hinton agar) and dissolve it in 1000 mL of distilled water. Sterilize it under high pressure (121°C, 20 min). After cooling to 50-60°C, add 1% rheumatoid artemisia stock solution (final concentration of rheumatoid artemisia: 1000 μM) to the experimental group, mix well and pour into culture dishes (20 mL / dish). Add 1% DMSO to the control group, mix well and place on a horizontal clean bench to dry for 20 min for later use.

[0034] (3) Drug combination sensitivity test: Salmonella typhimurium SL1344 was activated and cultured overnight in a 37°C constant temperature incubator. After 24 hours, a monoclonal strain with good growth was selected and shaken in LB liquid culture medium for about 12 hours to measure the OD600nm value. The Salmonella typhimurium SL1344 bacterial solution was adjusted to 0.5 McFarland turbidimetric value with culture medium. A sterile medical cotton swab was taken, dipped in the diluted bacterial solution, and evenly spread on the MH agar (Mueller Hinton agar) culture medium plate. Then, 6 commercial (Liofilchem, Ita) drug sensitivity paper strips (antibiotic contents were 30 μg chloramphenicol, 30 μg kanamycin, 15 μg azithromycin, 30 μg nalidixic acid, 5 μg ciprofloxacin and 30 μg tetracycline) were pasted on the MH agar plate containing 1000 μM rhein. Each antibiotic was pasted three times on the same culture dish, and then marked and placed in a 37°C constant temperature incubator for 24 hours. The next day, the diameter of the growth inhibition zone around each antibiotic filter paper was measured using a vernier caliper. The antimicrobial susceptibility of each antibiotic was determined according to the Antimicrobial Susceptibility Test of the Clinical and Laboratory Standards Institute (CLSI), and the diameter of the inhibition zone around the antibiotic was measured and scanned and recorded.

[0035] result( Figure 3 ) showed that compared with the use of each type of antibiotic alone, there was no significant difference in the diameter of the inhibition zone when emodin was used in combination with six commonly used antibiotics, including chloramphenicol, kanamycin, azithromycin, nalidixic acid and ciprofloxacin, that is, the combination of emodin and these antibiotics did not affect the antibacterial effect of the antibiotics.

[0036] Example 4 Addition of 50 μM emodin does not affect the proliferation of SW480 intestinal epithelial cells

[0037] (1) Drug treatment

[0038] 100 μL SW480 cells were plated in a 96-well plate and cultured in a cell culture incubator at 37°C with saturated humidity. When SW480 cells were stably attached to the wall and the density reached about 70%, the experimental group replaced the cell culture medium of the 96-well plate with a medium containing three working concentration gradients of low (10 μM), medium (50 μM) and high (100 μM) concentrations of emodin, and the control group replaced the cell culture medium of the 96-well plate with a medium containing DMSO1 / 1000. Five replicate wells were set up for each group of experiments. A circle of sterile water or PBS was placed around the 96-well plate to prevent the experimental group liquid from drying. Finally, the 96-well plate was placed in the incubator and waited until the treatment time.

[0039] (2) Cell proliferation rate determination

[0040] The next day, the drug-treated 96-well plate was taken out, 10 μL of CCK-8 solution was added to each experimental well, and the cells were incubated in a cell culture incubator for 1-2 h, and the absorbance was detected at 450 nm.

[0041] The results of cell proliferation rate determination are shown in Figure 4 , 50 μM emodin had no effect on the proliferation of SW480 intestinal epithelial cells.

[0042] Example 5: Under the premise of not affecting the proliferation of SW480 intestinal epithelial cells, 50 μM emodin was used in combination with 0.016 μg / mL ciprofloxacin and 2 μg / mL tetracycline, respectively, to significantly synergistically inhibit the intracellular invasion of SL1344.

[0043] (1) When SW480 cells grow to 95% density, remove the serum-containing DMEM in the culture dish, wash the adherent cells with an appropriate amount of PBS, and then use 0.25% trypsin to digest the cells. The cells will become bright circles under the microscope. Immediately add 1 mL of culture medium to collect the cells, centrifuge at 900-1000 rpm for 3 min, discard the supernatant, resuspend in 1 mL of sterile serum-containing culture medium, pipette 200 μL of the resuspended solution into 14 mL of culture medium and mix by pipetting, pipette 500 μL of the mixed solution and evenly spread it in a 24-well plate, and place it in a 37°C cell culture incubator for 6 to 8 h.

