Application of cyanocobalamine in preparation of Brucella bacteriostatic agent

Abstract

The invention belongs to novel medical application of known medicines, and particularly relates to application of cyanocobalamine in preparation of a Brucella bacteriostatic agent. In the specific embodiment of the invention, firstly, four main chemical forms (cyanocobalamin, hydroxycobalamin, methyl cobalamin and adenosine cobalamin) of the vitamin B12 are added into a Brucella culture medium to culture Brucella, and experimental results show that the four forms of vitamin B12 can obviously inhibit the growth of Brucella; wherein the inhibition effect of the cyanocobalamine is most obvious. Then, cyanocobalamin with different concentrations is added into a Brucella culture medium to culture Brucella, and the experimental result shows that the CFU of the Brucella is obviously reduced along with the increase of the concentration of the cyanocobalamin. Similarly, cyanocobalamine can also significantly inhibit intracellular proliferation of Brucella at the cellular level. The cyanocobalamine is a clinically common medicine, and no side effect is reported at present, so that the cyanocobalamine can be used for preparing the Brucella bacteriostatic agent and is clinically used for preventing and treating Brucella diseases.

Description

Technical Field

[0003] The invention belongs to new medical uses of known drugs, and specifically relates to the use of cyanocobalamin in the preparation of a Brucella antibacterial agent. Background Art

[0005] Vitamin B12 (cobalamin) is a water-soluble vitamin containing cobalt. It is the only vitamin containing metal elements. It is a red crystalline powder and a tetrapyrrole with multiple active and inactive forms. Vitamin B12 is essential for some physiological processes, including normal nervous system function and the development and maturation of red blood cells. It has antioxidant function, is a cofactor in mitochondrial energy metabolism, and contributes to DNA synthesis, methylation cycle and epigenetic regulation. Vitamin B12 is synthesized by intestinal microbiota or archaea. Humans cannot synthesize vitamin B12 and can obtain vitamin B12 by consuming animal-derived foods such as meat, eggs and milk, or fermented plant foods and supplements. Healthy adults need an average of 4-7 μg of vitamin B12 per day. Vitamin B12 obtained from food includes four main chemical forms, namely cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) (Wolffenbuttel et al 2023). The most common form of vitamin B12 in supplements is CNCbl.

[0006] There is currently no information on the use of cyanocobalamin in antibacterial activities, especially in inhibiting the proliferation of Brucella, nor is there any information on the use of cyanocobalamin in the prevention and treatment of brucellosis. Summary of the invention

[0007] The object of the present invention is to provide an application of cyanocobalamin in the preparation of a Brucella antibacterial agent, wherein the structural formula of the cyanocobalamin is as follows: Figure 5 .

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] In a specific embodiment of the present invention, cyanocobalamin at different concentrations was added to the culture medium to culture Brucella. The experimental results showed that the absorbance (OD) of Brucella increased with the increase of cyanocobalamin concentration. 600 ) and CFU significantly decreased. Similarly, cyanocobalamin can also significantly inhibit the intracellular proliferation of Brucella at the cellular level. Therefore, cyanocobalamin can be used in the preparation of Brucella antibacterial agent, and is used clinically to prevent and treat brucellosis.

[0010] The protection content of the present invention includes:

[0011] The application of cyanocobalamin in the preparation of brucella antibacterial agent includes using cyanocobalamin as one of the active ingredients or the only active ingredient for the preparation of brucella antibacterial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results are shown in Figure 2. The four main chemical forms of vitamin B12 were detected on Brucella S2 strain growing in TSB medium.

[0013] Figure 2 The results of the test of different concentrations of cyanocobalamin on the growth of Brucella S2 strain in TSB medium; A is the absorbance of the bacterial solution (OD 600 ), B is the CFU counting result.

[0014] Figure 3 The results are for the detection of cyanocobalamin on the growth of other strains of Brucella in TSB medium.

[0015] Figure 4 The results of the detection of cyanocobalamin on the proliferation of Brucella in IPEC-J2 cells infected with it; wherein: A is the cytotoxicity detection result of cyanocobalamin, B is the intracellular CFU statistical chart after Brucella infected IPEC-J2 cells, and C is the immunofluorescence detection chart after Brucella infected IPEC-J2 cells. Figure 5 It is the structural formula of cyanocobalamin. DETAILED DESCRIPTION

[0016] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art; the reagents or materials, unless otherwise specified, are all derived from commercial channels. The cyanocobalamin, methylcobalamin, adenosylcobalamin, and hydroxocobalamin used in the present invention are all commercially available products provided by MCE (MedChemExpress LLC). Brucella suis S2, a swine strain of the Brucella vaccine, and Brucella abortus A19, a bovine strain of the Brucella vaccine, are purchased from Chongqing Aolong Biological Products Co., Ltd. Brucella neotomae is preserved by the State Key Laboratory of Resource Exploration and Utilization of Huazhong Agricultural University (Liu Zhengfei et al. Monoclonal antibodies and applications of Brucella lipopolysaccharide from the Brucella neotomae strain. Application (patent) number: CN202110656015.3). The Brucella melitensis TZ strain used in the present invention was isolated and preserved by the State Key Laboratory of Resource Exploration and Utilization of Huazhong Agricultural University (Fang et al. Animal Diseases, 2023). Brucella LPS monoclonal antibody was prepared and preserved by the State Key Laboratory of Resource Exploration and Utilization of Huazhong Agricultural University (Liu Zhengfei et al. Monoclonal antibody and application of lipopolysaccharide of Brucella sarcoma. Application (patent) number: CN202110656015.3).

