CN114129635B - Antibacterial composition containing capsicum extract and its application - Google Patents
Antibacterial composition containing capsicum extract and its application Download PDFInfo
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Abstract
Antibacterial composition containing Capsici fructus extract and its application are provided. The invention discloses an antibacterial composition which comprises meropenem and a capsicum extract. When the capsicum extract and the meropenem are used together, the synergistic effect is achieved, the bacteriostatic effect on drug-resistant bacteria can be obviously improved, and the effect is superior to that of the respective single medicine. The invention provides a direction for researching the reversion of the drug-resistant bacteria by the combined use of Chinese and western medicines.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to an antibacterial composition containing meropenem and a capsicum extract and application thereof.
Background
Meropenem (meropenem) is a novel, artificially synthesized carbapenem antibiotic. The carbapenem antibiotics have wider antibacterial spectrum and antibacterial activity, are effective medicaments for clinically treating serious mixed infection and drug-resistant bacterial infection, and have higher treatment effect in clinical application. The meropenem has a good inhibition effect on escherichia coli and pseudomonas aeruginosa by inhibiting the synthesis of bacterial cell walls. However, as meropenem is frequently and widely used in clinical practice, some cases of unreasonable use gradually occur, which in some extent lead to the emergence of meropenem resistant bacteria, of which pseudomonas aeruginosa is one. The drug resistance of pseudomonas aeruginosa to meropenem has a positive correlation with the usage amount of carbapenem antibacterial drugs. The carbapenem drug-resistant strains have higher drug resistance rate to other antibacterial drugs than carbapenem-sensitive strains, which gradually reduces the clinical treatment benefit of meropenem drugs. To solve this problem, studies on how to inhibit drug-resistant bacteria are needed, as well as on the basis of the standardized and rational administration. There are studies reporting potential drugs for the treatment of carbapenem-resistant bacterial infections, indicating that the therapeutic drugs are mainly polymyxin, tigecycline, fosfomycin and aminoglycosides. However, chemical antibacterial drugs are prone to drug resistance, have long research periods, and may cause the appearance of multi-drug resistant bacteria ("superbacteria").
As a natural medicine, the traditional Chinese medicine has the advantages of low toxic and side effects, low drug resistance and the like. Research indicates that traditional Chinese medicines can directly inhibit or kill drug-resistant bacteria by interfering biochemical metabolism of the drug-resistant bacteria, or indirectly inhibit and kill bacteria by improving drug resistance of bacteria, and maintain organism balance to integrally regulate and treat drug-resistant bacteria infection. However, different Chinese herbs or Chinese herb extracts have different inhibitory effects on bacteria or drug-resistant bacteria, and have certain limitations.
For the experimental methods of combined bacteriostatic activity of natural drugs in vitro, in vivo and in vitro, the applicable methods of different types of natural drugs are different, so that a proper method is needed to screen out a proper traditional Chinese medicine or natural drug aiming at a specific flora or drug-resistant bacteria, thereby achieving the bacteriostatic or bactericidal effect on bacteria or drug-resistant bacteria.
Disclosure of Invention
The invention aims to provide an antibacterial composition which can play a role in inhibiting or killing drug-resistant bacteria.
In a first aspect, the present invention provides an antimicrobial composition comprising meropenem and capsicum extract.
In some embodiments, the capsicum extract is a capsicum alcohol extract; preferably, the capsicum extract is 60% -80% alcohol extract of capsicum, more preferably 70% alcohol extract of capsicum.
Herein, the 60% -80% alcohol extract of capsicum refers to a product obtained by extracting capsicum with 60% -80% alcohol (e.g. ethanol) and drying.
In some embodiments, the ratio of meropenem to the capsicum extract by weight in the antibacterial composition is (1-8) to (1000-4000).
Preferably, the weight part ratio of meropenem to the capsicum extract is (1-4): (1000-4000), further preferably (1-2): (1000-4000), more preferably the weight part ratio is 1.
In a second aspect, the present invention provides the use of the antibacterial composition in the manufacture of a medicament for antibacterial use.
In some embodiments, the bacterium is a meropenem-resistant bacterium.
In some embodiments, the drug-resistant bacteria include Escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas aeruginosa), klebsiella pneumoniae (Klebsiella pneumoniae), and Acinetobacter baumannii (Acinetobacter baumannii); preferably, the resistant bacterium is Escherichia coli.
The invention has the advantages of
In order to eliminate the drug resistance of bacteria, the invention starts with the traditional Chinese medicine extract, and screens the traditional Chinese medicine extract with stronger external bacteriostatic action on the meropenem drug-resistant bacteria by preparing a growth curve and a broth microdilution chessboard test. The result shows that surprisingly, when the capsicum extract is used in combination with meropenem, the capsicum extract has obvious inhibition effect on meropenem drug-resistant bacteria in vitro, and the effect is better than that of each drug which is used independently, so that the meropenem and the capsicum extract have obvious synergistic effect when used in combination. The relative dosage of each substance in the composition is greatly reduced, so that possible adverse reactions and side effects are reduced, the medication safety is improved, and the compliance of patients in taking can be increased, thereby ensuring the curative effect.
