CN114129635B - Antibacterial composition containing capsicum extract and its application - Google Patents

Abstract

Antibacterial composition containing capsicum extract and application thereof. The present invention discloses an antibacterial composition comprising meropenem and capsicum extract. When the pepper extract and meropenem are used in combination, there is a synergistic effect, and the bacteriostatic effect on drug-resistant bacteria can be significantly improved, and the effect is better than that of each alone. The invention provides a direction for studying the reversal of drug-resistant bacteria by the combination of traditional Chinese and western medicines.

Description

Antibacterial composition containing capsicum extract and its application

technical field

The invention relates to the technical field of biomedicine, in particular to an antibacterial composition comprising meropenem and capsicum extract and applications thereof.

Background technique

Meropenem (meropenem) is a new type of synthetic carbapenem antibiotics. Carbapenem antibiotics have a broad antibacterial spectrum and antibacterial activity, and are effective drugs for the clinical treatment of severe mixed infections and drug-resistant bacterial infections, and have a high therapeutic effect in clinical applications. Among them, meropenem has a good inhibitory effect on Escherichia coli and Pseudomonas aeruginosa by inhibiting the synthesis of bacterial cell walls. However, with the frequent and extensive use of meropenem in clinical practice, some irrational use has gradually occurred, which has led to the emergence of meropenem-resistant bacteria to a certain extent, and Pseudomonas aeruginosa is one of them. There is a positive correlation between the resistance of Pseudomonas aeruginosa to meropenem and the amount of carbapenem antibiotics used. The resistance rate of carbapenem-resistant strains to other antimicrobial agents is generally higher than that of carbapenem-sensitive strains, which makes the clinical benefit of meropenem gradually decrease. In order to solve this problem, it is necessary not only to standardize and rationalize drug use, but also to study how to inhibit drug-resistant bacteria. Studies have reported potential drugs for the treatment of carbapenem-resistant bacterial infections, pointing out that the main therapeutic drugs are polymyxin, tigecycline, fosfomycin and aminoglycosides. However, chemical antibacterial drugs are prone to drug resistance, and the research cycle is long, which may lead to the emergence of multi-drug resistant bacteria ("super bacteria").

As a class of natural medicine, traditional Chinese medicine not only has the advantages of low toxicity and side effects, but also low drug resistance. Studies have pointed out that traditional Chinese medicine can directly inhibit or kill drug-resistant bacteria by interfering with the biochemical metabolism of drug-resistant bacteria, or improve the drug resistance of bacteria to indirectly inhibit and kill bacteria, and maintain the balance of the body to regulate and treat drug-resistant bacterial infections as a whole. However, different Chinese medicines or Chinese medicine extracts have different inhibitory effects on bacteria or drug-resistant bacteria, and there are certain limitations.

For the experimental methods of in vitro, in vivo, and combined antibacterial activities of natural medicines, the applicable methods of different types of natural medicines are different. Traditional Chinese medicine or natural medicine, so as to play a bacteriostatic or bactericidal effect on bacteria or drug-resistant bacteria.

Contents of the invention

The object of the present invention is to provide an antibacterial composition, which can inhibit or sterilize drug-resistant bacteria.

In a first aspect, the present invention provides an antibacterial composition comprising meropenem and capsicum extract.

In some embodiments, the capsicum extract is an alcoholic extract of capsicum; preferably, the capsicum extract is a 60%-80% alcoholic extract of capsicum, more preferably a 70% ethanol extract of capsicum.

Herein, the 60%-80% alcohol extract of capsicum refers to the product obtained after the capsicum is extracted with 60%-80% alcohol (such as ethanol) and dried.

In some embodiments, in the antibacterial composition, the weight ratio of meropenem to the pepper extract is (1-8):(1000-4000).

Preferably, the weight ratio of meropenem to the pepper extract is (1-4):(1000-4000), more preferably (1-2):(1000-4000), more preferably the weight ratio 1:1000, 1:2000, 1:4000, 1:8000, 2:1000, 4:1000, 8:1000.

In a second aspect, the present invention provides the use of the antibacterial composition in the preparation of antibacterial drugs.

In some embodiments, the bacteria are meropenem-resistant bacteria.

In some embodiments, the drug-resistant bacteria include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Acinetobacter baumannii; preferably Preferably, the drug-resistant bacteria is Escherichia coli.

