Application of deoxycholic acid in preparation of anti-vihonor coccus product

Abstract

The invention discloses an application of deoxycholic acid in preparation of an anti-Vihonor product. The first test proves that deoxycholic acid has good activity of inhibiting atypical Wehonor coccus, specifically, the minimum inhibitory concentration required for inhibiting 50% of tested ATCC 17744 strain is measured, and the result shows that bacteria in a blank control hole containing no medicine grow obviously, and the MIC50 of deoxycholic acid for atypical Wehonor coccus is 154.2 mu g / ml. Therefore, the new application of deoxycholic acid provided by the invention provides reference for clinical prevention and control of epidemic diseases caused by Wehonor coccus.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and particularly relates to the application of deoxycholic acid in the preparation of products against Veillonella. Background Art

[0002] Atypical Veillonella is a Gram-negative anaerobic micrococcus, an obligate anaerobe, and part of the human oral, intestinal, and vaginal microbiota. Studies have shown that atypical Veillonella infection can cause retropharyngeal abscesses and have a harmful effect on the cervical microenvironment, but there are currently few studies and reports on atypical Veillonella infection. Bacterial resistance is a global problem. In order to reduce the use of antimicrobial drugs and prevent the emergence of drug-resistant bacteria, it has important reference value for the development of new drugs.

[0003] Bile acid is an effective ingredient in bile and one of the important components involved in the digestive function of bile. Its content change has important reference value in the diagnosis of liver disease, and it also has the effect of inhibiting tumors. Deoxycholic acid is one of the most important secondary bile acids in the intestine. At present, there are reports on the use of deoxycholic acid in the preparation of anti-Streptococcus mitis, anti-oral Streptococcus and anti-infantile Streptococcus products, but the above bacteria are all Gram-positive cocci, and there is no report on the use of deoxycholic acid in the preparation of atypical Veillonella products as Gram-negative cocci. In patent CN115531394 B, it is clearly shown through comparative examples that deoxycholic acid exhibits large differences in inhibitory activity against different types of streptococci. Specifically, when the concentration of deoxycholic acid is 200 μg / ml, its inhibition rate on the tested Streptococcus mitis is 96.75%, while its inhibition rate on the tested Streptococcus pharyngitis, which is also a streptococcus, is only 73.60%. Therefore, it is still unknown whether deoxycholic acid has an inhibitory effect on Gram-negative cocci, which Gram-negative cocci it has an inhibitory effect on, and what the inhibitory effect is. Summary of the invention

[0004] Based on the above, the purpose of the present invention is to provide the use of deoxycholic acid in the preparation of anti-Veillary Coccus products, wherein the deoxycholic acid has high antibacterial activity against Veillary Coccus, providing a new idea for the development of anti-Veillary Coccus products.

[0005] To achieve its purpose, the present invention adopts the following technical solution: The invention provides application of deoxycholic acid in preparing a product for resisting Veillonella atypica.

[0006] Preferably, the product is a bacteriostatic agent for Veillonella.

[0007] Preferably, the concentration of deoxycholic acid in the antibacterial agent is not less than 12.5 μg / ml.

[0008] Preferably, the Veillonella is an atypical Veillonella ATCC 17744 strain.

[0009] Preferably, the MIC50 of the deoxycholic acid against atypical Veillonella is 154.2 μg / ml.

[0010] The beneficial effects of the present invention are: The present invention provides the use of deoxycholic acid in the preparation of an anti-Veillonella product. The first test proved that deoxycholic acid has good activity in inhibiting atypical Veillonella. Specifically, by determining the minimum inhibitory concentration required to inhibit 50% of the tested ATCC 17744 strain, the results showed that the bacteria in the blank control well (i.e., without drug) grew significantly, while the MIC50 of deoxycholic acid against atypical Veillonella was 154.2 μg / ml. Therefore, the new use of deoxycholic acid provided by the present invention provides a reference for the clinical prevention and control of diseases caused by Veillonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the relationship between the concentration of deoxycholic acid and the growth inhibition rate of atypical Veillonella; Figure 2 This is the relationship between chloramphenicol concentration and growth inhibition rate of Streptococcus mitis; Figure 3 This is a picture showing the results of deoxycholic acid treatment causing cell membrane rupture of atypical Veillonella; Figure 4 This is a graph showing the results of deoxycholic acid treatment causing the leakage of nucleic acid content in atypical Veillonella; Figure 5 This is a graph showing the results of deoxycholic acid treatment causing leakage of protein contents of atypical Veillonella. DETAILED DESCRIPTION

[0012] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0013] In the following examples of the present invention, the deoxycholic acid was purchased from Sigma with a product number of 30960. The CAS number is 83-44-3 and the molecular formula is C 24 H 40 O4, molecular weight is 392.57 g / mol. Its structural formula is as follows: ; Before use, prepare the maximum concentration to 20 mg / mL with ethanol and dilute it by multiples.

[0014] The chloramphenicol was used as a reference substance, brand MACKLIN, product number 56757.

