Antibacterial composition containing ginseng leaf extract and application thereof

Abstract

The present invention relates to an antibacterial composition containing ginseng leaf extract and its application, and belongs to the technical field of antibacterial drugs. The present invention provides an antibacterial composition containing ginseng leaf extract, wherein the antibacterial composition includes a β-lactam antibiotic and a ginseng leaf extract. The present invention has a synergistic effect when the ginseng leaf extract is used in combination with a β-lactam antibiotic, which can significantly improve the antibacterial effect on bacteria (including drug-resistant bacteria), and the effect is better than that of each drug alone. The present invention provides a direction for studying the reversal of bacterial resistance by the combination of Western and Western medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial drugs, and particularly relates to an antibacterial composition containing ginseng leaf extract and application thereof. Background Art

[0002] As a class of natural medicines, Chinese herbal medicines not only have low toxicity and side effects, but also have multiple targets and low resistance. Studies have shown that Chinese herbal medicines can directly inhibit or kill drug-resistant bacteria by interfering with their biochemical metabolism, or reverse bacterial resistance to indirectly inhibit and kill bacteria. Chinese herbal medicines can also treat drug-resistant infections by enhancing the body's immune function and alleviating pathological damage caused by bacterial infection. However, different Chinese herbal medicines or herbal extracts have different inhibitory effects on bacteria or drug-resistant bacteria, and the development and utilization of Chinese herbal medicines for the treatment of bacterial infections (including drug-resistant bacteria) still has certain limitations. Summary of the Invention

[0003] The present invention aims to provide an antibacterial composition containing ginseng leaf extract and its application. The present invention combines ginseng leaf extract with β-lactam antibiotics to produce a synergistic effect, which can significantly improve the antibacterial effect on bacteria (including drug-resistant bacteria).

[0004] The present invention provides an antibacterial composition, which comprises beta-lactam antibiotics and ginseng leaf extract.

[0005] Preferably, the β-lactam antibiotics include one or more of ceftiofur, penicillin, amoxicillin and ampicillin.

[0006] Preferably, the β-lactam antibiotic is ceftiofur.

[0007] Preferably, the ginseng leaf extract comprises ginseng leaf alcohol extract.

[0008] Preferably, the preparation method of the ginseng leaf alcohol extract comprises the following steps: mixing ginseng leaves with an ethanol aqueous solution, refluxing extraction, filtering the filtrate, concentrating, and drying to obtain the ginseng leaf extract.

[0009] Preferably, the mass ratio of the β-lactam antibiotic to the ginseng leaf extract is (4-256):(97-6250).

[0010] Preferably, when the β-lactam antibiotic is ceftiofur, the mass ratio of ceftiofur to ginseng leaf extract is (4-64):(97-6250).

[0011] The present invention also provides the use of the antibacterial composition described in the above technical solution in the preparation of antibacterial drugs.

[0012] Preferably, the bacteria include one or more of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.

[0013] Preferably, the bacteria include drug-resistant bacteria; the drug-resistant bacteria include one or more of drug-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.

[0014] The present invention provides an antibacterial composition containing ginseng leaf extract. When the present invention uses ginseng leaf extract in combination with β-lactam antibiotics, it shows good synergistic effect, can significantly improve the antibacterial effect on bacteria (including drug-resistant bacteria), and the effect is better than each of them used alone. The present invention provides a direction for exploring the strategy of combined use of Chinese and Western medicine in reversing the drug resistance of drug-resistant bacteria. The experimental results show that through the microdilution checkerboard method experiment, it was found that ginseng leaf extract and β-lactam antibiotics (ceftiofur, penicillin, amoxicillin and ampicillin) have a synergistic effect when used in combination, can significantly improve each other's in vitro antibacterial effect on MRSA, and the effect is better than each of them used alone. The FICI of ceftiofur combined with ginseng leaf extract is the smallest, and the combined effect is the best. Growth curve experiments further showed that the combination of ginseng leaf extract and ceftiofur exhibited a good synergistic antibacterial effect. The combination of 0.78 mg / mL ginseng leaf extract and 4 μg / mL, 8 μg / mL and 16 μg / mL ceftiofur completely inhibited the proliferation of MRSA and lasted for 16 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is a graph showing the effect of 0.39 mg / mL ginseng leaf extract combined with ceftiofur on the growth of MRSA provided by the present invention; wherein GLE: ginseng leaf extract, CF: ceftiofur;

