Composition of vitamin B7 and compound amoxicillin and application thereof
By combining vitamin B7 with compound amoxicillin, the problem of compound amoxicillin's ineffectiveness against drug-resistant bacteria has been solved, achieving a more efficient bactericidal effect and a lower drug dosage, thus expanding its application.
Patent Information
- Application Number
- CN202511215269.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-12-16
AI Technical Summary
Existing compound amoxicillin is ineffective against some drug-resistant bacteria during the bactericidal process and is prone to developing drug resistance. New synergists need to be developed to expand its application.
Vitamin B7 is combined with compound amoxicillin to form a bactericidal and bacteriostatic composition, preferably with a concentration range of 0.01%-0.1% of vitamin B7 and 16-64 μg/mL of compound amoxicillin, for inhibiting foodborne bacteria such as drug-resistant Escherichia coli, with water as the solvent and an incubation temperature of 36.8℃~37.5℃.
It significantly enhances the bactericidal effect against drug-resistant Escherichia coli, reduces the concentration of compound amoxicillin used, and expands its application range.
Smart Images
Figure CN121129841A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of bactericidal and fungicidal products, and specifically discloses a composition of vitamin B7 and compound amoxicillin and application thereof. BACKGROUND
[0002] The bactericidal mechanism of compound amoxicillin mainly depends on two main components: amoxicillin and clavulanic acid. Among them, the main mechanism of action of amoxicillin is similar to that of traditional penicillins, and clavulanic acid acts as an auxiliary component to enhance the effect of amoxicillin.
[0003] The bactericidal mechanism of amoxicillin: inhibition of bacterial cell wall synthesis - amoxicillin binds to bacterial penicillin-binding proteins (PBPs) to inhibit bacterial cell wall synthesis. These penicillin-binding proteins are essential enzymes in the synthesis of bacterial cell walls. Amoxicillin can block the activity of these enzymes, causing the synthesis of bacterial cell walls to be interrupted, thereby making bacteria unable to maintain the integrity of the cell wall.
[0004] The role of clavulanic acid: Clavulanic acid is a beta-lactamase inhibitor that can effectively inhibit the activity of beta-lactamase produced by some bacteria. Clavulanic acid can prevent beta-lactamase from hydrolyzing amoxicillin, thereby protecting amoxicillin from attack by drug-resistant bacteria and enhancing the antibacterial effect of amoxicillin.
[0005] At the same time, compound amoxicillin has some unavoidable limitations, such as being ineffective against some drug-resistant bacteria and developing drug resistance on bacteria. In order to enhance the bactericidal effect of compound amoxicillin and reduce the dosage, it is essential to study the synergist. However, there are still few reported synergists at present, and the development of a compound amoxicillin synergist can greatly expand the application of compound amoxicillin in the field of bactericidal. SUMMARY
[0006] The purpose of the present application is to reduce the large-scale use of antibiotic compound amoxicillin, and a combination of vitamin B7 and compound amoxicillin is provided.
[0007] The purpose of the present application is achieved by the following technical solutions:
[0008] A bactericidal and fungicidal composition composed of vitamin B7 and compound amoxicillin.
[0009] Preferably, in the composition, the concentration of vitamin B7 is 0.01%-0.1%, and the concentration of compound amoxicillin is 16-64 μg / mL.
[0010] Preferably, the bacteriostatic object of the product is foodborne bacteria, such as Escherichia coli.
[0011] More preferably, the Escherichia coli is drug-resistant Escherichia coli.
[0012] Preferably, the solvent of the bactericidal and fungicidal product is water.
[0013] The application also protects the use of the bactericidal and fungicidal composition in the preparation of an E. coli inhibiting product.
[0014] Preferably, the E. coli is E. coli BW 25113 and / or E. coli drug-resistant bacteria LY9.
[0015] The application also protects a combined bactericidal method, in which compound amoxicillin and vitamin B7 are simultaneously incubated with a bacterial solution at a temperature of 36.8℃ to 37.5℃.
[0016] Preferably, the bacterial solution is an E. coli and / or Salmonella bacterial solution, and the concentration of the bacterial solution is 10 8 CFU / mL.