Beneficial effects of the present invention

In order to eliminate bacterial drug resistance, the present invention starts with extracts of traditional Chinese medicines, and screens extracts of traditional Chinese medicines with strong antibacterial effect on meropenem-resistant bacteria in vitro by preparing growth curves and broth microdilution checkerboard tests. As a result, it was surprisingly found that when the pepper extract was used in combination with meropenem, it had a significant inhibitory effect on meropenem-resistant bacteria in vitro, and the effect was better than that of each drug alone, so the combination of meropenem and the pepper extract There is an obvious synergistic effect. The relative amount of each substance in the composition is greatly reduced, thereby reducing possible adverse reactions and side effects, improving the safety of medication, and at the same time increasing the compliance of patients taking medication, thereby ensuring curative effect.

The combined use of the capsicum extract of the present invention and the meropenem antibiotic has the advantages of synergism and attenuation. Capsicum extract combined with meropenem antibiotics can inhibit meropenem-resistant bacteria. The present invention provides a new research idea and research and development direction for solving the increasingly serious drug resistance problem of meropenem.

The present invention uses a micro-broth dilution method to study the in vitro inhibitory effect of capsicum extract combined with meropenem on meropenem-resistant bacteria, which is of great research significance and is expected to provide reference for the development of new adjuvant drugs for meropenem in the treatment of severe infectious diseases and improve its therapeutic benefits.

Description of drawings

Figure 1 is the growth curve of pepper extract combined with meropenem.

Figure 2 is the growth curve of mulberry leaf extract combined with meropenem.

Figure 3 is the growth curve of Eucommia ulmoides leaf extract combined with meropenem.

Figure 4 is the growth curve of baicalin combined with meropenem.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

1. Bacteria and antibiotics

Meropenem-resistant Escherichia coli strain 20a02 (isolated and preserved from chicken farms by our laboratory).

Quality control strain Escherichia coli (ATCC25922) (purchased from China Veterinary Drug Control Institute).

Meropenem, powder, pure.

2. Chinese herbal extracts

Preparation of capsicum extract:

Take pepper, add 70% ethanol to extract twice, the first time is 2 hours, the second time is 1 hour, the extract is filtered, the filtrates are combined, concentrated and dried to obtain the product.

3. Preparation of drug-resistant bacteria and drugs

3.1 Recovery of resistant bacteria

The day before the experiment, the drug-resistant strains were inoculated on the agar medium, and cultured in a constant temperature and humidity incubator at 37°C for 24 hours.

3.2 Preparation of meropenem liquid medicine

Accurately weigh a certain amount of meropenem powder with an electronic analytical balance, and add pure water to dissolve it. The prepared concentration is 2560 μg/mL. In the ultra-clean workbench, use a syringe to draw the liquid medicine, filter it through a sterile filter membrane to filter and sterilize, use 2mLEP tubes to aliquot, and store in a -20°C refrigerator for later use.

3.3 Preparation of traditional Chinese medicine extract solution

According to the operating specifications of the weighing experiment, accurately weigh 40 mg of capsicum extract into a 10 mL EP tube with an electronic analytical balance, add 5 mL of pure water to prepare a solution of capsicum extract with a concentration of 8 mg/mL. Use a 5mL syringe to draw the extract solution in the ultra-clean workbench, pass it through a sterile filter membrane to filter and sterilize, and use 2mLEP tubes to aliquot it, and store it in a -20°C refrigerator for later use.

Record the results of the growth curve experiment, prepare a capsicum extract solution with a concentration of 32 mg/mL according to the above steps, and use it as a traditional Chinese medicine extract solution, and store it for future use after sterilization.

4. Determination of MIC of meropenem against drug-resistant Escherichia coli

Take out the meropenem liquid prepared in the refrigerator and use it when it melts to room temperature. Shake well before use. Dilute the meropenem solution to an initial concentration of 256 μg/mL, use a sterile 96-well polypropylene microtiter plate, and select 16 quality control bacteria according to the final concentration range of meropenem (0.008 μg/mL-0.06 μg/mL) The wells were numbered, and 100 μL of MHB broth medium was added to each well. Add 100 μL of 256 μg/mL meropenem drug solution to the first well, mix well, take 100 μL and add it to the second well, and then dilute to the 14th well, finally discard 100 μL, and then inoculate each well with the diluted bacterial solution 100 μL, the concentrations of the meropenem solution were: 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL, 0.125μg/mL, 0.0625μg/mL, 0.03125μg/mL, 0.015625μg/mL, 0.0078125μg/mL, no meropenem liquid was added to the 15th well, but 100μL of the bacterial solution was inoculated, as a positive control, no meropenem was added to the 16th well No bacterial solution was added to the medicinal solution as a negative control. Place in a constant temperature and humidity incubator for 24 hours at 37°C and observe the results. The inoculated bacteria were divided into quality control bacteria and experimental bacteria, and two parallel experiments were performed for verification.