The pepper extract and meropenem antibiotic are combined for use, so that the pepper extract has the advantages of synergism and attenuation. The capsicum extract can be used in combination with meropenem antibiotic to inhibit meropenem drug-resistant bacteria. The invention provides a new research thought and research and development direction for solving the increasingly serious drug resistance problem of meropenem.
The invention adopts a trace broth dilution method to research the in-vitro inhibition effect of the hot pepper extract and the meropenem drug on the meropenem drug-resistant bacteria, has great research significance, is expected to provide reference for developing a new auxiliary drug for treating severe infectious diseases by the meropenem, and improves the treatment benefit.
Drawings
Fig. 1 is a growth curve of capsicum extract in combination with meropenem.
FIG. 2 is a growth curve of the mulberry leaf extract in combination with meropenem.
FIG. 3 is a growth curve of folium Eucommiae extract in combination with meropenem.
FIG. 4 is a growth curve of baicalin in combination with meropenem.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
1. Bacterial and antibiotic
Meropenem resistant E.coli strain 20a02 (isolated from chicken houses and stored by the laboratory).
Quality control strain Escherichia coli (ATCC 25922) (purchased from China institute for veterinary medicine).
Meropenem, powder and pure product.
2. Chinese medicine extract
Preparation of a pepper extract:
extracting Capsici fructus with 70% ethanol twice (2 hr for the first time and 1 hr for the second time), filtering the extractive solution, mixing filtrates, concentrating, and drying.
3. Preparation of drug-resistant bacteria and drugs
3.1 Resuscitation of drug-resistant bacteria
One day before the experiment, the drug-resistant strain is plated and inoculated on an agar culture medium, and is cultured in a constant temperature and humidity incubator at 37 ℃ for 24 hours for later use.
3.2 preparation of Meropenem liquid medicine
Accurately weighing a certain amount of meropenem medicinal powder by using an electronic analytical balance, and adding pure water for dissolving. Making into solution with concentration of 2560 μ g/mL, sucking the solution with an injector in a clean bench, filtering with sterile filter membrane for sterilization, packaging with 2mL LEP tube, and storing in refrigerator at-20 deg.C.
3.3 preparation of Chinese medicine extract solution
According to the weighing experiment operation specification, 40mg of pepper extract is accurately weighed in a 10mL EP tube by an electronic analytical balance, and 5mL of pure water is added to prepare a pepper extract solution with the concentration of 8 mg/mL. The extract solution was aspirated in a 5mL syringe in a clean bench, filtered through sterile filter membrane for sterilization, and dispensed in 2mL LEP tubes and stored in a freezer at-20 ℃ until needed.
Recording the experimental result of the growth curve, preparing a capsicum extract solution with the concentration of 32mg/mL as a traditional Chinese medicine extract solution according to the steps, and storing for later use after degerming.
4. Determination of drug-resistant escherichia coli MIC of meropenem
Taking out the prepared meropenem liquid medicine in a refrigerator, and shaking up before use when the meropenem liquid medicine is melted to room temperature. Meropenem liquid medicine is diluted to an initial concentration of 256 mu g/mL, a sterile 96-well polypropylene microtiter plate is adopted, 16 wells are selected and numbered according to the quality control bacterium range of the final concentration of the Meropenem (0.008 mu g/mL-0.06 mu g/mL), and 100 mu L of MHB broth culture medium is added into each well. Adding 100 mu L of Meropenem liquid medicine of 256 mu g/mL into the first hole, uniformly mixing, adding 100 mu L of the Meropenem liquid medicine into the 2 nd hole, diluting to the 14 th hole in a multiple ratio, finally discarding 100 mu L, then inoculating 100 mu L of diluted liquid medicine into each hole, and obtaining the concentration of the Meropenem liquid medicine, wherein the concentration of the Meropenem liquid medicine is respectively as follows: 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL, 2 mu g/mL, 1 mu g/mL, 0.5 mu g/mL, 0.25 mu g/mL, 0.125 mu g/mL, 0.0625 mu g/mL, 0.03125 mu g/mL, 0.015625 mu g/mL, 0.0078125 mu g/mL, 100 mu L of the inoculum is inoculated in the 15 th well without adding the meropenem solution, and the 16 th well is used as a positive control without adding the meropenem solution or adding the inoculum as a negative control. Culturing at 37 deg.C for 24h in a constant temperature and humidity incubator, and observing the result. The inoculated bacterial liquid is subjected to quality control and experimental bacteria respectively, and two parallel experiments are carried out respectively for verification.