[0017] Compared with the prior art, the application has the following beneficial effects:
[0018] The application provides a composite bactericidal composition, which comprises compound amoxicillin and vitamin B7. The two components have limited bactericidal effects when used alone, but have obvious synergistic bactericidal effects when used in combination. Therefore, the two components are further added to a solvent in different concentrations to obtain a novel compound amoxicillin synergist. Compared with the bactericidal effect of compound amoxicillin alone, the application can more efficiently kill bacteria and reduce the concentration of compound amoxicillin, thereby further expanding the application of compound amoxicillin in bactericidal treatment. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 Figure 2 is a statistical result graph of the antibacterial effect in Example 2.
[0020] Figure 2 Figure 3 is a statistical result graph of the antibacterial effect in Example 3.
[0021] Figure 3 Figure 4 is a statistical result graph of the antibacterial effect in Example 4. DETAILED DESCRIPTION
[0022] The following specific embodiments of the application will be described in detail, which will fully and comprehensively illustrate the technical solutions of the application. It should be pointed out that the provided embodiments only represent a part of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.
[0023] The test method used in the embodiments of the present application is a conventional method unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified, and the commercially available raw materials are used after removing insoluble impurities, etc. by general filtration methods.
[0024] Determination principle: if the reduced bacterial amount of the experimental group is reduced by more than 1 logarithmic value on the basis of the reduction of the bacterial amount under the treatment of amoxicillin alone and the treatment of vitamin B7 alone, it indicates that the combination has a synergistic bacteriostatic effect.
[0025] Example 1 MIC determination of vitamin B7 and compound amoxicillin on Escherichia coli BW 25113 and Escherichia coli drug-resistant bacteria LY9
[0026] 1. Experimental materials:
[0027] (1) Test: cool the MH broth sterilized by high pressure for standby use.
[0028] (2) Vitamin B7: Solaybao brand, MW244.31; Compound amoxicillin: Mokang brand.
[0029] Test strain: Escherichia coli wild strain BW 25113.
[0030] 2. Preparation work before test:
[0031] (1) Subculture Escherichia coli wild strain BW 25113 and Escherichia coli drug-resistant bacteria LY9 on agar medium, and culture to an appropriate size.
[0032] (2) Weigh 51.2 mg of vitamin B7 in a centrifuge tube with a precision balance, add 10 mL of sterile water for dissolution, the concentration of vitamin B7 is 5120 μg / mL, vortex mixing and filtering, the filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterilized centrifuge tubes and stored in a-80℃ freezer.
[0033] (3) Weigh 51.2 mg of compound amoxicillin in a centrifuge tube with a precision balance, add 10 mL of sterile water for dissolution, the concentration of compound amoxicillin is 5120 μg / mL, vortex mixing and filtering, the filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterilized centrifuge tubes and stored in a-20℃ freezer.
[0034] 3. MIC determination experiment:
[0035] (1) Inoculate Escherichia coli BW 25113 and Escherichia coli drug-resistant bacteria LY9 in a centrifuge tube containing 5 mL of MH broth, and place it in a 37℃ shaking bed at 180 rpm for 3h, then take out the centrifuge tube;
[0036] (2) Use MH broth to dilute the incubated bacteria by 100 times, about 10 6 CFU / mL, standby;
[0037] (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 2nd to 12th wells;
[0038] (4) Add 5 μL of compound amoxicillin (5120 μg / mL) or 5 μL of vitamin B7 (5120 μg / mL) to the first well, mix well, and then transfer 100 μL to the second well, and so on. 100 μL is taken from the 10th well and discarded;
[0039] (5) Add 100 μL of diluted bacteria to the 1st to 11th wells, and add 200 μL of MH broth to the 12th well;
[0040] (6) The final concentration of compound amoxicillin in the 1st to 9th wells of the 96-well plate is 64, 32, 16, 8, 4, 2, 1 μg / mL, the 8th well is a growth control, and the 9th well is a blank control;
[0041] (7) The final concentration of vitamin B7 in the 1st to 9th wells of the 96-well plate is 512, 256, 128, 64, 32, 16, 8 μg / mL, the 8th well is a growth control, and the 9th well is a blank control;
[0042] (8) Each drug is set with 3 rows of repeats;
[0043] (9) Place the inoculated 96-well plate in a 37°C incubator for 16-18 hours, and read the results.
[0044] The results are shown in Table 1. For BW 25113, the MIC value of vitamin B7 is >512 μg / mL, and the MIC value of compound amoxicillin is 8 μg / mL. For LY9, the MIC value of vitamin B7 is >512 μg / mL, and the MIC value of compound amoxicillin is 64 μg / mL.
