Method for evaluating minimum inhibitory concentration of compound amoxicillin powder
By simulating the gastric acid and small intestinal environment, the minimum inhibitory concentration of compound amoxicillin powder was measured, which solved the problem of inaccurate evaluation results in the existing technology and achieved rapid and accurate product efficacy evaluation.
Patent Information
- Application Number
- CN202511993236.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-04-17
AI Technical Summary
Existing technologies make it difficult to quickly and accurately assess the minimum inhibitory concentration (MIC) of compound amoxicillin powder from different manufacturers, leading to discrepancies between assessment results and actual effects.
By simulating the gastric acid and small intestinal environment, the mixture was kept at 37-42℃ for 30-60 minutes, and the pH was adjusted using 0.01mol/L hydrochloric acid and 1mol/L sodium bicarbonate solution to simulate the changes of compound amoxicillin powder in the digestive tract and measure the minimum inhibitory concentration.
It enables rapid and accurate assessment of the minimum inhibitory concentration of compound amoxicillin powder, effectively distinguishing the effects of products from different manufacturers, conforming to actual application environments, and reducing costs and workload.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of compound amoxicillin technology, and more specifically to a method for evaluating the minimum inhibitory concentration of compound amoxicillin powder. Background Technology
[0002] With the vigorous development of my country's animal husbandry industry, the demand for veterinary drugs has shown a steady growth trend. More and more compound amoxicillin powders from different brands are entering the market. The ingredients of compound amoxicillin drugs from different manufacturers are quite similar, and some even use raw materials from the same supplier, leaving many users unsure how to choose. Currently, in order to more effectively evaluate compound amoxicillin drugs from different manufacturers, establishing a rapid and effective method for evaluating the efficacy of compound amoxicillin powder is key to solving this problem.
[0003] Currently, methods for evaluating this drug include drug sensitivity testing, active ingredient content determination, water solubility, blood drug concentration detection, and comparison of clinical application effects. While blood drug concentration detection and comparison of clinical application effects are instructive for clinical drug use, both involve extensive animal testing, are labor-intensive, have long testing cycles, and are very costly, making them unavailable to many institutions. On the other hand, methods such as drug sensitivity testing, active ingredient content determination, and water solubility determination are relatively simpler, easier to perform, have shorter time cycles, and lower costs, but the test results often differ from real-world clinical outcomes due to variations in the testing environment, leading to discrepancies in evaluation results.
[0004] Antimicrobial susceptibility testing includes methods such as the disk assay and minimum inhibitory concentration (MIC). The MIC is the minimum concentration of a drug required to inhibit the growth of pathogenic microorganisms. By testing the MIC of a drug against standard candidate strains or a specific strain, the sensitivity of these strains to the drug can be assessed. It is primarily used to evaluate bacterial sensitivity to drugs and the antimicrobial activity of antimicrobial agents, and is an important evaluation indicator. Therefore, how to quickly and accurately assess the MIC of compound amoxicillin powder from different manufacturers has become a problem that troubles many users. Summary of the Invention
[0005] In view of this, the present invention provides a method for evaluating the minimum inhibitory concentration of compound amoxicillin powder, which is simple to operate and can quickly and accurately evaluate the effect of compound amoxicillin powder.
[0006] This invention provides a method for evaluating the minimum inhibitory concentration (MIC) of compound amoxicillin powder, comprising the following steps: (1) To simulate the gastric acid environment, different compound amoxicillin powder samples were added to obtain different sample solutions; (2) Simulate the small intestinal environment using different sample solutions after step (1); (3) Take samples of different sample solutions after step (2), measure the minimum inhibitory concentration, and evaluate the effect of compound amoxicillin powder samples.
[0007] Optionally, the pH value of the gastric acid environment is 1 to 4.
[0008] Optionally, the gastric acid environment is simulated using 0.01 mol / L hydrochloric acid.
[0009] Optionally, the gastric acid environment in step (1) may also include a temperature of 37~42℃ and a gastric acid environment time of 30~60min.
[0010] Optionally, the pH value of the small intestinal environment is 6-8.
[0011] Optionally, the small intestinal environment is simulated using a 1 mol / L sodium bicarbonate solution.
[0012] Optionally, the small intestinal environment in step (2) also includes a temperature of 37~42℃ and a time of 30~60min.
[0013] Optionally, in step (2), water bath heating is used to maintain the temperature of the small intestine environment.
[0014] The above-described technical solution of the present invention has at least the following beneficial effects: This method simulates the changes of drugs passing through the digestive tract of livestock and poultry, successively simulating the pH value and temperature conditions of the gastric acid environment and the small intestinal environment. It simulates the influence of the digestive tract environment on compound amoxicillin during actual application, and evaluates the effect by detecting the minimum inhibitory concentration of the treated compound amoxicillin powder. The method is simple to operate, low in cost, and has strong resolution, and can effectively and accurately distinguish and evaluate the effects of compound amoxicillin powder products from different manufacturers. Detailed Implementation
[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.