5. Drawing of growth curves
Taking out the prepared pepper extract solution and meropenem which are frozen and stored in a refrigerator, and melting to room temperature for use. Diluting meropenem in a clean bench toThe final concentration is 1/4 of the MIC of the drug-resistant bacteria, and the capsicum extract solution is not diluted; then, the bacteria cultured overnight on agar plate are picked up and inoculated into nutrient broth for proliferation, and the bacteria are diluted to 1.5X 10 6 CFU/mL。
The experiments were divided into four groups: adding no medicine group, meropenem group, capsicum extract group and capsicum extract combined meropenem group, and adding 100 mu L broth and 100 mu L bacteria diluent into a sterile 96-hole polypropylene microtiter plate without adding medicine group; adding Meropenem liquid and broth into Meropenem group, wherein each of the Meropenem liquid and broth is 50 mu L, and each of the Meropenem liquid and broth is 100 mu L; adding 50 μ L each of the Capsici fructus extract solution and broth and 100 μ L of bacteria diluent into Capsici fructus extract group; the Capsici fructus extract and meropenem group are added with 50 μ L of meropenem liquid and Capsici fructus extract solution and 100 μ L of bacteria diluent respectively. Each set was replicated in triplicate, and 200. Mu.L of broth was added dropwise to each well around the periphery of the experimental area to seal and prevent contamination. Culturing at 37 ℃ for 24h in a constant-temperature and constant-humidity incubator, measuring the OD value of each experimental hole at 5 time points (0 h, 1h, 6h, 12h and 24 h) in total, calculating the average value of each time point of each group when the wavelength is 600nm, drawing a bacterial growth curve according to data, and calculating the bacteriostatic rate.
6. Broth microdilution chessboard method for determining in vitro antibacterial activity
According to the results of the growth curve, the combined bacteriostasis rate of the pepper extract and the meropenem is observed to be more than 50 percent. Newly preparing a pepper extract solution with the concentration of 32mg/mL, diluting meropenem to the final concentration of 2 times of the MIC of the drug-resistant bacteria, and diluting the prepared pepper extract solution and meropenem liquid medicine in a sample injector by 7 concentration gradients in broth in a multiple ratio respectively.
In a sterile 96-well polypropylene microtiter plate, 50 mu L of meropenem liquid medicine is dripped into 7 wells in each horizontal row, the last well is not added with medicine, and the final concentration of each well from left to right is as follows: 64. Mu.g/mL, 32. Mu.g/mL, 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0. Mu.g/mL; 50 mu.L of pepper extract solution is dripped into 7 vertical holes, the last hole is not added with medicine, and the final concentration of each hole from top to bottom is 8mg/mL, 4mg/mL, 2mg/mL, 1/2mg/mL, 1/4mg/mL, 1/8mg/mL and 0mg/mL. Finally, 100 mu L of bacterial diluent is dripped into each hole, and the mixture is put into a constant temperature and humidity incubator to be cultured for 18 to 24 hours. The following day the Minimum Inhibitory Concentration (MIC) was determined as the lowest concentration at which no bacteria grew under visual observation. Index values of Fractional Inhibitory Concentration (FICI) to confirm whether the tested pepper extract and meropenem had synergistic effect, FICI = (MIC pepper extract combination/MIC pepper extract alone) + (MIC meropenem combination/MIC meropenem alone).
The judgment standard is as follows: when the FICI is less than or equal to 0.5, the FICI and the FICI are judged to have synergistic action; when the 0.5-woven FICI woven fabrics are 4.0, the two are judged to have unrelated functions; when FICI is greater than 4.0, the two compounds are judged to have antagonistic action.
7 results and analysis
7.1 MIC of meropenem
When the growth of the bacteria after 24h of culture was observed, the medium in the wells became cloudy or precipitates formed, indicating that the bacteria were growing, and the medium was marked with "+" or when the medium was clear and transparent, no bacteria were found, indicating that no bacteria were growing, and marked with "-".
The MIC result of the quality control bacteria is shown in the table 1, and the MIC of the meropenem medicine to the quality control bacteria is 0.015625 mu g/mL and is within the quality control range. The MIC results of the experimental bacteria are shown in Table 2, and the MIC of meropenem to the experimental bacteria is 32 mug/mL.
TABLE 1 bacterial growth results of quality control bacteria
TABLE 2 bacterial growth results of the test bacteria
7.2 growth Curve drawing of combination of Capsici fructus extract and Meropenem
A growth curve graph is drawn according to the measured OD value, and the measured that the combined application of the capsicum extract and the meropenem has better bacteriostasis (figure 1), the bacteriostasis rate is more than 65 percent, and the bacteriostasis rate result is shown in a table 3.