[0045] Table 1 MIC of component species on E. coli and Salmonella
[0046] Example 2 Evaluation of the combined bactericidal effect of vitamin B7 concentration and compound amoxicillin on E. coli BW 25113
[0047] 1. Experimental materials:
[0048] (1) Test: Cool the high-pressure sterilized LB broth, PBS buffer, and ultrapure water for standby use.
[0049] (2) Vitamin B7: Solabia brand, MW 244.31; Compound amoxicillin: Mokang brand.
[0050] Test strain: Escherichia coli wild strain BW 25113 (laboratory preservation).
[0051] 2. Preparation work before test:
[0052] (1) The Escherichia coli wild strain BW 25113 was subcultured on agar medium and cultured to an appropriate size.
[0053] (2) 51.2 mg of Vitamin B7 was weighed on a precision balance in a centrifuge tube, 10 mL of sterile water was added for dissolution, the concentration of Vitamin B7 was 5120 μg / mL, vortex mixing was performed, and filtration was performed. The filtrate was physically sterilized through a filter membrane with a diameter of 0.22 μm, and was divided into sterilized centrifuge tubes and stored in a -80°C freezer.
[0054] (3) 51.2 mg of compound amoxicillin was weighed on a precision balance in a centrifuge tube, 10 mL of sterile water was added for dissolution, the concentration of compound amoxicillin was 5120 μg / mL, vortex mixing was performed, and filtration was performed. The filtrate was physically sterilized through a filter membrane with a diameter of 0.22 μm, and was divided into sterilized centrifuge tubes and stored in a -20°C freezer.
[0055] 3. Bactericidal experiment:
[0056] (1) A single colony of Escherichia coli BW 25113 was inoculated in a centrifuge tube containing 20 mL of MH broth, and was placed in a 37°C shaking bed at 180 rpm for 3 h, and then the centrifuge tube was taken out.
[0057] (2) The bacterial solution was diluted with MH broth medium to a bacterial amount of 10 6 CFU / mL for standby.
[0058] (3) The system used in this experiment was 5 mL, and the bacterial solution step was (2):
[0059] A) Control group: 4.95 mL of MH broth + 0.05 mL of bacterial solution;
[0060] B) 16 μg / mL Vitamin B7 group: 4.934 mL of MH broth + 15.625 μL of VB7 + 0.05 mL of bacterial solution;
[0061] C) 32 μg / mL Vitamin B7 group: 4.918 mL of MH broth + 31.25 μL of VB7 + 0.05 mL of bacterial solution;
[0062] D) 64 μg / mL Vitamin B7 group: 4.887 mL of MH broth + 62.5 μL of VB7 + 0.05 mL of bacterial solution;
[0063] E) Single drug compound amoxicillin group: 4.946 mL MH broth + 3.125 μL AMC + 0.05 mL bacterial solution;
[0064] F) Combined 16 μg / mL vitamin B7 group: 4.931 mL MH broth + 3.125 μL AMC + 15.625 μL VB7 + 0.05 mL bacterial solution;
[0065] G) Combined 32 μg / mL vitamin B7 group: 4.915 mL MH broth + 3.125 μL AMC + 31.25 μL VB7 + 0.05 mL bacterial solution;
[0066] H) Combined 64 μg / mL vitamin B7 group: 4.884 mL MH broth + 3.125 μL AMC + 62.5 μL VB7 + 0.05 mL bacterial solution
[0067] (4) This experiment sets 8 groups:
[0068] Escherichia coli BW25113: ① control group; ② 16 μg / mL vitamin B7 group; ③ 32 μg / mL vitamin B7 group ④ 64 μg / mL vitamin B7 group; ⑤ single drug compound amoxicillin group; ⑥ combined 16 μg / mL vitamin B7 group; ⑦ combined 32 μg / mL vitamin B7 group; ⑧ combined 64 μg / mL vitamin B7 group.
[0069] (5) After the system is prepared, it is cultured at 37°C, 180 rpm shaking bed for 3h.
[0070] (6) At 3h, 100 μL of bacterial solution was taken from each tube and added to a 2ml centrifuge tube containing 900 μL of PBS buffer for ten-fold gradient dilution. After dilution, 25 μL was taken and dropped on LB agar medium, which was incubated in a 37°C incubator for 16-18h for colony counting. The experimental results were statistically analyzed after three biological repeats, and the growth rate was displayed.
