Drug combination composition for resisting MRSA infection and application thereof
By optimizing the administration time and concentration of the combined pharmaceutical composition of berberine and erythromycin, the problem of poor therapeutic effect of MRSA infection in the existing technology is solved, and significant antibacterial effect and drug resistance reduction are achieved.
Patent Information
- Application Number
- CN202411539097.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2025-01-24
AI Technical Summary
The prior art is difficult to effectively treat MRSA infection, and traditional antibiotic use is poor and can easily lead to the intensification of drug resistance.
A combined pharmaceutical composition of berberine and erythromycin was used to determine the effective concentration range of the drug through microbiological experiments and optimize the administration time to improve the antibacterial effect and reduce drug resistance.
Significantly inhibit the growth of MRSA, delay its entry into the long -term period of life, enhance the intervention effect of MRSA biofilms, improve the treatment effect and reduce the adverse reaction of drugs.
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Figure CN119345215A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical drugs, and in particular to a combined drug composition for resisting MRSA infection and application thereof. Background Art
[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is an important clinical pathogen that has attracted much attention due to its resistance to multiple antibiotics. The treatment of MRSA infection has always been a difficult problem in clinical practice. The use of traditional antibiotics often fails to achieve ideal therapeutic effects and is prone to further aggravation of drug resistance. Therefore, it is particularly important to find new treatment strategies to deal with MRSA infection.
[0003] In recent years, drug combination has gradually gained attention as a new treatment strategy. Drug combination can improve the antibacterial effect and reduce the development of drug resistance through the synergistic effect between different drugs. Among many drugs, berberine and erythromycin have attracted much attention due to their unique antibacterial mechanism and wide antibacterial spectrum.
[0004] Berberine is an active ingredient extracted from the Chinese herbal medicine Coptis chinensis, which has a variety of pharmacological effects such as broad-spectrum antibacterial, anti-inflammatory, and antioxidant. Studies have shown that berberine can achieve antibacterial effects by inhibiting the synthesis of bacterial DNA, RNA, and protein. In addition, berberine can also enhance the body's immunity, promote the disappearance of inflammation, and has a good therapeutic effect on a variety of infectious diseases.
[0005] Erythromycin is a macrolide antibiotic that exerts its antibacterial effect mainly by inhibiting bacterial protein synthesis. Erythromycin has a certain antibacterial effect on a variety of Gram-positive and Gram-negative bacteria, and is not easy to develop drug resistance. However, with the widespread use of erythromycin, its drug resistance has gradually increased, and the effect of using erythromycin alone to treat MRSA infections is limited.
[0006] Given the unique antibacterial mechanisms and broad antibacterial spectrum of berberine and erythromycin, researchers have begun to explore the possibility of using the two together to fight MRSA infections. By using berberine and erythromycin together, the synergistic effect between the two can be exerted to improve the antibacterial effect and reduce the development of drug resistance. In addition, combined use can also reduce the dosage of a single drug and reduce adverse reactions and toxicity of the drug.
[0007] However, the effect of combined medication is affected by many factors, such as drug concentration, administration time, etc. Therefore, when berberine and erythromycin are used together to fight MRSA infection, parameters such as drug concentration and administration time need to be optimized to achieve the best therapeutic effect.
[0008] In order to solve the above problems, the applicant proposes a combined drug composition for anti-MRSA infection and its application. Summary of the invention
[0009] The purpose of the present invention is to provide a method for solving the problems in the prior art.
[0010] To achieve the above object, the present invention provides the following technical solution: a combined drug composition for anti-MRSA infection, the composition comprising berberine and erythromycin as active ingredients, wherein:
[0011] The concentration of berberine ranged from 1.95 μg / mL to 62.5 μg / mL;
[0012] Erythromycin concentrations range from 250 μg / mL to 2000 μg / mL;
[0013] The above concentrations are all fractional multiples of the minimum inhibitory concentration for MRSA determined based on microbiological experiments.
[0014] Optionally, the berberine and erythromycin are used in combination at different dosing time intervals to optimize the anti-MRSA effect, specifically including:
[0015] When berberine is administered before erythromycin, the time window for erythromycin administration is no more than 3 hours from berberine administration;
[0016] When erythromycin was administered before berberine, berberine maintained a stable anti-MRSA effect within 8 hours after erythromycin administration.