[0044] Prepare LB agar plates, wait for the agar plates to dry, and then activate Salmonella typhimurium SL1344 by three-zone streak method. The next day, pick a single clone strain and inoculate it into LB liquid medium for about 16 hours. After shaking the bacterial solution, take 151 μL and transfer it to 5 mL sterile liquid medium and shake the bacteria for 3 hours before infection.

[0045] Pour LB agar medium into a square culture dish and blow dry it under a clean bench for 20 minutes before use.

[0046] (2) SW480 cell infection

[0047] Observe the growth of SW480 cells under a microscope and take photos to record the cell phenotype. Discard the original cell culture medium, wash twice with PBS, and wash away the antibiotics in the complete culture medium. The experimental group was added with a medium without dual antibodies containing only 50μM rhein, a medium without dual antibodies containing only the minimum inhibitory concentration of antibiotics, or a medium without dual antibodies containing both the minimum inhibitory concentration of antibiotics and 50μM rhein, with more than 3 replicates set for each group; the control group was added with a medium without dual antibodies containing 1 / 1000 DMSO, and incubated in the incubator for 24h to wait for infection.

[0048] The SL1344 bacterial suspension of the transfer culture was placed in a cell culture medium without dual antibodies, and the infection multiplicity MOI was 1:50 (20 μL of bacterial suspension was added to 1 mL of culture medium). After mixing, it was diluted 10 times with the culture medium, evenly transferred to a 24-well plate, and centrifuged at 700 r / m for 5 minutes to promote infection. After culturing in the incubator for 1 hour, the supernatant was discarded, and the culture medium containing 100 μg / mL gentamicin was replaced and incubated for another 1 hour. The supernatant culture medium was discarded, and 100 μL of 1% Triton X-100 was added to each test well. After lysis on ice for about 30 minutes, the cells were scraped off with a soft-head cell scraper and collected into a 1.5 mL EP tube. Each experimental group and control group were sequentially diluted 10 times with sterile PBS or physiological saline. After dilution 10 times, 10 μL of the suspension of each dilution was aspirated and inoculated into a square culture dish and placed at room temperature for 10 minutes to wait for drying. It was placed in a 37°C constant temperature incubator for overnight culture. The entire dilution process was performed with a filter tip pipette. The bacteria were counted the next day. The intracellular invasion rate represents the ability of Salmonella to infect cells. The larger the value, the stronger the infection ability, and the smaller the value, the weaker the infection ability. Intracellular invasion rate = bacterial count of experimental group / bacterial count of control group.

[0049] Results Figure 5 50μM emodin combined with 0.016μg / mL ciprofloxacin and 2μg / mL tetracycline showed a significant synergistic inhibitory effect on SL1344 invading intestinal epithelial cells SW480, especially when emodin was used in combination with tetracycline, the inhibitory effect was more significant.

[0050] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of emodin and antibiotics in the preparation of a drug for inhibiting salmonella from invading intestinal epithelial cells, characterized in that: The antibiotic is ciprofloxacin or tetracycline.

2. Use of a composition comprising emodin and an antibiotic in the preparation of a drug for inhibiting the invasion of Salmonella into intestinal epithelial cells, characterized in that: The antibiotic is ciprofloxacin or tetracycline.

3. The use of rheum officinale and antibiotics in the preparation of a drug for preventing and treating salmonella infection, characterized in that: The antibiotic is ciprofloxacin or tetracycline.

4. Use of a composition comprising emodin and an antibiotic in the preparation of a medicament for preventing and treating Salmonella infection, characterized in that: The antibiotic is ciprofloxacin or tetracycline.

5. A drug for inhibiting the invasion of Salmonella into intestinal epithelial cells or preventing and treating Salmonella infection, characterized in that: The invention comprises rhein and antibiotics; the antibiotics are ciprofloxacin or tetracycline.

6. The drug according to claim 5, characterized in that: It also contains excipient ingredients that are acceptable in pharmaceuticals.

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