[0015] The present invention adopts the internationally common microbroth two-fold dilution method to test the in vitro antibacterial activity of deoxycholic acid, investigates its antibacterial activity against atypical Veillonella, and evaluates the experimental system with the reference substance chloramphenicol.

[0016] Example 1: Test strain and its cultivation method 1. Strain: Atypical Veillonella ATCC 17744.

[0017] 2. Source of strains: purchased from Beijing Biobio Biotechnology Co., Ltd.

[0018] 3. Culture conditions: Reinforced Clostridium Medium (RCM) + 1.5% (60% sodium lactate), incubate at 37°C for 24h-48h.

[0019] 4. Start inoculation and cultivation of bacteria Wipe the ampoule tube containing the atypical Veillonella strain ATCC 17744 with cotton wool soaked in 75% alcohol, heat the top with a flame, drop a small amount (2-3 drops) of sterile water on the heated top to break it, knock off the broken top of the ampoule tube with a file or tweezers and pass the opening of the freeze-dried tube over the flame, use a sterile pipette to draw 0.3 ml of sterile water, drop it into the freeze-dried tube, and gently shake it until it dissolves. Pipette all the bacterial suspension and inoculate it on RCM +1.5% (60% sodium lactate) solid culture medium. After culturing at 37°C for 24 hours, pick a single colony and culture it in liquid culture medium under the same culture conditions.

[0020] 5. Preparation of bacterial working solution When the OD600 value of atypical Veillonella reached about 0.85 (about 109 CFU / ml), after gradient dilution (0, 10, 100, 1000, 10000, 100000, 1000000, 10000000 times) with RCM + 1.5% (60% sodium lactate) liquid medium, 100 μL of the bacterial suspension was inoculated on the solid culture medium. After culturing for 24 h, the number of viable bacteria was counted and the concentration of the bacterial suspension was calculated. The suspension was then diluted to a final concentration of about 2×10 6 CFU / ml. All bacterial operations were carried out under sterile conditions. After the experiment, all consumables were sterilized and then discarded.

[0021] 6. Preparation of the control group bacterial solution The control group used Streptococcus mitis ATCC49456 stored at -80°C. After recovery, when the OD600 value reached about 0.5 (about 10 8CFU / ml), and after gradient dilution with THB broth (0, 10, 100, 1 000, 10000, 100000, 10000), 100 μL of the bacterial suspension was inoculated on solid culture medium. After culturing for 24 h, the number of viable bacteria was counted and the concentration of the bacterial suspension was calculated. The suspension was then diluted to a final concentration of about 2×10 6 CFU / ml. All bacterial operations were carried out under sterile conditions. After the experiment, all consumables were sterilized and then discarded.

[0022] Example 2: Determination of the minimum inhibitory concentration required to inhibit 50% of the test bacteria (MIC50) 1. Test method: Use the broth microdilution method to determine MIC50.

[0023] The experiment was divided into a positive control group (bacterial suspension without drugs), a negative control group (culture medium containing solvent ethanol) and an experimental group (containing deoxycholic acid bacterial suspensions of different concentrations). Three parallel groups were set up for each of the above groups. The deoxycholic acid experimental solution with a concentration of 400 μg / ml was diluted in the liquid culture medium in equal times to make the final concentrations of 400 μg / ml, 200 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml and 0 μg / ml. The concentration was 2×10 6 CFU•mL -1 atypical Veillonella suspension and each group of drug solution were inoculated in a sterile 96-well plate at a ratio of 1:1, and the final concentration of the bacterial suspension was 1×10 6 CFU / ml (containing 10 5 CFU / well) and incubated under culture conditions for 24 h.

[0024] At the same time, the reference substance chloramphenicol was set to have a final concentration of 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, and 0 μg / ml, respectively, with a concentration of 2×10 6 CFU / ml Streptococcus mitis suspension and chloramphenicol solution of each group were inoculated in a sterile 96-well plate at a ratio of 1:1, and the final concentration of the bacterial suspension was 1×10 6 CFU / ml (containing 10 5 CFU / well) and incubated under culture conditions for 24 h.

[0025] After the incubation, the plate was observed with the naked eye against a black background. The lowest drug concentration at which no bacteria grew was the minimum inhibitory concentration (MIC) of deoxycholic acid. The MIC determination experiment for all bacterial species was repeated 3 times. The OD600 value of the suspended bacteria was measured, and the calculation method of the inhibition rate of the test sample and the control at different concentrations was as follows: Inhibition rate (%) = 1- (OD value of test sample-OD value of negative control) / (OD value of positive control-OD value of negative control) × 100%. MIC50 was calculated by curve fitting function.

[0026] 2. Result judgment The test results are shown in Table 1-2, and the inhibition rate is shown in Figure 1-2 The test is only meaningful when there is significant bacterial growth in the positive control wells (i.e., those containing no drug).