[0017] Optionally, the combined use of berberine and erythromycin can delay the time for MRSA to enter the logarithmic growth phase, as shown in the following:
[0018] When berberine was administered at 0 hours and erythromycin was administered at 4 hours, the time for MRSA to enter the logarithmic growth phase was delayed to 32 hours.
[0019] Optionally, the composition has a strong intervention effect on MRSA biofilm, specifically manifested as:
[0020] When berberine and erythromycin were used together at a concentration of 1 / 2 of their respective MICs, the intervention effect on the biofilm of the MRSA standard strain ATCC43300 was the most significant, and its OD value was similar to that of the blank control group.
[0021] Alternatively, when berberine and erythromycin were administered at intervals, their intervention effect on MRSA biofilm was weakened compared with simultaneous administration, but was still better than the single-drug group, as shown in the following:
[0022] When erythromycin was administered at 0 hour, berberine was administered at 4 hours, 6 hours, and 8 hours, and when berberine was administered at 0 hour and erythromycin was administered at 8 hours, the OD values of each group were smaller than that of the negative control group, but higher than that of the group administered with simultaneous administration.
[0023] Use of the combined pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing MRSA infection.
[0024] A method for preventing MRSA infection, comprising administering the combined pharmaceutical composition according to any one of claims 1 to 5 to a patient.
[0025] Beneficial effects: 1. Improve antibacterial effect:
[0026] The present invention determines the MIC value of berberine and erythromycin against MRSA by microbroth dilution method, and determines the effective concentration range of the combined use of the two. The experimental results show that at a specific concentration, the combined use of berberine and erythromycin can significantly inhibit the growth of MRSA, and the effect is better than using either drug alone.
[0027] When berberine and erythromycin are used together at the optimal administration time, the antibacterial effect can be further improved. Experiments show that when berberine is administered at 0 hours and erythromycin is administered within 4 hours, it can significantly delay the time for MRSA to enter the logarithmic growth phase, thereby effectively controlling MRSA infection.
[0028] 2. Enhance the ability to intervene in MRSA biofilm formation:
[0029] MRSA often forms biofilms to evade the host's immune defense and the bactericidal effect of antibiotics. The present invention investigates the intervention effect on MRSA biofilms by combining berberine with erythromycin at gradient concentrations. The experimental results show that the combined drug group has a significant intervention effect on MRSA biofilms compared with the negative control group, can inhibit biofilm formation, and enhance the bactericidal effect of the drug.
[0030] In addition, the present invention also explores the effect of different administration times on the effect of combined drug intervention on biofilm. The experiment shows that when berberine and erythromycin are administered at a specific time interval, although the intervention effect is slightly worse than that of simultaneous administration, it can still significantly inhibit the growth of MRSA and the formation of biofilm. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 It is a schematic diagram of the growth curve of the MRSA standard strain ATCC43300 of the embodiment of the present invention;
[0032] Figure 2 This is a schematic diagram comparing the OD values of the intervention effect of erythromycin and berberine combined with erythromycin on MRSA biofilm in an embodiment of the present invention;
[0033] Figure 3 The effect of erythromycin and berberine on bacterial growth (OD) at different administration time combinations in the present invention is shown in Figure 2 595nm Schematic diagram of the impact of (value measurement). DETAILED DESCRIPTION
[0034] The following describes the preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.
[0035] Example 1
[0036] As an important pathogen, MRSA has developed resistance to multiple antibiotics, which has brought severe challenges to clinical anti-infection treatment. With the widespread use of antibiotics, the drug resistance of MRSA has continued to increase, and new treatment methods are urgently needed to deal with this problem. This study aims to explore the application of berberine combined with erythromycin in anti-MRSA infection, determine the MIC value of the drug against MRSA by microbroth dilution method, study the effect of different administration times on the anti-MRSA efficacy of erythromycin and berberine combined, and the intervention effect of the combined administration of the two on the growth curve and biofilm formation of the MRSA standard strain ATCC43300, so as to provide new treatment strategies for clinical practice.
[0037] The main purpose of the present invention is to evaluate the efficacy of berberine combined with erythromycin in anti-MRSA infection, and to provide a theoretical basis and practical guidance for clinical medication by determining the MIC value, studying the effect of different administration times on the efficacy, and the intervention effect on the growth curve of MRSA standard strains and biofilms.