[0016] Seven different brands of compound amoxicillin powder were randomly selected from the market as samples, numbered 1, 2, 3, 4, 5, 6, and 7. Examples 1-7 tested the minimum inhibitory concentration (MIC) of the samples against *Haemophilus parasuis* using the technical solutions provided by this invention, while comparative examples 1-7 tested the MIC of the samples against *Haemophilus parasuis* using existing technical solutions. Examples 8-14 tested the MIC of the samples against *Streptococcus* using the technical solutions provided by this invention, while comparative examples 8-14 tested the MIC of the samples against *Streptococcus* using existing technical solutions.
[0017] Example 1 Weigh 2.0g of sample 1 into a 100ml volumetric flask, dilute to the mark with 0.01mol / L hydrochloric acid solution, incubate at 37℃ for 60min, then add 1mol / L sodium bicarbonate solution to adjust the pH to 6.0, mix well, continue incubating at 37℃ for 60min, and then take a sample to obtain the test sample solution.
[0018] Take a sterile 96-well plate and add 100 μL of sterile nutrient broth to each well. Add 100 μL of the test sample solution to the first well, and serially dilute at a rate of 0.5 times (100 μL to 100 μL). Continue diluting until the 10th well is reached, mix well, and then discard 100 μL. Then add bacterial suspension (containing Haemophilus parasuis at a concentration of 1.5 x 10⁻⁶) to wells 1-10. 8 100 μL of bacterial culture was added to well 1-10. The amoxicillin concentrations in these wells were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 μg / ml, respectively. 100 μL of bacterial culture was added to well 11 as a positive control, and 100 μL of broth was added to well 12 as a negative control.
[0019] The above-mentioned well plates were placed in a constant temperature incubator at 37℃ and incubated for 16 hours. The concentration at which no bacteria grew in the last well was the minimum inhibitory concentration.
[0020] Example 2 Weigh 2.0g of sample No. 2 into a 100ml volumetric flask, dilute to the mark with 0.01mol / L hydrochloric acid solution, incubate at 37℃ for 60min, then add 1.0ml of 1mol / L sodium bicarbonate solution to adjust the pH to 6.0, mix well, and continue incubating at 37℃ for 60min before taking a sample to obtain the test sample solution.
[0021] Take a sterile 96-well plate and add 100 μL of sterile nutrient broth to each well. Add 100 μL of the test sample solution to the first well, and serially dilute at a rate of 0.5 times (100 μL to 100 μL). Continue diluting until the 10th well is reached, mix well, and then discard 100 μL. Then add bacterial suspension (containing Haemophilus parasuis at a concentration of 1.5 x 10⁻⁶) to wells 1-10. 8100 μL of bacterial culture was added to well 1-10. The amoxicillin concentrations in these wells were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 μg / ml, respectively. 100 μL of bacterial culture was added to well 11 as a positive control, and 100 μL of broth was added to well 12 as a negative control.
[0022] The above-mentioned well plates were placed in a constant temperature incubator at 37℃ and incubated for 16 hours. The concentration at which no bacteria grew in the last well was the minimum inhibitory concentration.
[0023] Example 3 Weigh 2.0g of sample No. 3 into a 100ml volumetric flask, dilute to the mark with 0.01mol / L hydrochloric acid solution, incubate at 37℃ for 60min, then add 1.0ml of 1mol / L sodium bicarbonate solution to adjust the pH to 6.0, mix well, and continue incubating at 37℃ for 60min before taking a sample to obtain the test sample solution.
[0024] Take a sterile 96-well plate and add 100 μL of sterile nutrient broth to each well. Add 100 μL of the test sample solution to the first well, and serially dilute at a rate of 0.5 times (100 μL to 100 μL). Continue diluting until the 10th well is reached, mix well, and then discard 100 μL. Then add bacterial suspension (containing Haemophilus parasuis at a concentration of 1.5 x 10⁻⁶) to wells 1-10. 8 100 μL of bacterial culture was added to well 1-10. The amoxicillin concentrations in these wells were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 μg / ml, respectively. 100 μL of bacterial culture was added to well 11 as a positive control, and 100 μL of broth was added to well 12 as a negative control.
[0025] The above-mentioned well plates were placed in a constant temperature incubator at 37℃ and incubated for 16 hours. The concentration at which no bacteria grew in the last well was the minimum inhibitory concentration.
[0026] Example 4 Weigh 2.0g of sample No. 4 into a 100ml volumetric flask, dilute to the mark with 0.01mol / L hydrochloric acid solution, incubate at 37℃ for 60min, then add 1.0ml of 1mol / L sodium bicarbonate solution to adjust the pH to 6.0, mix well, and continue incubating at 37℃ for 60min before taking a sample to obtain the test sample solution.
