Application of 12-ketolithocholic acid in preparation of anti-Vihonor product

Abstract

The invention discloses an application of 12-ketolithocholic acid in preparation of an anti-Vihonor coccus product. The first test proves that the 12-ketolithocholic 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 the 12-ketolithocholic acid for the atypical Wehonor coccus is 294.7 mu g / ml. Therefore, the new application of the 12-ketolithocholic 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 relates to the technical field of biopharmaceuticals, and in particular to application of 12-ketolithocholic acid in the preparation of an anti-Veillonella product. 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. The change in its content has important reference value in the diagnosis of liver disease, and it also has the effect of inhibiting tumors. 12-Ketolithocholic acid is one of the important secondary bile acid derivatives in the intestine. There are reports on the use of 12-ketolithocholic acid in the preparation of anti-Streptococcus mitis, anti-oral Streptococcus and anti-pharyngitis Streptococcus products, but the above bacteria are all Gram-positive cocci, and there are no reports on the use of 12-ketolithocholic acid in the preparation of atypical Veillonella products as Gram-negative cocci. In patent CN 115531394 B, it is clearly shown through comparative examples that deoxycholic acid, which is also a secondary bile acid derivative, shows great differences in the 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%. By analogy, based on the reports that only 12-ketolithocholic acid has an inhibitory effect on Gram-positive cocci, it is still unknown whether 12-ketolithocholic 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 an application of 12-ketolithocholic acid in the preparation of anti-Veillonella products. The 12-ketolithocholic acid has high antibacterial activity against Veillonella, providing a new idea for the development of anti-Veillonella products.

[0005] To achieve its purpose, the present invention adopts the following technical solution: The invention provides application of 12-ketolithocholic acid in preparing a product for resisting Veillonella atypica.

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

[0007] Preferably, the concentration of 12-ketolithocholic 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 12-ketolithocholic acid against atypical Veillonella is 294.7 μg / ml.

[0010] The beneficial effects of the present invention are: The present invention provides the use of 12-ketolithocholic acid in the preparation of an anti-Veillonella product. The first test proved that 12-ketolithocholic 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 12-ketolithocholic acid against atypical Veillonella was 294.7 μg / ml. Therefore, the new use of 12-ketolithocholic acid provided by the present invention provides a reference for the clinical prevention and control of epidemics caused by Veillonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the relationship between the concentration of 12-ketolithocholic acid and the growth inhibition rate of atypical Veillonella; Figure 2 This is the relationship between chloramphenicol concentration and the growth inhibition rate of Streptococcus mitis. 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 12-ketolithocholic acid is branded as Avanti, with a product number of 700239P and a molecular weight of 390.56 g / mol. Its structural formula is as follows: ; Before use, prepare the maximum concentration to 50 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 12-ketolithocholic acid, investigates its antibacterial activity against atypical Veillonella, and evaluates the experimental system with 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 onto 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, draw 0.3 ml of sterile water with a sterile pipette, drop it into the freeze-dried tube, and shake it gently until it dissolves. Pipette all the bacterial suspension, inoculate it on RCM +1.5% (60% sodium lactate) solid culture medium, culture 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 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 8 CFU / 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 6CFU / 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 12-ketolithocholic acid bacterial suspensions of different concentrations). Three parallel groups were set up for each of the above groups. The 12-ketolithocholic acid experimental solution with a concentration of 400 μg / ml was diluted in the liquid culture medium in double dilutions 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 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 105 CFU / well) and cultured 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 12-ketolithocholic 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 1-2The 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 determination of the effect of different concentrations of 12-ketolithocholic acid on the growth of atypical Veillonella

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

[0029] The statistical results showed that the MIC50 of 12-ketolithocholic acid against the tested atypical Veillonella was 294.7 μg / ml, indicating that 12-ketolithocholic 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.

Claims

1. 12-Ketolithocholic Acid in the Preparation of Anti-Veillonella Veillonella atypica application in products.

2. The 12-ketolithocholic 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. The 12-ketolithocholic 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 12-ketolithocholic acid in the antibacterial agent is not less than 12.5 μg / ml.

4. The 12-ketolithocholic 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. The 12-ketolithocholic acid as claimed in any one of claims 1 to 4 is used in the preparation of anti-Veillonella Veillonella atypica The application of the product is characterized in that The MIC50 of the 12-ketolithocholic acid against atypical Veillonella is 294.7 μg / ml.

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