[0038] MIC value determination by broth microdilution method
[0039] Preparation of drug solution:
[0040] Berberine: Berberine was dissolved in DMSO (12.5%) to prepare solutions of different concentrations.
[0041] Erythromycin: Erythromycin was dissolved in methanol (12.5%) to prepare solutions of different concentrations.
[0042] Preparation of microbroth dilution plates:
[0043] Add different concentrations of drug solution to each row or column of the microbroth dilution plate.
[0044] Add an appropriate amount of MH broth to each well to ensure a consistent total volume.
[0045] Inoculation of bacteria:
[0046] The MRSA standard strain ATCC43300 was inoculated into a micro-broth dilution plate to make the bacterial concentration in each well uniform.
[0047] Cultivation and observation:
[0048] Place the microbroth dilution plate in an incubator at appropriate temperature and humidity.
[0049] Observe the growth of bacteria and record the growth of bacteria in each well to determine the MIC value. The effect of different administration times on the efficacy of erythromycin combined with berberine against MRSA Experimental design:
[0050] Different combinations of administration times were set, such as berberine was administered at 0 h and erythromycin was administered at different times (0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h); erythromycin was administered at 0 h and berberine was administered at different times (0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h).
[0051] Different concentrations of erythromycin and berberine were used for co-administration at each dosing time combination.
[0052] Bacterial culture and observation:
[0053] The drugs were added to the culture medium according to the set dosing time combination.
[0054] The MRSA standard strain ATCC43300 was inoculated into the culture medium.
[0055] Observe the bacterial growth and record the bacterial growth in each well.
[0056] Data Analysis:
[0057] Based on the observation results, the effects of different administration time combinations on the anti-MRSA efficacy of erythromycin and berberine were analyzed.
[0058] The bacterial growth under different concentration combinations was compared to determine the optimal administration time and concentration combination.
[0059] Effect of combined administration of berberine and erythromycin on the growth curve of MRSA standard strain ATCC43300
[0060] Experimental design:
[0061] Set different administration time combinations, such as berberine at 0h and erythromycin at 4h, 6h, and 8h.
[0062] Optimal concentrations of erythromycin and berberine were used for co-administration at each dosing time combination.
[0063] Bacterial culture and observation:
[0064] The drugs were added to the culture medium according to the set dosing time combination.
[0065] The MRSA standard strain ATCC43300 was inoculated into the culture medium.
[0066] Measure the bacterial concentration at regular intervals (such as every 2 hours) and record the growth curve.
[0067] Data Analysis:
[0068] According to the growth curve, the effects of different administration time combinations on the growth of MRSA standard strain ATCC43300 were analyzed.
[0069] Compare the growth curves under different drug administration time combinations to determine the optimal drug administration time combination.
[0070] Intervention study of erythromycin and berberine on MRSA biofilm
[0071] A biofilm of MRSA standard strain ATCC43300 was prepared.
[0072] Different drug concentration combinations were set, such as gradient concentrations of erythromycin (1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, 1 / 32MIC) and gradient concentrations of berberine (1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, 1 / 32MIC) for combined medication.
[0073] Set different dosing time combinations, such as simultaneous administration and interval administration (erythromycin is administered at 0 hours, berberine is administered at 4 hours, 6 hours, and 8 hours; berberine is administered at 0 hours, erythromycin is administered at 8 hours, etc.).
[0074] Biofilm intervention experiment
[0075] MRSA was added to culture medium containing different combinations of drug concentrations and administration times.
[0076] Cultivate at an appropriate temperature for a period of time to allow the drug to fully act on the bacteria.
[0077] Remove the bacterial solution, wash with 200μL pure water 3 times, add 200μL methanol to each well for 20 minutes to fix the biofilm. Remove the methanol, add 100μL 1% crystal violet solution to each well, stain for 5 minutes, wash the excess dye with pure water, add 200μL 33% acetic acid solution to each well, and use a microplate reader to measure the OD value at 595nm to evaluate the intervention effect of the drug on the biofilm.
[0078] Data analysis
[0079] According to the measured OD values, the intervention effects of different drug concentration combinations and administration time combinations on MRSA biofilm were analyzed.
[0080] The OD values of each group were compared with those of the negative control group and the blank control group to determine the optimal drug concentration combination and administration time combination.
