Application of 12-ketolithocholic acid in the preparation of anti-Veillonella products

Abstract

The present invention discloses the application of 12-ketolithocholic acid in the preparation of anti-Veillonella products. The first test showed that 12-ketolithocholic acid has good activity in inhibiting atypical Veillonella. Specifically, by measuring the minimum inhibitory concentration required to inhibit 50% of the tested ATCC 17744 strain, the results showed that the bacteria in the blank control wells without drugs grew significantly, while the MIC50 of 12-ketolithocholic acid for 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.

Description

Technical Field

[0001] The present 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 that is an obligate anaerobe and is part of the human oral, intestinal, and vaginal microbiome. Studies have shown that atypical Veillonella infection can cause retropharyngeal abscesses and adversely affect the cervical microenvironment, but research and reports on atypical Veillonella infection are limited. Bacterial resistance is a global problem, and this study provides valuable insights into the development of new drugs to reduce antimicrobial use and prevent the emergence of resistant bacteria.

[0003] Bile acid is an active ingredient in bile and one of the important components involved in bile digestion. Changes in its content have 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 currently 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 its inhibitory activity against different types of streptococci. Specifically, at a concentration of 200 μg / ml, deoxycholic acid had an inhibitory rate of 96.75% against Streptococcus mitis, a test strain, while its inhibitory rate against Streptococcus pharyngitis, also a Streptococcus, was only 73.60%. By analogy, while 12-ketolithocholic acid has only been reported to inhibit Gram-positive cocci, it remains unknown whether, which Gram-negative cocci it inhibits, and the extent of its inhibitory effect. Summary of the Invention

[0004] Based on the above, the purpose of the present invention is to provide the use 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 solutions:

[0006] The present invention provides application of 12-ketolithocholic acid in preparing a product for resisting Veillonella atypica.

[0007] Preferably, the product is a bacteriostatic agent for Veillonella.

[0008] Preferably, the concentration of 12-ketolithocholic acid in the antibacterial agent is not less than 12.5 μg / ml.

[0009] Preferably, the Veillonella is an atypical Veillonella ATCC 17744 strain.

[0010] Preferably, the MIC50 of the 12-ketolithocholic acid against atypical Veillonella is 294.7 μg / ml.

[0011] The beneficial effects of the present invention are:

[0012] The present invention provides the use of 12-ketolithocholic acid in the preparation of an anti-Veillonella product. Initial experiments demonstrated that 12-ketolithocholic acid exhibits excellent activity against atypical Veillonella. Specifically, determination of the minimum inhibitory concentration required to inhibit 50% of the tested ATCC 17744 strain revealed significant bacterial growth in blank control wells (i.e., drug-free), while the MIC50 of 12-ketolithocholic acid against atypical Veillonella was 294.7 μg / ml. Therefore, the novel use of 12-ketolithocholic 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

[0013] Figure 1 This is a graph showing the relationship between the concentration of 12-ketolithocholic acid and the growth inhibition rate of atypical Veillonella;

[0014] Figure 2 This is a graph showing the relationship between chloramphenicol concentration and the growth inhibition rate of Streptococcus mitis. DETAILED DESCRIPTION

[0015] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] In the following examples of the present invention, the 12-ketolithocholic acid is branded Avanti, product number 700239P, and has a molecular weight of 390.56 g / mol. Its structural formula is as follows:

[0017] ;

[0018] Before use, prepare the maximum concentration to 50 mg / mL with ethanol and dilute it in 1 / 2 times.

[0019] The chloramphenicol was used as a reference substance, brand MACKLIN, product number 56757.

[0020] The present invention adopts the internationally common microbroth two-fold dilution method to test the in vitro antibacterial activity of 12-ketolithocholic acid, examines its antibacterial activity against atypical Veillonella, and uses chloramphenicol as a reference substance to evaluate the experimental system.

[0021] Example 1: Test strain and its cultivation method

[0022] 1. Strain: Atypical Veillonella ATCC 17744.

[0023] 2. Source of bacteria: purchased from Beijing Biobowei Biotechnology Co., Ltd.

[0024] 3. Culture conditions: Reinforced Clostridium Medium (RCM) + 1.5% (60% sodium lactate), incubate at 37°C for 24-48 hours.

