CN113975304A

CN113975304A - Antibacterial composition containing isatis root extract and application thereof

Info

Publication number: CN113975304A
Application number: CN202111266652.6A
Authority: CN
Inventors: 李基云; 陈小军; 孙志良
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2021-10-28
Filing date: 2021-10-28
Publication date: 2022-01-28

Abstract

An antibacterial composition containing radix Isatidis extract and its application are provided. The invention discloses an antibacterial composition which comprises meropenem and isatis root extracts. When the isatis root 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

Antibacterial composition containing isatis root extract and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to an antibacterial composition containing meropenem and isatis root extracts 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 unreasonable use situations gradually occur, which result in the emergence of meropenem resistant bacteria to some extent, and pseudomonas aeruginosa is one of the meropenem resistant bacteria. 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 easy to generate drug resistance, the research period is long, and multiple drug resistant bacteria ("superbacteria") can appear.

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 inhibit or kill drug-resistant bacteria.

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

In some embodiments, the isatis root extract is an aqueous isatis root extract; preferably, the isatis root extract is prepared by a water extraction and alcohol precipitation method.

In some embodiments, the isatis root extract of the present invention can be prepared by the following method: decocting radix Isatidis in water for 2-4 times, each for 1-2 hr, mixing filtrates, concentrating, adding ethanol, standing for precipitation, collecting supernatant, recovering ethanol, concentrating, and drying.

In some embodiments, the isatis root extract of the present invention may be prepared with reference to the "chinese pharmacopoeia", or may be a commercially available product.

In some embodiments, the antibacterial composition has a ratio of meropenem to the radix Isatidis extract by weight of (1-8): (1000-4000).

Preferably, the weight ratio of the meropenem to the isatis root extract is (1-4): 1000-4000, more preferably (1-2): 1000-4000, and more preferably the weight ratio is 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 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 (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 results surprisingly found that the isatis root extract has obvious inhibition effect on meropenem resistant bacteria in vitro when being used together with meropenem, and the effect is better than that of each drug which is used independently, so that the meropenem and the isatis root extract have obvious synergistic effect when being used together. 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 combination of the isatis root extract and the meropenem antibiotic has the advantages of synergy and attenuation. The isatis root 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 method adopts a trace broth dilution method to research the in-vitro inhibition effect of the isatis root 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 graph showing the growth curve of the combination of isatis root extract and meropenem.

FIG. 2 is a growth curve of the mulberry leaf extract in combination with meropenem.

FIG. 3 is a growth curve of eucommia ulmoides 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 (ATCC25922) (purchased from China veterinary medicine inspection institute).

Meropenem, powder and pure product.

2. Chinese medicinal extract

Preparation of the isatis root extract:

decocting radix isatidis twice in water for 2 hours for the first time and 1 hour for the second time, filtering decoction, combining filtrates, concentrating until the relative density is 1.20(50 ℃), adding ethanol until the ethanol content reaches 60%, standing for precipitation, taking supernatant, recovering ethanol, concentrating until the relative density is 1.32-1.35 (60 ℃), drying, and crushing to obtain the isatis root extract.

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 powder by using an electronic analytical balance, and adding pure water to dissolve. Making into solution with concentration of 2560 μ g/mL, placing in a clean bench, sucking the solution with an injector, filtering with a sterile filter membrane for sterilization, packaging with 2mLEP tube, and storing in a refrigerator at-20 deg.C.

3.3 preparation of Chinese medicine extract solution

According to the weighing experiment operation specification, 40mg of isatis root extract is accurately weighed in a 10mL EP tube by an electronic analytical balance, and 5mL of pure water is added to prepare isatis root 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 the isatis root extract solution with the concentration of 32mg/mL according to the steps to be used as the traditional Chinese medicine extract solution, 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 the initial concentration of 256 mu g/mL, sterile 96-hole polypropylene microtiter plates are adopted, 16 holes are selected according to the quality control bacterium range (0.008 mu g/mL-0.06 mu g/mL) of the final concentration of the Meropenem and are numbered, and 100 mu L of MHB broth culture medium is added into each hole. 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 isatis root extract solution and meropenem which are frozen and stored in a refrigerator, and melting the isatis root extract solution and the meropenem to room temperature for use. Diluting meropenem in an ultra-clean workbench to a final concentration of 1/4 concentration of drug-resistant bacteria MIC, and not diluting the isatis root extract solution; 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 10⁶CFU/mL. The experiments were divided into four groups: adding no medicine group, meropenem group, radix Isatidis extract group, and radix Isatidis extract combined with meropenem group, adding broth 100 μ L and bacteria diluent 100 μ L into sterile 96-well polypropylene microtiter plate without medicine group; adding meropenem liquid and broth into the meropenem group, wherein each of the meropenem liquid and broth is 50 mu L, and each of the broth and the broth is 100 mu L; adding radix Isatidis extract solution and broth each 50 μ L and bacteria diluent 100 μ L into radix Isatidis extract group; the radix Isatidis extract and meropenem group are added with 50 μ L of meropenem liquid medicine and 50 μ L of radix Isatidis extract solution and 100 μ L of bacteria diluent respectively. Each set was repeated three times, and 200. mu.L of broth was dropped into each well at the periphery of the experimental area to prevent contamination. Culturing at 37 deg.C for 24h in a constant temperature and humidity incubator, measuring OD value of each experimental well at 5 time points (0h, 1h, 6h, 12h, and 24h) with wavelength of 600nm, calculating average value of each time point, and analyzing dataAnd drawing a growth curve graph of bacteria, and calculating the bacteriostasis 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 isatis root extract and the meropenem is observed to be more than 50 percent. And (3) preparing an isatis root extract solution with the concentration of 32mg/mL again, diluting meropenem to the final concentration of 2 times of the MIC (minimal inhibitory concentration) of the drug-resistant bacteria, and diluting the prepared isatis root extract solution and meropenem liquid medicine in a sample injector by using broth for 7 concentration gradients.