[0081] MIC value determination by broth microdilution method
[0082] The MIC values of berberine and erythromycin against the MRSA standard strain ATCC43300 were 62.5 μg / mL and 2000 μg / mL, respectively, as determined by the broth microdilution method.
[0083] Effect of different administration time on the efficacy of erythromycin combined with berberine against MRSA
[0084] The experimental results showed that when berberine was administered at 0h and erythromycin was administered at different times, the effect of the combination of the two on inhibiting the MRSA standard strain ATCC43300 gradually weakened as the erythromycin administration time was delayed. When erythromycin was administered at 0h and berberine was administered at different times, the effect of the combination of the two on inhibiting the MRSA standard strain ATCC43300 was relatively stable within the investigated administration time. This shows that the administration time of erythromycin has a greater impact on the effect of combined medication, while the administration time of berberine has a relatively small impact on the effect.
[0085] Effect of combined administration of berberine and erythromycin on the growth curve of MRSA standard strain ATCC43300
[0086] The experimental results showed that the combined administration of berberine and erythromycin can significantly delay the time for the MRSA standard strain ATCC43300 to enter the logarithmic growth phase. For example, when berberine is administered at 0h and erythromycin is administered at 4h, the time to enter the logarithmic growth phase is delayed to 32h; when berberine is administered at 0h and erythromycin is administered at 6h, the time to enter the logarithmic growth phase is delayed to 24h; when berberine is administered at 0h and erythromycin is administered at 8h, the time to enter the logarithmic growth phase is delayed to 12h later. This shows that combined medication can significantly inhibit the growth rate of MRSA.
[0087] Intervention of erythromycin and berberine on MRSA biofilm
[0088] The experimental results showed that the combination of gradient concentrations of erythromycin and gradient concentrations of berberine had a strong intervention effect on the biofilm of the MRSA standard strain ATCC43300. Compared with the negative control group, the OD values of the combined drugs in each group were smaller, and the OD value of the combination group of erythromycin with a concentration of 1 / 2MIC and berberine with a concentration of 1 / 2MIC was similar to the OD value of the blank control group. In addition, the intervention effects of different combinations of dosing time on biofilms were also different. The OD value of the simultaneous dosing group was the smallest, and the intervention effect on biofilms was the most significant; while the OD value of the interval dosing group was relatively high, but still lower than the OD value of the negative control group, and still had a certain intervention effect on biofilms. Experimental operation to investigate the effect of dosing time on drug efficacy
[0089] Take the MRSA bacterial solution in the logarithmic growth phase and add TSB medium to dilute the bacterial solution to 1x10 6 cfu / ml, add the diluted bacterial solution to a 24-well plate and add the drug solution, add 500μL of bacterial solution and 250μL of drug solution to each well, and set up 3 parallel groups, and set up a Chinese medicine monomer control group, a blank culture medium control group and a negative control group. Place in a constant temperature incubator for culture, add 250μL of another drug solution at different observation times, culture for 18h, and the microplate reader is at OD 595nm The OD value was measured at 4 °C to investigate the effect of different administration times of berberine combined with erythromycin on the drug
[0090] Berberine was administered at 0h, and erythromycin was administered at different times
[0091] Erythromycin at a concentration of 1 / 2MIC (1000 μg / mL) was combined with berberine at gradient concentrations (1 / 2MIC (31.25 μg / mL), 1 / 4MIC (15.63 μg / mL), 1 / 8MIC (7.81 μg / mL), 1 / 16MIC (3.91 μg / mL), 1 / 32MIC (1.95 μg / mL), 1 / 64MIC (0.98 μg / mL)). The combination of the two inhibited the erythromycin inhibitory effect as the interval between erythromycin administration increased (0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h). The effect on MRSA standard strain ATCC43300 was weakened. When erythromycin at a concentration of 1 / 4 MIC (500 μg / mL) was used in combination with 1 / 2 berberine concentration (31.25 μg / mL), the inhibitory effect of the two on MRSA standard strain ATCC43300 weakened with the increase of the interval between erythromycin administration (0h, 1h, 2h, 3h, 4h, 6h, 8h). When the erythromycin concentration was fixed and the berberine concentration was reduced or the berberine concentration was fixed and the erythromycin concentration was reduced, the inhibitory effect of the two on MRSA standard strain ATCC43300 was weakened.
