Application of combination of dipentene and florfenicol in inhibition of escherichia coli

Abstract

The invention relates to the technical field of biomedicine, in particular to application of combination of dipentene and florfenicol in inhibition of escherichia coli. According to the application of the combination of the dipentene and the florfenicol in inhibiting the escherichia coli, the dipentene is a mixture composed of R-limonene and S-limonene, it is found that when the dipentene and the florfenicol are used together, the single superposition effect is not achieved, and when the dipentene and the florfenicol are used together, the synergistic sterilization effect on the escherichia coli is achieved; while escherichia coli is inhibited, the use amount of antibiotics is reduced, so that drug residues are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically relates to the use of the combination of dipentene and florfenicol in inhibiting Escherichia coli. Background Art

[0002] With the continuous development of medical technology, reducing the use of antibiotics has become an important trend in the global medical field. This is because the overuse of antibiotics may lead to an increase in bacterial drug resistance, thereby reducing the treatment effect and causing a series of public health problems. Therefore, searching for natural drugs to replace antibiotics has become a research hotspot. Natural drugs have received increasing attention due to their wide sources and relatively small toxic and side effects.

[0003] Dipentene is a natural colorless liquid, a mixture composed of R-limonene (dextrorotatory) and S-limonene (levorotatory). It has a lemon aroma and has been proven to have various biological activities such as anti-inflammatory, antibacterial, and antiviral, so it is considered a potential natural bactericidal drug. However, the current application of dipentene mainly focuses on the use of single components, that is, the use of R-limonene or S-limonene alone.

[0004] For example, in the literature "In Vitro Antimicrobial and Antibiofilm Activity of S-(-)-Limonene and R-(+)-Limonene against Fish Bacteria", it is disclosed that the combination of R-limonene and florfenicol can inhibit Aeromonas hydrophila, and the combination of S-limonene and florfenicol can inhibit Aeromonas hydrophila. However, this technology only shows the bactericidal effects contributed by R / S-limonene alone and florfenicol alone, and its bactericidal effect is still limited. Summary of the Invention

[0005] The purpose of the present invention is to avoid the deficiencies in the prior art and provide the use of the combination of dipentene and florfenicol in inhibiting Escherichia coli, and the combination of dipentene and florfenicol can significantly improve the inhibitory effect on Escherichia coli.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] Provide the use of the combination of dipentene and florfenicol in inhibiting Escherichia coli, and the dipentene is a mixture composed of R-limonene and S-limonene.

[0008] In some embodiments, the Escherichia coli includes Escherichia coli BW25113 and Escherichia coli multi-drug resistant strain NDM5.

[0009] In some embodiments, the concentration of dipentene is 0.05 - 0.7%, and the concentrations of florfenicol are 1 - 256 μg / mL respectively.

[0010] In some embodiments, the concentration of dipentene is 0.078% or 0.625%, and the concentration of florfenicol is any one of 1 μg / mL and 256 μg / mL.

[0011] In some embodiments, the dipentene and the florfenicol are simultaneously applied to the Escherichia coli.

[0012] The beneficial effects of the use of the combination of dipentene and florfenicol in inhibiting Escherichia coli according to the present invention:

[0013] In the present invention, dipentene and florfenicol are combined to inhibit Escherichia coli. Dipentene contains R-limonene and S-limonene. When dipentene and florfenicol act on Escherichia coli jointly, they play a synergistic effect, can significantly inhibit Escherichia coli, and provide an effective way to inhibit Escherichia coli. Compared with the traditional method, the present invention develops a new use of the combination of dipentene and florfenicol, and discovers that the combination of dipentene and florfenicol does not always have a single additive effect. When dipentene and florfenicol are combined, they have a synergistic bactericidal effect on Escherichia coli, and while inhibiting Escherichia coli, the amount of antibiotics used is reduced, thereby reducing drug residues. Description of the Drawings

[0014] Figure 1 It is a statistical result graph of antibacterial activity against Escherichia coli BW25113 in Example 2.