[0017] Figure 2 This is a graph showing the effect of 0.58 mg / mL ginseng leaf extract combined with ceftiofur on the growth of MRSA provided by the present invention; wherein GLE: ginseng leaf extract, CF: ceftiofur;

[0018] Figure 3This is a graph showing the effect of 0.78 mg / mL ginseng leaf extract combined with ceftiofur on the growth of MRSA provided by the present invention; wherein, GLE: ginseng leaf extract, CF: ceftiofur. DETAILED DESCRIPTION

[0019] The present invention provides an antibacterial composition comprising a β-lactam antibiotic and a ginseng leaf extract. In the present invention, the β-lactam antibiotic preferably comprises one or more of ceftiofur, penicillin, amoxicillin, and ampicillin. In the present invention, the β-lactam antibiotic is preferably ceftiofur. In order to eliminate bacterial resistance, the present invention starts with Chinese herbal extracts and screens out Chinese herbal extracts with strong antibacterial effects on drug-resistant bacteria when used in combination with β-lactam antibiotics through microbroth dilution method, checkerboard method, and growth curve test. The present invention finds that when ginseng leaf extract is used in combination with β-lactam antibiotics, it has a significant inhibitory effect on drug-resistant bacteria, and the effect is better than that of each substance used alone, showing a significant synergistic effect. The relative amount of each substance in the composition is greatly reduced, thereby reducing possible adverse reactions and side effects, improving medication safety, reducing treatment costs, and at the same time increasing patient compliance, thereby ensuring efficacy.

[0020] In the present invention, the ginseng leaf extract includes a ginseng leaf alcohol extract. In the present invention, the ginseng leaf extract is preferably prepared with reference to the relevant standards of the 2020 edition of the Chinese Pharmacopoeia, or may be a commercially available product. In the present invention, the preparation method of the ginseng leaf alcohol extract preferably includes the following steps: mixing ginseng leaves with an ethanol aqueous solution, refluxing extraction, filtering the filtrate, concentrating, and drying to obtain a ginseng leaf extract. In the present invention, the ethanol aqueous solution preferably contains 50% by mass of ethanol.

[0021] In the present invention, the mass ratio of the β-lactam antibiotic to the ginseng leaf extract is preferably (4-256):(97-6250), more preferably (4-128):(97-6250), and most preferably (4-128):(390-6250). In the present invention, when the β-lactam antibiotic is penicillin, the mass ratio of penicillin to the ginseng leaf extract is preferably (8-128):(97-6250), more preferably (16-64):(97-6250), further preferably (16-64):(1560-6250), and more preferably 16:6250, 32:3125, or 64:1560. In the present invention, when the antibacterial composition of the present invention is used, the concentration of penicillin is preferably 8-128 μg / mL, more preferably 16-64 μg / mL; the concentration of ginseng leaf extract is preferably 0.097-6.25 mg / mL, more preferably 1.56-6.25 mg / mL.

[0022] In the present invention, when the β-lactam antibiotic is amoxicillin, the mass ratio of amoxicillin to ginseng leaf extract is preferably (16-256):(97-6250), more preferably (32-128):(97-6250), further preferably (32-128):(390-6250), more preferably 32:1560, 64:780 or 128:390. In the present invention, when the antibacterial composition of the present invention is used, the concentration of amoxicillin is preferably 16-256 μg / mL, more preferably 32-128 μg / mL; the concentration of ginseng leaf extract is preferably 0.097-6.25 mg / mL, more preferably 0.39-6.25 mg / mL.