[0092] Table 4
[0093]
[0094] Note: “-” indicates bacterial growth inhibition and clear medium; “+” indicates bacterial growth and turbid medium
[0095] Erythromycin was administered at 0h, and berberine was administered at different times
[0096] Erythromycin at a concentration of 1 / 2MIC (1000 μg / mL) was combined with berberine at gradient concentrations (1 / 2MIC (31.25 μg / mL), 1 / 4MIC (15.63 μg / mL), 1 / 8MIC (7.81 μg / mL), 1 / 16MIC (3.91 μg / mL), 1 / 32MIC (1.95 μg / mL), 1 / 64MIC (0.98 μg / mL)). The berberine administration intervals investigated (0h, 1h, 2h, 3h, 4h, 6h, 8h) The combined use of both had stable effects on inhibiting the MRSA standard strain ATCC43300. When erythromycin at a concentration of 1 / 4 MIC (500 μg / mL) was used in combination with berberine at a concentration of 1 / 2 (31.25 μg / mL), the inhibitory effect of the two on the MRSA standard strain ATCC43300 weakened with the increase of the interval between berberine administration (0h, 1h, 2h, 3h, 4h, 6h, 8h). When the concentration of berberine or berberine was fixed and the concentration of erythromycin was reduced, the inhibitory effect of the two on the MRSA standard strain ATCC43300 weakened.
[0097] Table 5
[0098]
[0099]
[0100] Note: “-” indicates bacterial growth inhibition and clear medium; “+” indicates bacterial growth and turbid medium
[0101] The present invention determines the MIC values of berberine and erythromycin against the MRSA standard strain ATCC43300 by the microbroth dilution method, and studies the effects of different administration times on the anti-MRSA efficacy of combined medication and the intervention effect of combined medication on the MRSA growth curve and biofilm. The experimental results show that the combined use of berberine and erythromycin can significantly inhibit the growth rate of MRSA and delay its entry into the logarithmic growth phase; at the same time, the combination of the two also has a strong intervention effect on the formation of MRSA biofilm. These findings provide a new treatment strategy for the clinical treatment of MRSA infection.
[0102] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.
[0103] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A combined drug composition for use against MRSA infection, characterized in that: The composition comprises berberine and erythromycin as active ingredients, wherein: The concentration of berberine ranged from 1.95 μg / mL to 62.5 μg / mL; Erythromycin concentrations range from 250 μg / mL to 2000 μg / mL; The above concentrations are all multiples of the minimum inhibitory concentration for MRSA determined based on microbiological experiments.
2. The combined pharmaceutical composition according to claim 1, characterized in that: The berberine and erythromycin are used in combination at different dosing time intervals to optimize the anti-MRSA effect, specifically including: When berberine is administered before erythromycin, the time window for erythromycin administration is no more than 3 hours from berberine administration; When erythromycin was administered before berberine, berberine maintained a stable anti-MRSA effect within 8 hours after erythromycin administration.
3. The combined pharmaceutical composition according to claim 2, characterized in that: The combined use of berberine and erythromycin can delay the time for MRSA to enter the logarithmic growth phase, which is specifically manifested as follows: When berberine was administered at 0 hours and erythromycin was administered at 4 hours, the time for MRSA to enter the logarithmic growth phase was delayed to 32 hours.
4. The combined pharmaceutical composition according to claim 1, characterized in that: The composition has a strong intervention effect on MRSA biofilm, which is specifically manifested as follows: When berberine and erythromycin were used together at a concentration of 1 / 2 of their respective MICs, the intervention effect on the biofilm of the MRSA standard strain ATCC43300 was the most significant, and its OD value was similar to that of the blank control group.
5. The combined pharmaceutical composition according to claim 4, characterized in that: When berberine and erythromycin were administered at intervals, their intervention effect on MRSA biofilm was weakened compared with simultaneous administration, but still better than the single-drug group, as shown in the following: When erythromycin was administered at 0 hour, berberine was administered at 4 hours, 6 hours, and 8 hours, and when berberine was administered at 0 hour and erythromycin was administered at 8 hours, the OD values of each group were smaller than that of the negative control group, but higher than that of the group administered with simultaneous administration.
6. Use of the combined pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing MRSA infection.
7. A method for preventing MRSA infection, characterized in that: The method comprises administering to a patient the combination composition of any one of claims 1 to 5.