[0015] Figure 2 It is a statistical result graph of antibacterial activity against Escherichia coli multi-drug resistant strain NDM5 in Example 3. Detailed Embodiments

[0016] The preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Although the preferred embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0017] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0018] The florfenicol (FFC) used is a product of Guangzhou Weigu Technology Co., Ltd., with MW 358.21;

[0019] The dipentene (Lim) is a product of Guangzhou Weigu Technology Co., Ltd.

[0020] Judgment principle: If the amount of bacteria reduced in the experimental group is further reduced by 2 logarithmic values on the basis of the reduction of the amount of bacteria treated with florfenicol alone and dipentene alone, it indicates that florfenicol and dipentene have a synergistic antibacterial effect.

[0021] Example 1

[0022] Determination of the minimum inhibitory concentration (MIC) of florfenicol and dipentene against Escherichia coli BW25113 (abbreviated as E. coli BW25113) and the multidrug-resistant strain NDM5 of Escherichia coli

[0023] 1. Experimental materials:

[0024] (1) Test: Cool the autoclaved MH broth for later use.

[0025] (2) Florfenicol: A product of Guangzhou Weigu Technology Co., Ltd., MW 358.21

[0026] Dipentene: A product of Guangzhou Weigu Technology Co., Ltd.

[0027] Test strains: Escherichia coli wild strain BW25113; Escherichia coli multidrug-resistant strain NDM5.

[0028] 2. Preparation before the test:

[0029] (1) Subculture Escherichia coli wild strain BW25113 and Escherichia coli multidrug-resistant strain NDM5 on agar medium and culture until the appropriate size.

[0030] (2) Weigh 0.0512 g of florfenicol powder with a precision balance in a centrifuge tube, add 10 mL of absolute ethanol for dissolution, the concentration of florfenicol is 5120 μg / mL, vortex and mix well for filtration, filter the filtrate through a 0.22 μm diameter filter membrane for physical sterilization, aliquot into sterilized centrifuge tubes, and store in a -20 °C freezer.

[0031] 3. MIC determination experiment:

[0032] (1) Inoculate Escherichia coli BW25113 and Escherichia coli multidrug-resistant strain NDM5 in centrifuge tubes containing 4 mL of MH broth, place them in a shaker at 37 °C and shake at 180 rpm for 4 h, then take out the centrifuge tubes;

[0033] (2) Dilute the incubated bacteria 100-fold with MH broth to about 10 6 CFU / mL for later use;

[0034] (3) Take a sterile 96-well plate, add 180 μL of MH broth medium to the first well, and add 100 μL of MH broth medium to the second to twelfth wells;

[0035] (4) Add 20 μL of dipentene or 20 μL of florfenicol (512 μg / mL) to the first well. After pipetting evenly, aspirate 100 μL and transfer it to the second well, and so on. Discard 100 μL from the tenth well;

[0036] (5) Add 100 μL of the diluted bacterial solution to wells 1 to 11, and add 200 μL of MH broth to the twelfth well;

[0037] (6) The final concentrations (%) of dipentene in wells 1 to 10 of the 96-well plate are 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, 0.0195%, 0.00975%. Well 11 is the growth control, and well 12 is the blank control;

[0038] (7) The final concentrations (μg / mL) of florfenicol in wells 1 to 10 of the 96-well plate are 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5. Well 11 is the growth control, and well 12 is the blank control;

[0039] (8) Set 3 replicates for each drug;

[0040] (9) Place the inoculated 96-well plate in an incubator at 37 °C for 16 - 18 h, and read the results.

[0041] The results are shown in Table 1. The MIC value of dipentene for BW25113 is 0.3125%, and the MIC value of florfenicol for BW25113 is 2 μg / mL; the MIC value of dipentene for NDM5 is 2.5%, and the MIC value of florfenicol for NDM5 is > 256 μg / mL.