[0027] Table 1 Absorbance measurement results of the effect of different concentrations of deoxycholic acid on the growth of atypical Veillonella

[0028] Table 2 Absorbance measurement results of the effect of different concentrations of chloramphenicol on the growth of Streptococcus mitis

[0029] The results in Table 1-2 show that the bacteria in the positive control wells (i.e., without drugs) grow normally, and the medium control medium wells have no bacteria growth. The MIC value of the reference substance chloramphenicol against Streptococcus mitis is 4μg / ml, which meets the standard of chloramphenicol against Streptococcus mitis ≤4μg / ml in the "Implementation Standards for Antimicrobial Susceptibility Testing: Information Supplement of the 11th Edition" M100-S11 of the National Committee for Clinical Laboratory Standards (NCCLS) in 2001, indicating that this experimental method is reliable.

[0030] The statistical results showed that the MIC50 of deoxycholic acid against the tested atypical Veillonella was 154.2 μg / ml, indicating that deoxycholic acid has good inhibitory activity against atypical Veillonella. The research results are expected to provide a reference for the clinical prevention and control of atypical Veillonella.

[0031] Example 3: Determination of cell membrane rupture caused by deoxycholic acid 1. Test methods Set up a control group, a solvent control group, and an experimental group. Take the bacterial solution in the middle of logarithmic growth and add it to 5 ml of culture medium at a ratio of 1:100. Add DCA to the experimental group to reach the concentration of MIC value, add an equal volume of solvent to the solvent control group, and do no additional treatment to the control group. Place it in a 37° anaerobic incubator for 8-9 hours, aspirate the bacterial solution, centrifuge and discard the supernatant, wash with 1 mL of PBS buffer containing 1% BSA, and then stain. Add 100 μL of Syto9 and PI solution to fully resuspend the bacteria, and incubate at 4°C in the dark for 30 minutes. After the incubation is completed, wash 3 times with 1 mL of PBS buffer containing 1% BSA. Finally, resuspend the bacterial precipitate with 100 μL of buffer, prepare slides for laser confocal microscopy observation and photography.

[0032] 2. Interpretation of results Syto9 staining results were positive, shown in green in the figure, PI staining results were positive, shown in red in the figure, and Syto9 and PI co-localization was shown in yellow. Figure 3 .

[0033] Figure 3 The results showed that compared with the untreated group and the solvent control group, the number of PI-positive bacteria increased significantly after deoxycholic acid treatment, and the number of Syto9 and PI co-localized bacteria increased, indicating that deoxycholic acid treatment would destroy the bacterial cell membrane and increase the number of dead bacteria.

[0034] Example 4: Determination of leakage of test bacteria caused by deoxycholic acid 1. Test methods Take 60mL of bacterial suspension in the middle of logarithmic growth, centrifuge at 12000g for 10min, discard the supernatant, add 24ml sterile PBS to resuspend, and mix thoroughly. The resuspended bacterial suspension is divided into two groups by volume, set as the experimental group and the solvent control group. Each group aspirates 2ml of bacterial suspension and centrifuges at 12000g for 5min. After passing the obtained supernatant through a 0.22 um pore size filter membrane, aspirate 200μL and add it to a 96-well plate to measure the absorbance values ​​at 260nm and 280nm as the absorbance value at 0 min. Next, one group adds DCA to its concentration to reach the MIC value as the experimental group. The other group adds an equal volume of solvent as the solvent control group. The shaking bottles of the bacterial suspensions of the experimental and control groups were placed in an anaerobic incubator at 37°. Every 40 minutes, 2 mL of the bacterial suspension was centrifuged at 12000 g for 5 minutes. The supernatant was passed through a 0.22 μm pore size filter membrane, and 200 μL was added to a 96-well plate to measure the absorbance values ​​at 260 nm and 280 nm. The absorbance measurement experiment at all time points was repeated for 3 wells.

[0035] 2. Result judgment Deoxycholic acid treatment induced the leakage of nucleic acid contents of atypical Veillonella. Figure 4 , deoxycholic acid treatment induced leakage of protein contents of atypical Veillonella Figure 5 .

[0036] Figure 4 , Figure 5 The results showed that compared with the solvent control group, deoxycholic acid significantly caused the leakage of nucleic acid and protein contents of atypical Veillonella, affecting bacterial growth.

Claims

1. Deoxycholic acid in the preparation of anti-Veillonella Veillonella atypica application in products.

2. Deoxycholic acid as claimed in claim 1 is used in the preparation of anti-Veillonella Veillonella atypica The application of the product is characterized in that The product is an antibacterial agent for Veillonella.

3. Deoxycholic acid as claimed in claim 2 is used in the preparation of anti-Veillonella Veillonella atypica The application of the product is characterized in that The concentration of deoxycholic acid in the antibacterial agent is not less than 12.5 μg / ml.

4. Deoxycholic acid as claimed in claim 3 is used in the preparation of anti-Veillonella Veillonella atypica The application of the product is characterized in that The Veillonella is an atypical Veillonella ATCC 17744 strain.

5. Deoxycholic acid as claimed in any one of claims 1 to 4 in the preparation of anti-Veillonella Veillonella atypica The application of the product is characterized in that The MIC50 of the deoxycholic acid against atypical Veillonella is 154.2 μg / ml.

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