5. Drawing of growth curve

Take out the prepared capsicum extract solution and meropenem that have been frozen in the refrigerator, and let them thaw to room temperature for use. In the ultra-clean workbench, dilute meropenem so that the final concentration is 1/4 of the MIC for drug-resistant bacteria, and the pepper extract solution is not diluted; then pick a single colony and inoculate the bacteria that have been cultured overnight on the agar plate Nutrient broth was grown and bacteria were diluted to 1.5 x 10 ⁶ CFU/mL.

The experiment was divided into four groups: no drug group, meropenem group, capsicum extract group, capsicum extract combined with meropenem group. In a sterile 96-well polypropylene microtiter plate, the no drug group was added with 100 μL of broth and 100 μL of bacterial diluent; 50 μL of meropenem liquid and broth and 100 μL of bacterial diluent were added to the meropenem group; 50 μL of pepper extract solution and broth and 100 μL of bacterial diluent were added to the pepper extract group; In the ropenem group, 50 μL of meropenem liquid and capsicum extract solution and 100 μL of bacterial dilution were added. Three replicates were set up for each group, and 200 μL of broth was added dropwise to each well outside the experimental area to prevent contamination. Place in a constant temperature and humidity incubator for 24 hours at 37°C, and measure the OD value of each experimental well at 5 time points (0h, 1h, 6h, 12h, 24h). The average value, and then draw the growth curve of bacteria according to the data, and calculate the bacteriostatic rate.

6. Determination of Antibacterial Activity in Vitro by Broth Microdilution Checkerboard Method

According to the results of the growth curve, it was observed that the combined antibacterial rate of the capsicum extract and meropenem was >50%. Reconstitute the capsicum extract solution with a concentration of 32mg/mL, dilute the meropenem to make the final concentration twice the concentration of the MIC against drug-resistant bacteria, and put the prepared capsicum extract solution and meropenem liquid in the injector, respectively. Dilute 7 concentration gradients with broth.

In a sterile 96-well polypropylene microtiter plate, 50 μL of meropenem solution was added dropwise to each horizontal row of 7 wells, and no drug was added to the last well. The final concentrations of each well from left to right were: 64 μg/mL, 32 μg/mL , 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, 0 μg/mL; 50 μL of capsicum extract solution was added dropwise to each vertical row of 7 wells, and no drug was added to the last well, from top to bottom The final concentration of each well is 8mg/mL, 4mg/mL, 2mg/mL, 1mg/mL, 1/2mg/mL, 1/4mg/mL, 1/8mg/mL, 0mg/mL. Finally, 100 μL of bacterial dilution was added dropwise to each well, and placed in a constant temperature and humidity incubator for 18-24 hours. The lowest concentration at which no bacterial growth was observed with the naked eye on the second day was the minimum inhibitory concentration (MIC). Fractional inhibitory concentration (FICI) index value to confirm whether the tested pepper extract has a synergistic effect with meropenem, FICI=(MIC pepper extract combined/MIC pepper extract alone)+(MIC meropenem combined/MIC meropenem single use).

The judging criteria are: when FICI≤0.5, it is judged that the two have synergistic effect; when 0.5<FICI<4.0, it is judged that the two have irrelevant effect; when FICI>4.0, it is judged that the two have antagonistic effect.

7 Results and Analysis

7.1 MIC of Meropenem

Observe the growth of bacteria after 24 hours of cultivation. If the culture medium in the well becomes cloudy or precipitates form, it indicates that there is bacterial growth, which is recorded as "+". If the medium is clear and transparent and no bacteria can be seen, it indicates that there is no bacterial growth. "-".

The MIC results of the quality control bacteria are shown in Table 1. The MIC of meropenem against the quality control bacteria was 0.015625 μg/mL, which was within the quality control range. The MIC results of the experimental bacteria are shown in Table 2. It can be seen that the MIC of meropenem against the experimental bacteria is 32 μg/mL.