[0017] Embodiment 1:

[0018] Effects of four main chemical forms of vitamin B12 on the growth of Brucella S2 strain in TSB medium

[0019] A single colony of Brucella S2 strain was picked and inoculated into TSB liquid medium, cultured at 180 rpm and 37 °C for 24 h, and 100 μL of the bacterial solution was transferred to TSB medium supplemented with 200 nM cyanocobalamin, methylcobalamin, adenosylcobalamin, hydroxocobalamin and an equal volume of ultrapure water, respectively, and cultured at 180 rpm and 37 °C. The OD of the bacterial solution was measured at 12 h and 24 h. 600 Value, test results see Figure 1 .

[0020] result Figure 1 The results showed that all four forms of vitamin B12 could significantly inhibit the growth of Brucella, among which cyanocobalamin had the most significant inhibitory effect. Therefore, we chose cyanocobalamin to continue the follow-up study.

[0021] Embodiment 2:

[0022] Effects of different concentrations of cyanocobalamin on the growth of Brucella S2 strain in TSB medium

[0023] A single colony of Brucella S2 strain was picked and inoculated into TSB liquid medium, cultured at 180 rpm and 37 °C for 24 h, and 100 μL of the bacterial solution was transferred to TSB medium supplemented with 0, 50, 500, and 5000 nM cyanocobalamin, respectively, and cultured at 180 rpm and 37 °C. The OD of the bacterial solution was measured at 12 h and 24 h. 600 The CFU was determined at 12h. The test results are shown in Figure 2 , Tables 1 and 2.

[0024] The results showed that cyanocobalamin could inhibit the proliferation of Brucella in a concentration-dependent manner, and the higher the concentration of cyanocobalamin, the slower the growth of Brucella.

[0025] Table 1 OD of the bacterial solution after cyanocobalamin acted on Brucella 600 value

[0026]

[0027] Table 2 CFU of Brucella after cyanocobalamin acted on the culture solution for 12 hours

[0028]

[0029] Embodiment 3:

[0030] Effects of cyanocobalamin on the growth of other strains of Brucella in TSB medium

[0031] Single colonies of B. bovis A19, B. melitensis M5, B. melitensis TZ and B. neotomae were selected and inoculated into TSB liquid medium, cultured at 180 rpm and 37 °C for 24 h, and 100 μL of the bacterial solution was transferred to TSB medium supplemented with 0, 2, 20, 200 and 2000 nM cyanocobalamin, respectively, cultured at 180 rpm and 37 °C, and the OD of the bacterial solution was measured at 12 h and 24 h. 600 Value, test results see Figure 3 , Table 3.

[0032] The results showed that cyanocobalamin had an inhibitory effect on the proliferation of Brucella strains in a concentration-dependent manner.

[0033] Table 3 OD of the bacterial solution after cyanocobalamin acted on Brucella strains for 24 hours 600 value

[0034]

[0035] Embodiment 4:

[0036] Effect of cyanocobalamin on the proliferation of Brucella in IPEC-J2 cells

[0037] Take the porcine small intestinal epithelial cells IPEC-J2 frozen in liquid nitrogen and shake them quickly in a 42°C water bath until they are completely thawed. Centrifuge at 500×g for 5 minutes, remove the supernatant, resuspend in 10% DMEM medium and transfer to a T25 culture flask, gently blow and mix, and place in a 37°C CO2 incubator for culture. The CCK-8 kit was used to measure the effect of cyanocobalamin on cell activity, and the optimal experimental concentration was screened. Specifically: The IPEC-J2 cells in the 96-well plate grew to about 80%, incubated with different concentrations (2, 20, 200, 2000nM) of cyanocobalamin, and incubated in a 37°C CO2 incubator for 24 hours. Add 10μL of CCK-8 solution to each well under light-proof conditions and incubate in a 37°C CO2 incubator for 1 hour, and use an enzyme reader to measure the OD 450 The effects of different concentrations of cyanocobalamin on cell viability are shown in Figure 2. Figure 4 As shown in A, the results show that low concentrations of cyanocobalamin are not toxic to cells.

[0038] Porcine intestinal epithelial cells IPEC-J2 were cultured at 1×10 5 cells / well were inoculated into 24-well plates and cultured in a 37°C CO2 incubator for 24 h. IPEC-J2 cells were infected with the S2 strain at an MOI of 500. IPEC-J2 cells were incubated with gentamicin for 1 h to kill extracellular bacteria, and then the cell culture medium was replaced with DMEM medium containing 2% FBS, 500 nM cyanocobalamin, and 100 ng IF (intrinsic factor, which promotes the absorption of cyanocobalamin).

[0039] At 24 hpi, cells were lysed with 0.2% Triton X-100, and intracellular bacteria were released and CFU counted. Figure 4 Figure B shows that the addition of cyanocobalamin to DMEM medium can significantly inhibit the intracellular growth of Brucella. Figure 4 This result was further confirmed by indirect immunofluorescence in C, where lipopolysaccharide (LPS) indicated the location of Brucella and DAPI indicated the cell nucleus. Figure 4 It is obvious from species C that the number of Brucella was significantly reduced when cyanocobalamin was added to the DMEM medium.

[0040] All the above experiments were repeated 3 times independently, and the results are the average values.

Claims

1. Application of cyanocobalamin in inhibiting the proliferation of Brucella.

2. Application of cyanocobalamin in the prevention and treatment of brucellosis.

3. The use according to claim 1, wherein the Brucella is Brucella suis S2 strain, Brucella bovis A19 strain, Brucella melitensis M5 strain, Brucella melitensis TZ strain and Brucella sarcoma.

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