TABLE 3 antibacterial ratio of meropenem in combination with capsicum extract
7.3 Broth microdilution chessboard results observations
Broth microdilution chessboard experiments were performed on the pepper extracts to further verify the cooperative relationship between the pepper extracts and meropenem drugs. The observed bacterial growth results for each well of the board are tabulated, "-" indicates no bacterial growth and "+" indicates bacterial growth.
The results of bacterial growth of the pepper extract in combination with meropenem are shown in table 4, from which it can be seen that:
the MIC of the capsicum extract is more than 8mg/mL when used alone, and the MIC of the capsicum extract is 2mg/mL when combined;
the MIC of meropenem is 32 mug/mL when used singly and 2 mug/mL when combined;
therefore, FICI = 2/> 8+2/32, and the two are judged to have synergistic effect.
TABLE 4 antibacterial Effect of meropenem in combination with Capsicum extracts
8. Conclusion and discussion
Growth curve drawing experiments and broth microdilution chessboard experiments show that the pepper extract and meropenem have synergistic effect when being used together, can remarkably improve the in vitro antibacterial effect on drug-resistant bacteria, and the effect is better than that of the respective independent application.
The experiment result shows that when the capsicum extract and the meropenem are used together, the proportion relationship is in a certain range, and the synergistic effect is good. When the combined administration of the capsicum extract and the antibiotic is researched, the optimal proportion relation between the capsicum extract and the antibiotic is researched through a large number of experiments, and a good bacteriostatic formula is obtained.
The pepper extract screened by the experiment can be combined with meropenem to inhibit the growth of drug-resistant bacteria, so that a direction is provided for further researching the action mechanism of the combination of Chinese and western medicines on bacteria, and reference significance is provided for researching the in-vitro bacteriostatic action of the Chinese herbal medicine extract on other drug-resistant bacteria.
Comparative example
In the process of screening the traditional Chinese medicine extracts, the applicant combines a plurality of traditional Chinese medicine extracts and meropenem and then compares the antibacterial effect, and the result shows that not every combination has a good antibacterial effect or synergistic effect. In some cases, even though the traditional Chinese medicine extract has an antibacterial effect, the traditional Chinese medicine extract does not obviously show a good antibacterial effect after being used with meropenem, and does not show an effective inhibition effect or synergistic effect on meropenem drug-resistant bacteria after being used in combination.
According to the experiment method of the invention, the mulberry leaf extract, the eucommia bark leaf extract and the baicalin do not show synergistic effect when being respectively combined with meropenem. Their growth curves are shown in FIGS. 2, 3 and 4, respectively, and FICI is between 0.5 and 4.0, indicating no synergistic effect.
And (4) conclusion:
when the traditional Chinese medicine extract and the meropenem are researched for combined administration, the remarkable inhibition effect on meropenem resistant bacteria in vitro is unexpectedly found when the capsicum extract is used in combination with the meropenem, and the effect is superior to that of each independent drug, so that the meropenem and the capsicum extract have remarkable synergistic effect when used in combination. In the antibacterial composition, the combination of the capsicum extract and the meropenem antibiotic has the advantages of synergy and attenuation. Therefore, the relative dosage of each substance in the composition can be greatly reduced, so that possible adverse reactions and side effects are reduced, the medication safety is improved, and the compliance of patients in taking can be improved, thereby ensuring the curative effect.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (2)
1. The antibacterial composition is characterized by consisting of meropenem and a hot pepper extract, wherein the hot pepper extract is a 60-80% alcohol extract of hot pepper, and the weight ratio of the meropenem to the hot pepper extract in the antibacterial composition is (1-2) to (1000-4000); the bacterium is Escherichia coli (Escherichia coli).
2. Use of the antibacterial composition of claim 1 for the preparation of a medicament for antibacterial use.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103054833A (en) * | 2012-12-28 | 2013-04-24 | 湖南农业大学 | A formula of an antimicrobial oil emulsion microcapsule for veterinary use |
| CN104610109A (en) * | 2009-11-12 | 2015-05-13 | 西格纳姆生物科学公司 | ANTI-BACTERIAL AGENTS and composites thereof |
| KR102042151B1 (en) * | 2018-12-18 | 2019-11-07 | 한국식품연구원 | A composition as a prebiotic for improving intestinal microflora containing extract from pepper leaves |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104610109A (en) * | 2009-11-12 | 2015-05-13 | 西格纳姆生物科学公司 | ANTI-BACTERIAL AGENTS and composites thereof |
| CN103054833A (en) * | 2012-12-28 | 2013-04-24 | 湖南农业大学 | A formula of an antimicrobial oil emulsion microcapsule for veterinary use |
| KR102042151B1 (en) * | 2018-12-18 | 2019-11-07 | 한국식품연구원 | A composition as a prebiotic for improving intestinal microflora containing extract from pepper leaves |
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