[0071] The results are shown in Figure 1 and Table 2. Compared with the 1 / 2MIC AMC group and the VB7 single drug group, the combined group results show that the synergistic effect of compound amoxicillin and vitamin B7 has obvious effect on the sterilization of Escherichia coli BW25113.
[0072] Table 2 Bactericidal effect on Escherichia coli BW25113
[0073] Example 3 Evaluation of the combined bactericidal effect of vitamin B7 and compound amoxicillin on Escherichia coli BW25113
[0074] 1. Experimental materials:
[0075] (1) Test: Cool the LB broth, PBS buffer, and ultrapure water sterilized by high pressure for standby use.
[0076] (2) Vitamin B7: Solaybao brand, MW 244.31; Compound amoxicillin: Mokang brand.
[0077] Test strain: Escherichia coli wild strain BW 25113 (laboratory preservation).
[0078] 2. Preparation work before test:
[0079] (1) Subculture the Escherichia coli wild strain BW 25113 on agar medium and culture to appropriate size.
[0080] (2) Weigh 5.12 mg of vitamin B7 in a centrifuge tube, add 10 mL of sterile water for dissolution, the concentration of vitamin B7 is 5120 μg / mL, vortex mixing and filtering, the filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterilized centrifuge tubes and stored in a -80℃ freezer.
[0081] (3) Weigh 5.12 mg of compound amoxicillin in a centrifuge tube, add 10 mL of sterile water for dissolution, the concentration of compound amoxicillin is 5120 μg / mL, vortex mixing and filtering, the filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterilized centrifuge tubes and stored in a -20℃ freezer.
[0082] 3. Bactericidal test:
[0083] (1) Inoculate Escherichia coli BW 25113 single colony in a centrifuge tube containing 20 mL of MH broth, and place it in a 37℃ shaking bed at 180 rpm for 3h, then take out the centrifuge tube.
[0084] (2) Dilute the bacterial liquid to 10 6 CFU / mL with MH broth medium for standby use.
[0085] (3) This experiment uses a system of 5 mL, and the bacterial liquid step is (2):
[0086] A) Control group: 4.95 mL of MH broth + 0.05 mL of bacterial liquid;
[0087] B) Single drug vitamin B7 group: 4.95 mL of MH broth + 25 μL of VB7 + 0.05 mL of bacterial liquid;
[0088] C) Single drug compound amoxicillin group: 4.95 mL MH broth + 3.125 μL AMC + 0.05 mL bacterial solution;
[0089] D) Combined group: 4.95 mL MH broth + 3.125 μL AMC + 25 μL VB7 + 0.05 mL bacterial solution.
[0090] (4) This experiment sets up 4 groups:
[0091] Escherichia coli BW25113: ① control group; ② single compound amoxicillin group; ③ single vitamin B7 group ④ combined drug group
[0092] (5) After the system is prepared, it is cultured at 37°C, 180 rpm shaking bed for 3h.
[0093] (6) At 3h, 6h, 9h, 24h, 100 μL of bacterial solution was taken from each tube and added to a 2ml centrifuge tube containing 900 μL PBS buffer for ten-fold gradient dilution. After dilution, 25 μL was taken and dropped on LB agar medium, which was incubated in a 37°C incubator for 16-18h for colony counting. The experimental results were statistically analyzed after three biological repeats, and the results were shown as log values.
[0094] The results are shown in Table 3 and Figure 2 As shown in Table 3 and
[0095] Table 3 3h Vitamin B7 and compound amoxicillin combined for killing effect of Escherichia coli wild strain BW25113
[0096] Example 4 Evaluation of the combined bactericidal effect of vitamin B7 and compound amoxicillin on Escherichia coli resistant strain LY9
[0097] 1. Experimental materials:
[0098] (1) Test: cool the high-pressure sterilized LB broth, PBS buffer, ultrapure water for standby use.
[0099] (2) Vitamin B7: Solaybao brand, MW244.31; Compound amoxicillin: Mokang brand.
[0100] Test strain: Escherichia coli clinical drug-resistant strain LY9 (laboratory preservation).
[0101] 2. Preparation before the test:
[0102] (1) Subculture Escherichia coli clinical drug-resistant strain LY9 on agar medium and culture to appropriate size.