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 isatis root 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, 1mg/mL, 1/2mg/mL, 1/4mg/mL, 1/8mg/mL and 0 mg/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. Grading the index value of the inhibitory concentration (FICI) to confirm whether the tested radix isatidis extract and meropenem have synergistic effect, wherein FICI is (MIC combined radix isatidis extract/MIC single radix isatidis extract) + (MIC combined meropenem/MIC single meropenem).

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 0.5< FICI <4.0, the two are judged to have unrelated effects; when FICI is greater than 4.0, the two are judged to have antagonistic action.

7 results and analysis

7.1 MIC of meropenem

The growth of the bacteria after 24h of culture was observed, and the medium became cloudy or had a precipitate formed in the wells, indicating that there was bacterial growth, and was marked as "+", and if the medium was clear, no bacteria could be seen, indicating that there was no bacterial growth, was marked as "-".

The MIC results of the quality control bacteria are shown in 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 experimental bacteria

7.2 drawing of growth curves of combination of Isatis root extract and Meropenem

A growth curve graph is drawn according to the measured OD value, and the measured isatis root extract and meropenem combined drug has a good bacteriostatic action (figure 1), the bacteriostatic rate is more than 75%, and the bacteriostatic rate result is shown in Table 3.

TABLE 3 antibacterial ratio of meropenem in combination with Isatis root extract

7.3 Broth microdilution chessboard results observations

Broth microdilution chessboard experiments are carried out on the isatis root extract so as to further verify the cooperative relationship between the isatis root extract and the meropenem medicament. 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 combination of isatis root extract and meropenem are shown in table 4, and it can be seen from table 4 that:

the MIC of the isatis root extract is more than 8mg/mL when used singly, and the MIC of the isatis root extract is 1/4mg/mL when combined;

the MIC of meropenem is 32 mug/mL when used singly, and the MIC of meropenem is 1 mug/mL when combined;

therefore, the FICI is (1/4)/> 8+1/32, and the FICI is less than 0.5, so that the two are judged to have synergistic action.

TABLE 4 antibacterial Effect of meropenem in combination with Isatis root extract

8. Conclusion and discussion

Growth curve drawing experiments and broth microdilution chessboard experiments show that the radix isatidis extract and meropenem have synergistic effect when being used together, can obviously improve the in vitro antibacterial effect on drug-resistant bacteria, and the effect is better than that of the respective independent medicines.

The experiment results show that when the isatis root extract and the meropenem are used together, the proportion relationship is in a certain range, and the isatis root extract and the meropenem have good synergistic effect. When the combined application of the isatis root extract and the antibiotic is researched, the optimal proportion relation between the isatis root extract and the antibiotic is researched through a large number of experiments, and a good bacteriostatic formula is obtained.

The isatis root 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. The growth curves are shown in the graphs of fig. 2-4 respectively, and the FICI is between 0.5 and 4.0, which shows that no synergistic effect is generated.

And (4) conclusion:

when the combination of the traditional Chinese medicine extract and the meropenem is researched, the remarkable inhibition effect on meropenem resistant bacteria in vitro is unexpectedly found when the isatis root extract is used in combination with the meropenem, and the effect is superior to that of each drug which is used independently, so that the meropenem and the isatis root extract have remarkable synergistic effect when used in combination. In the antibacterial composition, the combination of the isatis root 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

1. An antibacterial composition, characterized in that it comprises meropenem and isatis root extract.

2. The antibacterial composition as claimed in claim 1, wherein said extract of radix isatidis is an aqueous extract of radix isatidis.

3. The antibacterial composition of claim 1, wherein the isatis root extract is prepared by water extraction and alcohol precipitation.

4. The antibacterial composition according to any one of claims 1 to 3, wherein the weight ratio of meropenem to the radix Isatidis extract in the antibacterial composition is (1-8): (1000-4000).

5. The antibacterial composition according to claim 4, wherein the weight ratio of meropenem to the radix Isatidis extract in the antibacterial composition is (1-4): (1000-4000).

6. The antibacterial composition according to claim 5, wherein the weight ratio of meropenem to the radix Isatidis extract in the antibacterial composition is (1-2): (1000-4000).

7. Use of the antibacterial composition according to any one of claims 1 to 6 for the manufacture of a medicament for antibacterial use.

8. Use according to claim 7, characterized in that the bacterium is a meropenem-resistant bacterium.

9. Use according to claim 8, characterized in that the drug-resistant bacteria comprise Escherichia coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Klebsiella pneumoniae (Klebsiella pneumoniae) and Acinetobacter baumannii (Acinetobacter baumannii).

10. Use according to claim 9, characterized in that the resistant bacterium is Escherichia coli (Escherichia coli).