Table 1 Bacterial growth results of quality control bacteria

Table 2 Bacterial growth results of experimental bacteria

7.2 Drawing the growth curve of capsicum extract combined with meropenem

The growth curve was drawn according to the measured OD value. It was found that the combination of capsicum extract and meropenem had a good antibacterial effect (Figure 1), and the antibacterial rate was greater than 65%. The results of the antibacterial rate are shown in Table 3.

Table 3 The antibacterial rate of meropenem combined with capsicum extract

7.3 Observation of results of broth microdilution checkerboard method

A broth microdilution checkerboard experiment was performed on the pepper extract to further verify the cooperative relationship between the pepper extract and meropenem. The observed bacterial growth results in each well of the checkerboard are expressed in a table, "-" represents no bacterial growth, and "+" represents bacterial growth.

The bacterial growth results of the combination of capsicum extract and meropenem are shown in Table 4, from which it can be seen that:

The MIC of capsicum extract when used alone is >8mg/mL, and the MIC when combined is 2mg/mL;

The MIC of meropenem when used alone is 32 μg/mL, and the MIC when combined is 2 μg/mL;

Therefore, FICI=2/>8+2/32, it is determined that the two have a synergistic effect.

Table 4 Antibacterial effect of meropenem combined with capsicum extract

8. Conclusion and Discussion

Through the growth curve drawing experiment and the broth microdilution checkerboard experiment, it was found that the combination of capsicum extract and meropenem had a synergistic effect, which could significantly improve the antibacterial effect of each other on drug-resistant bacteria in vitro, and the effect was better than that of each drug alone .

Combined with the results of this experiment, it shows that when the capsicum extract is combined with meropenem, the ratio relationship is within a certain range, and it has a good synergistic effect. When the present invention explores the combination of capsicum extract and antibiotics, the optimal ratio relationship between the two is studied through a large number of experiments, and a good antibacterial formula is obtained.

The capsicum extract screened out in this experiment can combine with meropenem to inhibit the growth of its drug-resistant bacteria, which provides a direction for further research on the mechanism of action of the combination of Chinese and Western medicine on bacteria, and is also useful for exploring the in vitro inhibitory effect of Chinese herbal medicine extracts on other drug-resistant bacteria. The role of bacteria is of reference significance.

comparative example

In the process of screening Chinese medicine extracts, the applicant compared the antibacterial effects of various Chinese medicine extracts combined with meropenem, and found that not every combination has a good antibacterial effect or synergistic effect. In some cases, even though the traditional Chinese medicine extract itself has antibacterial effect, it does not show obvious antibacterial effect when combined with meropenem, nor does it show effective inhibition of meropenem-resistant bacteria after combined use effect or synergy.

According to the same method as the experimental method of the present invention, when mulberry leaf extract, eucommia leaf extract, and baicalin were used in combination with meropenem, no synergistic effect was shown. Their growth curves are shown in Fig. 2, Fig. 3 and Fig. 4 respectively, and the FICIs are all between 0.5 and 4.0, which indicates that there is no synergistic effect.

in conclusion:

When the present invention was studying the combination of traditional Chinese medicine extracts and meropenem, it was unexpectedly found that when the pepper extract was used in combination with meropenem, it had a significant inhibitory effect on meropenem-resistant bacteria in vitro, and the effect was better than that of each drug alone. Therefore, the combination of meropenem and the pepper extract has obvious synergistic effect. In the antibacterial composition of the present invention, the combined use of the pepper extract and the meropenem antibiotic has the advantages of synergism and attenuation. In this way, the relative dosage of each substance in the composition will be greatly reduced, thereby reducing possible adverse reactions and side effects, improving the safety of medication, and at the same time increasing the compliance of patients taking medication, thereby ensuring the curative effect.

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (2)

1. Antibacterial composition, characterized in that, the antibacterial composition is composed of meropenem and capsicum extract, wherein the capsicum extract is 60%-80% alcoholic extract of capsicum, in the antibacterial composition, The weight ratio of meropenem to the pepper extract is (1-2):(1000-4000); the bacterium is Escherichia coli. 2. the purposes of the antibacterial composition described in claim 1 in the medicine that is used for antibacterial preparation.

Images