[0027] Take a sterile 96-well plate and add 100 μL of sterile nutrient broth to each well. Add 100 μL of the test sample solution to the first well, and serially dilute at a rate of 0.5 times (100 μL to 100 μL). Continue diluting until the 10th well is reached, mix well, and then discard 100 μL. Then add bacterial suspension (containing Haemophilus parasuis at a concentration of 1.5 x 10⁻⁶) to wells 1-10. 8100 μL of bacterial culture was added to well 1-10. The amoxicillin concentrations in these wells were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 μg / ml, respectively. 100 μL of bacterial culture was added to well 11 as a positive control, and 100 μL of broth was added to well 12 as a negative control.
[0028] The above-mentioned well plates were placed in a constant temperature incubator at 37℃ and incubated for 16 hours. The concentration at which no bacteria grew in the last well was the minimum inhibitory concentration.
[0029] Example 5 The only difference from Example 1 is that Sample 5 is used instead of Sample 1; all other steps and operations are the same.
[0030] Example 6 The only difference from Example 1 is that Sample 6 is used instead of Sample 1; all other steps and operations are the same.
[0031] Example 7 The only difference from Example 1 is that Sample 7 is used instead of Sample 1; all other steps and operations are the same.
[0032] Examples 8-14 correspond to Examples 1-7, respectively. The only difference is that the strains in Examples 8-14 are streptococci instead of Haemophilus parasuis in Examples 1-7. All other steps and operations are the same.
[0033] Comparative Example 1 Weigh 2.0g of sample 1 into a 100ml volumetric flask, dilute to the mark with purified water, mix well and set aside to obtain the sample solution.
[0034] Take a sterile 96-well plate and add 100 μL of sterile broth to each well. Add 100 μL of sample solution to the first well, and serially dilute 0.5 times (100 μL to 100 μL) until the 10th well is reached. Mix well and discard 100 μL. Then add bacterial suspension (1.5 x 10⁻⁶) to wells 1-10. 8 100 μL of bacterial culture was added to well 1-10. The amoxicillin concentrations in these wells were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 μg / ml, respectively. 100 μL of bacterial culture was added to well 11 as a positive control, and 100 μL of broth was added to well 12 as a negative control.
[0035] The above-mentioned well plates were placed in a constant temperature incubator at 37℃ and incubated for 16 hours. The concentration at which no bacteria grew in the last well was the minimum inhibitory concentration.
[0036] Comparative Example 2 Weigh 2.0g of sample No. 2 into a 100ml volumetric flask, dilute to the mark with purified water, mix well and set aside to obtain the sample solution.
[0037] Take a sterile 96-well plate and add 100 μL of sterile broth to each well. Add 100 μL of sample solution to the first well, and serially dilute 0.5 times (100 μL to 100 μL) until the 10th well is reached. Mix well and discard 100 μL. Then add bacterial suspension (1.5 x 10⁻⁶) to wells 1-10. 8 100 μL of bacterial culture was added to well 1-10. The drug concentrations in wells 1-10 were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 μg / ml (calculated as amoxicillin), respectively. 100 μL of bacterial culture was added to well 11 as a positive control, and 100 μL of broth was added to well 12 as a negative control.
[0038] The above-mentioned well plates were placed in a constant temperature incubator at 37℃ and incubated for 16 hours. The concentration at which no bacteria grew in the last well was the minimum inhibitory concentration.
[0039] Example 3 Weigh 2.0g of sample No. 3 into a 100ml volumetric flask, dilute to the mark with purified water, mix well and set aside to obtain the sample solution.
[0040] Take a sterile 96-well plate and add 100 μL of sterile broth to each well. Add 100 μL of sample solution to the first well, and serially dilute 0.5 times (100 μL to 100 μL) until the 10th well is reached. Mix well and discard 100 μL. Then add bacterial suspension (1.5 x 10⁻⁶) to wells 1-10. 8 100 μL of bacterial culture was added to well 1-10. The drug concentrations in wells 1-10 were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 μg / ml (calculated as amoxicillin), respectively. 100 μL of bacterial culture was added to well 11 as a positive control, and 100 μL of broth was added to well 12 as a negative control.
[0041] The above-mentioned well plates were placed in a constant temperature incubator at 37℃ and incubated for 16 hours. The concentration at which no bacteria grew in the last well was the minimum inhibitory concentration.
[0042] Comparative Example 4 Compared with Comparative Example 1, the only difference is that Sample 4 is used instead of Sample 1; all other steps and operations are the same.
[0043] Comparative Example 5 Compared with Comparative Example 1, the only difference is that Sample 5 is used instead of Sample 1; all other steps and operations are the same.