[0042] Table 1 MIC of florfenicol and dipentene against standard bacteria and drug-resistant bacteria

[0043]

[0044] Example 2

[0045] Evaluation of the combined bactericidal effect of florfenicol and dipentene against Escherichia coli BW25113

[0046] 1. Experimental materials:

[0047] (1) Test: Cool the autoclaved LB broth, MH broth, and ultrapure water for later use.

[0048] (2) Florfenicol: Product of Guangzhou Weigu Technology Co., Ltd., MW 358.21

[0049] Dipentene: Product of Guangzhou Weigu Technology Co., Ltd.

[0050] Test strain: Escherichia coli wild strain BW25113 (preserved in the laboratory).

[0051] 2. Preparatory work before the test:

[0052] (1) Subculture the Escherichia coli wild strain BW25113 on agar medium until it grows to an appropriate size.

[0053] (2) Weigh 0.0512 g of florfenicol powder with a precision balance into a centrifuge tube, add 10 mL of absolute ethanol to dissolve it. The concentration of florfenicol is 5120 μg / mL. Vortex and mix well, then filter. Filter the filtrate through a 0.22 μm diameter filter membrane for physical sterilization, aliquot it into sterilized centrifuge tubes, and store it in a -20°C freezer.

[0054] 3. Bactericidal experiment:

[0055] (1) Inoculate a single colony of Escherichia coli BW25113 into a centrifuge tube containing 20 mL of LB broth, place it in a shaker at 37°C and shake at 180 rpm for 3 h, then take out the centrifuge tube.

[0056] (2) Centrifuge the cultured bacterial solution at 5500 rpm for 3 min, resuspend it with an equal volume of MH broth, and the bacterial count is 10 8 CFU / mL for standby.

[0057] (3) The system used in this experiment is 15 mL, and the added bacterial solution is the bacterial solution in (2):

[0058] A) Control group: 11.85 mL of MH broth + 3 mL of MH broth + 0.15 mL of bacterial solution;

[0059] B) Single drug group: 11.85 mL of MH broth + 1.5 mL of A / B drug + 1.5 mL of MH broth + 0.15 mL of bacterial solution;

[0060] C) Combined group: 11.85 mL of MH broth + 1.5 mL of A drug + 1.5 mL of B drug + 0.15 mL of bacterial solution.

[0061] (4) This test is set up with 4 groups:

[0062] Escherichia coli BW25113: ① Control group; ② Single florfenicol group: 1 / 2 MIC (1 μg / mL) florfenicol; ③ Single dipentene group: 1 / 4 MIC (0.078%) dipentene; ④ Combined drug group: 1 / 4 MIC dipentene + 1 μg / mL florfenicol;

[0063] (5) After the system is prepared, it is cultured on a shaker at 37 °C and 180 rpm for 24 h.

[0064] (6) At 24 h, 100 μL of the bacterial liquid is pipetted from each tube and added to a 2 mL centrifuge tube containing 900 μL of PBS buffer for ten-fold serial dilution. After dilution, 25 μL is pipetted and dropped onto the LB agar medium, and then incubated in an incubator at 37 °C for 16 - 18 h for colony counting. The experimental results are statistically analyzed after three biological replicates.

[0065] The results are shown in Table 2 and Figure 1 As shown, the bacteria in the growth control group grew normally, indicating that Escherichia coli BW25113 could grow normally under these experimental conditions; in the single-drug group, there was no significant decrease in Escherichia coli BW25113 compared with the single-drug treatment group, indicating that the inhibitory effect of the single-drug treatment group on Escherichia coli BW25113 was not obvious; the results of the combined group showed that the synergistic effect of florfenicol and dipentene had a significant bactericidal effect on Escherichia coli BW25113.