[0103] (2) Weigh 5.12 mg of vitamin B7 in a centrifuge tube, add 10 mL of sterile water for dissolution, the concentration of vitamin B7 is 5120 μg / mL, vortex mixing for filtration, the filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterile centrifuge tubes and stored in a -80°C freezer.
[0104] (3) Weigh 5.12 mg of compound amoxicillin in a centrifuge tube, add 10 mL of sterile water for dissolution, the concentration of compound amoxicillin is 5120 μg / mL, vortex mixing for filtration, the filtrate is physically sterilized through a filter membrane with a diameter of 0.22 μm, and is divided into sterile centrifuge tubes and stored in a -20°C freezer.
[0105] 3. Bactericidal experiment:
[0106] (1) Inoculate Escherichia coli clinical drug-resistant strain LY9 single colony in a centrifuge tube containing 20 mL of MH broth, and place it in a 37°C shaker at 180 rpm for 3 h, then take out the centrifuge tube.
[0107] (2) Dilute the bacterial solution with MH broth medium to a bacterial amount of 108 CFU / mL for standby.
[0108] (3) This experiment uses a system of 5 mL, and adds bacterial solution in (2):
[0109] A) Control group: 4.95 mL of MH broth + 0.05 mL of bacterial solution;
[0110] B) Single drug vitamin B7 group: 4.925 mL of MH broth + 25 μL of VB7 + 0.05 mL of bacterial solution;
[0111] C) Single drug compound amoxicillin group: 4.95 mL of MH broth + ½ MIC μL of AMC + 0.05 mL of bacterial solution;
[0112] D) Combined group: 4.95 mL of MH broth + ½ MIC μL of AMC + 25 μL of VB7 + 0.05 mL of bacterial solution.
[0113] (4) This test sets up 4 groups:
[0114] Escherichia coli BW25113: ① Control group; ② Amoxicillin alone group; ③ Vitamin B7 alone group; ④ Combined drug group
[0115] (5) After the system is prepared, it is shaken and cultured at 37℃ and 180 rpm for 3 hours.
[0116] (6) At 3h, 6h, 9h and 24h, 100 μL of bacterial culture was taken from each tube and added to a 2ml centrifuge tube containing 900μL of PBS buffer for a tenfold serial dilution. After dilution, 25μL was taken and dropped onto LB agar medium and incubated in a 37°C incubator for 16-18h. Colony counting was performed, and the experimental results were statistically analyzed after three biological replicates.
[0117] The results are shown in Table 4 and Figure 3 As shown, bacteria grew normally in the blank control group, indicating that the clinically resistant strain LY9 of Escherichia coli could grow normally under the experimental conditions. In the single-drug group, the clinically resistant strain LY9 of Escherichia coli was not significantly reduced compared with the vitamin B7-only group, indicating that the vitamin B7-only group had no significant inhibitory effect on the clinically resistant strain LY9 of Escherichia coli. Similarly, the results also showed that the use of compound amoxicillin alone had a significant inhibitory effect on the clinically resistant strain LY9 of Escherichia coli. The results of the combined group showed that the synergistic effect of compound amoxicillin and vitamin B7 had a significant bactericidal effect on the clinically resistant strain LY9 of Escherichia coli.
[0118] Table 4. Bactericidal effect of 3h vitamin B7 and compound amoxicillin combined on LY9.
[0119] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.
Claims
1. A bactericidal and bacteriostatic composition, characterized in that, It consists of compound amoxicillin and vitamin B7.
2. The bactericidal and bacteriostatic composition according to claim 1, characterized in that, The concentration of amoxicillin in the compound amoxicillin in the composition is 0.01%-0.1%, and the concentration of vitamin B7 is 16-64 μg / mL.
3. The bactericidal and bacteriostatic composition according to claim 1, characterized in that, The solvent for the composition is water.
4. The use of the bactericidal and bacteriostatic composition of claim 1 in the preparation of products that inhibit Escherichia coli and / or Salmonella.
5. The application according to claim 4, characterized in that, The Escherichia coli is Escherichia coli BW 25113 and / or drug-resistant Escherichia coli LY9.
6. A combined sterilization method, wherein compound amoxicillin and vitamin B7 are simultaneously incubated with bacterial solution at an incubation temperature of 36.8℃~37.5℃.
7. The combined sterilization method according to claim 6, characterized in that, The bacterial solution is Escherichia coli, and the bacterial concentration is 10. 8 CFU / mL.