[0044] Comparative Example 6 Compared with Comparative Example 1, the only difference is that Sample 6 is used instead of Sample 1; all other steps and operations are the same.
[0045] Comparative Example 7 Compared with Comparative Example 1, the only difference is that Sample 7 is used instead of Sample 1; all other steps and operations are the same.
[0046] Comparative Examples 8-14 correspond to Comparative Examples 1-7, respectively. The only difference is that the strains in Comparative Examples 8-14 are streptococci instead of Haemophilus parasuis in Comparative Examples 1-7. All other steps and operations are the same.
[0047] Table 1 is obtained by statistically analyzing the test data of Examples 1 to 7, Table 2 is obtained by analyzing the test data of Comparative Examples 1 to 7, Table 3 is obtained by analyzing the test data of Examples 8 to 14, and Table 4 is obtained by analyzing the test data of Comparative Examples 8 to 14.
[0048] Table 1. Results of minimum inhibitory concentrations (MICs) against Haemophilus parasuis in Examples 1-7
[0049] Table 2. Results of minimum inhibitory concentrations against Haemophilus parasuis in Comparative Examples 1–7
[0050] As shown in Table 1, the MIC value of sample 1 was 15.63 μg / mL, samples 2, 3, 6, and 7 were 31.25 μg / mL, sample 5 was 62.5 μg / mL, and sample 4 was 125 μg / mL. This experimental result effectively and accurately distinguishes the antibacterial effects of the samples, indicating that sample 1 had the best antibacterial effect and a significant advantage.
[0051] As shown in Table 2, except for sample 4 with a MIC value of 31.25 μg / mL, the MIC values of the other samples were all 15.63 μg / mL, indicating that the test results had poor discriminatory effect on the antibacterial effect of the samples.
[0052] A comparison of Table 1 and Table 2 shows that the evaluation method provided by this invention is more effective.
[0053] Table 3 shows the minimum inhibitory concentrations (MICs) against Streptococcus in samples from Examples 8-14.
[0054] Table 4. Results of minimum inhibitory concentrations against Streptococcus in Comparative Examples 8–14
[0055] Table 3 shows that the MIC values for samples 1 and 7 were 15.63 μg / mL, for samples 2, 3, and 6 they were 31.25 μg / mL, for sample 5 it was 62.5 μg / mL, and for sample 4 it was 125 μg / mL. The experimental results effectively and accurately distinguish the antibacterial effects of the samples, indicating that samples 1 and 7 had the best antibacterial effects and showed a significant advantage.
[0056] As shown in Table 4, the MIC values of samples 4 and 6 were 31.25 μg / mL, while the MIC values of the remaining samples were all 15.63 μg / mL. This indicates that the test results had poor discriminatory power in terms of the antibacterial effect of the samples.
[0057] In summary, traditional MIC tests are not ideal and cannot clearly distinguish the advantages of different samples. The evaluation method provided by this invention can effectively distinguish the advantages of different samples and conforms to the actual application environment of the samples, resulting in more accurate and effective results.
[0058] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for evaluating the minimum inhibitory concentration (MIC) of compound amoxicillin powder, characterized in that, Includes the following steps: (1) To simulate the gastric acid environment, different compound amoxicillin powder samples were added to obtain different sample solutions; (2) Simulate the small intestinal environment using different sample solutions after step (1); (3) Take samples of different sample solutions after step (2), measure the minimum inhibitory concentration, and evaluate the effect of compound amoxicillin powder samples.
2. The method for evaluating the minimum inhibitory concentration of compound amoxicillin powder according to claim 1, characterized in that, The pH value of the gastric acid environment is 1~4.
3. The method for evaluating the minimum inhibitory concentration of compound amoxicillin powder according to claim 1, characterized in that, The gastric acid environment was simulated using 0.01 mol / L hydrochloric acid.
4. The method for evaluating the minimum inhibitory concentration of compound amoxicillin powder according to claim 1, characterized in that, In step (1), the gastric acid environment also includes a temperature of 37~42℃ and a time of 30~60min.
5. The method for evaluating the minimum inhibitory concentration of compound amoxicillin powder according to claim 1, characterized in that, The pH value of the small intestinal environment is 6-8.
6. The method for evaluating the minimum inhibitory concentration of compound amoxicillin powder according to claim 1, characterized in that, The small intestinal environment was simulated using a 1 mol / L sodium bicarbonate solution.
7. The method for evaluating the minimum inhibitory concentration of compound amoxicillin powder according to claim 1, characterized in that, In step (2), the small intestinal environment also includes a temperature of 37~42℃ and a time of 30~60min.
8. The method for evaluating the minimum inhibitory concentration of compound amoxicillin powder according to claim 7, characterized in that, In step (2), water bath heating is used to maintain the temperature of the small intestine environment.