[0066] Table 2 Bactericidal effect of the combination of florfenicol and dipentene on Escherichia coli wild strain BW25113

[0067]

[0068] Example 3

[0069] Evaluation of the combined bactericidal effect of florfenicol and dipentene on Escherichia coli resistant strain NDM5

[0070] 1. Experimental materials:

[0071] (1) Experiments: The autoclaved LB broth, MH broth, and ultrapure water were cooled and reserved.

[0072] (2) Florfenicol: A product of Guangzhou Weigu Technology Co., Ltd., MW 358.21

[0073] Dipentene: A product of Guangzhou Weigu Technology Co., Ltd.

[0074] Test strain: Escherichia coli resistant strain NDM5 (preserved in the laboratory).

[0075] 2. Preparation before the test:

[0076] (1) The Escherichia coli resistant strain NDM5 was subcultured on the agar medium until it reached an appropriate size.

[0077] (2) Weigh 0.0512 g of florfenicol powder with a precision balance and place it in a centrifuge tube. Add 10 mL of absolute ethanol to dissolve it. The concentration of florfenicol is 5120 μg / mL. Vortex and mix well, then filter. Filter the filtrate through a 0.22 μm filter membrane for physical sterilization, and aliquot it into sterilized centrifuge tubes, and store it in a -20°C freezer.

[0078] 3. Bactericidal experiment:

[0079] (1) Inoculate a single colony of Escherichia coli resistant bacterium NDM5 into a centrifuge tube containing 20 mL of LB broth, and place it in a shaker at 37°C and shake at 180 rpm for 3 h, then take out the centrifuge tube.

[0080] (2) Centrifuge the cultured bacterial solution at 5500 rpm for 3 min, and resuspend it with an equal volume of MH broth. The bacterial amount is 10 8 CFU / mL, and set aside for use.

[0081] (3) The system used in this experiment is 15 mL, and the added bacterial solution is the bacterial solution in (2):

[0082] A) Control group: 11.85 mL of MH broth + 3 mL of MH broth + 0.15 mL of bacterial solution;

[0083] B) Single drug group: 11.85 mL of MH broth + 1.5 mL of A / B drug + 1.5 mL of MH broth + 0.15 mL of bacterial solution;

[0084] C) Combined group: 11.85 mL of MH broth + 1.5 mL of A drug + 1.5 mL of B drug + 0.15 mL of bacterial solution.

[0085] (4) This experiment is set up with 4 groups:

[0086] Escherichia coli multidrug-resistant bacterium NDM5: ① Control group; ② Single florfenicol group: 1 / 2 MIC (256 μg / mL) of florfenicol; ③ Single dipentene group: 1 / 4 MIC (0.625%) of dipentene; ④ Combined drug group: 1 / 4 MIC of dipentene + 1 μg / mL of florfenicol;

[0087] (5) After the system is prepared, incubate it in a shaker at 37°C and 180 rpm for 24 h.

[0088] (6) At 24 h, pipette 100 μL of the bacterial solution from each tube into a 2 mL centrifuge tube containing 900 μL of PBS buffer for ten-fold serial dilution. After dilution, pipette 25 μL and drop it on the LB agar medium, and incubate it in a 37°C incubator for 16 - 18 h, then perform colony counting. The experimental results are statistically analyzed after three biological replicates.

[0089] The results are shown in Table 3 and Figure 2As shown, the bacteria grew normally in the growth control group, indicating that Escherichia coli multi-drug resistant bacterium NDM5 could grow normally under the experimental conditions; in the single drug group, Escherichia coli multi-drug resistant bacterium NDM5 did not decrease significantly compared with the single drug treatment group, indicating that the inhibitory effect of the single drug treatment group on Escherichia coli multi-drug resistant bacterium NDM5 was not obvious; the results of the combined group showed that the synergistic effect of florfenicol and dipentene had a significant bactericidal effect on Escherichia coli multi-drug resistant bacterium NDM5.