[0025] 4. Start inoculation and culture of bacteria

[0026] Wipe the ampoule containing the atypical Veillonella strain ATCC 17744 with cotton wool soaked in 75% alcohol. Heat the tip of the ampoule over a flame and drip a small amount (2-3 drops) of sterile water onto the heated tip to rupture it. Use a file or tweezers to knock off the ruptured tip of the ampoule. Pass the lyophilized tube opening over a flame. Use a sterile pipette to draw 0.3 ml of sterile water into the lyophilized tube and gently shake until dissolved. Aspirate the entire bacterial suspension and inoculate onto RCM + 1.5% (60% sodium lactate) solid medium. Incubate at 37°C for 24 hours. Then, select a single colony and culture it in liquid medium under the same conditions.

[0027] 5. Preparation of bacterial working solution

[0028] When the OD600 value reached about 0.85 (about 109 CFU / ml), the suspension was diluted in RCM + 1.5% (60% sodium lactate) liquid medium (0, 10, 100, 1000, 100000, 1000000, 10000000 times), and 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 make the final concentration of the bacterial suspension about 2×10 6 CFU / ml. All bacterial manipulations were performed under sterile conditions. After the experiment, all consumables were sterilized and discarded.

[0029] 6. Preparation of control group bacterial solution

[0030] The control group used Streptococcus mitis ATCC49456 stored at -80℃; after recovery, when the OD600 value reached about 0.5 (about 108 CFU / ml), and after gradient dilution in THB broth (0, 10, 100, 1000, 100000, 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 approximately 2 × 10 6 CFU / ml. All bacterial manipulations were performed under sterile conditions. After the experiment, all consumables were sterilized and discarded.

[0031] Example 2: Determination of the minimum inhibitory concentration required to inhibit 50% of the test bacteria (MIC50)

[0032] 1. Test method: MIC50 was determined by microbroth dilution method.

[0033] The experiment was divided into a positive control group (bacterial suspension without drug), a negative control group (medium containing solvent ethanol), and an experimental group (containing bacterial suspensions containing different concentrations of 12-ketolithocholic acid). Three parallel groups were set up for each of the above groups. The 12-ketolithocholic acid experimental solution with a concentration of 400μg / ml was diluted in the liquid culture medium in equal folds to make the final concentrations of 400μg / ml, 200μg / ml, 100μg / ml, 50μg / ml, 25μg / ml, 12.5μg / ml, and 0μg / ml. The concentration was 2×10 6 CFU / mL atypical Veillonella suspension and each group of drug solution were inoculated into sterile 96-well plates 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.

[0034] 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 into sterile 96-well plates at a ratio of 1:1, respectively, to make the final concentration of the bacterial suspension 1×10 6 CFU / ml (containing 10 5 CFU / well) and incubated under culture conditions for 24 h.

[0035] After the incubation period, visual observation was performed against a black background. The lowest drug concentration at which no bacterial growth was observed within the plate was the minimum inhibitory concentration (MIC) of 12-ketolithocholic acid. The MIC determination experiment was repeated three times for all bacterial species. The OD600 value of the suspended bacteria was measured. The inhibition rate of the test sample and the control at different concentrations was calculated as follows:

[0036] Inhibition rate (%) = 1 - (test sample OD value - negative control OD value) / (positive control OD value - negative control OD value) × 100%. MIC50 was calculated using curve fitting.

[0037] 2. Result judgment

[0038] 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 bacterial growth is evident in the positive control wells (i.e., those containing no drug).

[0039] Table 1 Absorbance determination of the effect of different concentrations of 12-ketolithocholic acid on the growth of atypical Veillonella

[0040]

[0041] Table 2 Absorbance determination of the effect of different concentrations of chloramphenicol on the growth of Streptococcus mitis

[0042]

[0043] The results in Table 1-2 show normal bacterial growth in the positive control wells (i.e., drug-free), and no bacterial growth in the vehicle control wells. The MIC of the control substance, chloramphenicol, against S. mitis was 4 μg / mL, which meets the standard of ≤ 4 μg / mL for chloramphenicol against S. mitis as specified in the National Committee for Clinical Library Standards (NCCLS) 2001 "Performance Standards for Antimicrobial Susceptibility Testing: Information Supplement, 11th Edition," M100-S11, demonstrating the reliability of this experimental method.

[0044] 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 Antibacterial Agents against 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 according to 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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