[0090]

[0091] Table 3 Bactericidal effect of the combination of florfenicol and dipentene on Escherichia coli multi-drug resistant bacterium NDM5

[0092] The results are shown in Table 3 and Figure 2 As shown, the bacteria grew normally in the growth control group, indicating that Escherichia coli multi-drug resistant bacterium NDM5 could grow normally under the experimental conditions; in the single drug group, Escherichia coli multi-drug resistant bacterium NDM5 did not decrease significantly compared with the single drug treatment group, indicating that the inhibitory effect of the single drug treatment group on Escherichia coli multi-drug resistant bacterium NDM5 was not obvious; the results of the combined group showed that the synergistic effect of florfenicol and dipentene had a significant bactericidal effect on Escherichia coli multi-drug resistant bacterium NDM5.

[0093] From Experimental Examples 2 to 3, it can be seen that the antibacterial effects of florfenicol and dipentene used alone on standard strains and drug-resistant strains were weak, and the bactericidal amount was 0.5 log - 1 log when used alone. After their combined use, there was an obvious synergistic antibacterial effect. The present invention can more efficiently inhibit Escherichia coli and can replace the use of antibiotics.

[0094] Example 4

[0095] This example provides the use of the combination of dipentene and florfenicol in inhibiting Escherichia coli, and the dipentene is a mixture composed of R-limonene and S-limonene.

[0096] Specifically, dipentene - racemic limonene;

[0097] Structure: A mixture composed of R-limonene (dextrorotatory) and S-limonene (levorotatory), belonging to monoterpene compounds;

[0098] CAS number: 138 - 86 - 3;

[0099] R / S-limonene - single enantiomer limonene;

[0100] Structure: R-limonene (D-limonene): dextrorotatory body, with a citrus fragrance, CAS number: 5989 - 27 - 5;

[0101] S - Limonene (L - Limonene): The levorotatory form, with a smell more like pine or pungent, CAS No.: 5989 - 54 - 8;

[0102] Enantiomers: Mirror image isomers of each other, with opposite optical activities.

[0103] Specifically, the Escherichia coli is Escherichia coli BW25113, Escherichia coli multi - drug resistant strain NDM5.

[0104] The concentration of dipentene is 0.05 - 0.7%, preferably, the concentration of dipentene is 0.078% and 0.625%, and the concentration of florfenicol is 1 - 256 μg / mL, preferably, the concentration of florfenicol is any one of 1 μg / mL and 256 μg / mL. In practical applications, other concentrations of dipentene and florfenicol can be selected.

[0105] The dipentene and the florfenicol are simultaneously applied to the Escherichia coli. Specifically, dipentene and florfenicol are simultaneously added to the Escherichia coli.

[0106] Furthermore, an Escherichia coli inhibitor is also disclosed, which is composed of dipentene and florfenicol, and the dipentene is a mixture composed of R - limonene and S - limonene.

[0107] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of dipentene in combination with florfenicol in inhibiting Escherichia coli, wherein the dipentene is a mixture composed of R-limonene and S-limonene.

2. Use of dipentene in combination with florfenicol for inhibiting Escherichia coli according to claim 1, characterized in that, The Escherichia coli includes Escherichia coli BW25113 and Escherichia coli multi-drug resistant strain NDM5.

3. Use of dipentene in combination with florfenicol for inhibiting Escherichia coli according to claim 1, characterized in that, The concentration of dipentene is 0.05 - 0.7%, and the concentration of florfenicol is 1 - 256 μg / mL.

4. Use of dipentene in combination with florfenicol for inhibiting Escherichia coli according to claim 1, characterized in that, The concentration of dipentene is 0.078% or 0.625%, and the concentration of florfenicol is any one of 1 μg / mL and 256 μg / mL.

5. Use of dipentene in combination with florfenicol for inhibiting Escherichia coli according to claim 1, characterized in that, The dipentene and the florfenicol are simultaneously administered to the Escherichia coli.

6. An Escherichia coli inhibitor, characterized in that, It is composed of dipentene and florfenicol, wherein the dipentene is a mixture composed of R-limonene and S-limonene.

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