[0023] In the present invention, when the β-lactam antibiotic is ampicillin, the mass ratio of ampicillin to ginseng leaf extract is preferably (16-256):(97-6250), more preferably (32-128):(97-6250), further preferably (32-128):(390-6250), and more preferably 32:1560, 64:780, or 128:390. In the present invention, when the antibacterial composition of the present invention is used, the concentration of ampicillin is preferably 16-256 μg / mL, more preferably 32-128 μg / mL; the concentration of ginseng leaf extract is preferably 0.097-6.25 mg / mL, more preferably 0.39-6.25 mg / mL.

[0024] In the present invention, when the β-lactam antibiotic is ceftiofur, the mass ratio of ceftiofur and ginseng leaf extract is preferably (4-64):(97-6250), more preferably (4-32):(97-6250), further preferably (4-32):(390-6250), more preferably 4:780, 8:780 or 16:780. In the present invention, when the antibacterial composition of the present invention is used, the concentration of ceftiofur is preferably 4-64 μg / mL, more preferably 4-32 μg / mL; the concentration of ginseng leaf extract is preferably 0.097-6.25 mg / mL, more preferably 0.39-0.78 mg / mL. The ginseng leaf extract of the present invention combined with the ceftiofur antibiotic has the advantages of synergistic and toxicity reduction. The use of ginseng leaf extract combined with ceftiofur antibiotic can inhibit methicillin-resistant Staphylococcus aureus (MRSA). The present invention provides a new research idea and development direction for solving the increasingly serious drug resistance problem of ceftiofur and Staphylococcus aureus. Specifically, the present invention uses the microbroth dilution method to study the in vitro inhibitory effect of ginseng leaf extract combined with ceftiofur on methicillin-resistant Staphylococcus aureus (MRSA). Ceftiofur, as a third-generation β-lactam antibiotic, has the advantages of a broad antibacterial spectrum and long-lasting efficacy. Restoring the sensitivity of ceftiofur to MRSA and returning it to the front line of antibiotics has important practical significance. The antibacterial composition of the present invention is expected to provide a reference for the preparation of drugs using ceftiofur to treat severe infectious diseases and improve the therapeutic effect.

[0025] The present invention also provides the use of the antibacterial composition described in the above technical solution in the preparation of an antibacterial drug. In the present invention, the antibacterial preferably refers to antibacterial. In the present invention, the antibacterial preferably refers to inhibiting or killing bacteria. In the present invention, the bacteria preferably include one or more of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Acinetobacter baumannii. In the present invention, the bacteria preferably include drug-resistant bacteria; the drug-resistant bacteria preferably include one or more of drug-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Acinetobacter baumannii. In the present invention, the drug-resistant bacteria are more preferably drug-resistant Staphylococcus aureus. In the present invention, the drug-resistant Staphylococcus aureus is preferably methicillin-resistant Staphylococcus aureus (MRSA). Ginseng leaf extract exhibited a synergistic effect when combined with β-lactam antibiotics (ceftiofur, penicillin, amoxicillin, and ampicillin), significantly enhancing each other's in vitro antibacterial activity against MRSA, surpassing the efficacy of each agent alone. The combination of ceftiofur and ginseng leaf extract exhibited the lowest FICI, demonstrating the best combined efficacy. The combination of ginseng leaf extract and ceftiofur demonstrated a strong synergistic antibacterial effect, with 0.78 mg / mL ginseng leaf extract and 4, 8, and 16 μg / mL ceftiofur completely inhibiting MRSA proliferation for up to 16 hours.

[0026] To further illustrate the present invention, the antibacterial composition containing ginseng leaf extract and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Bacteria and antibiotics

[0029] The test strain MRSAUSA300 and the quality control strain Staphylococcus aureus ATCC29213 were purchased from the China Veterinary Drug Administration.

[0030] Ceftiofur sodium, penicillin sodium, amoxicillin sodium, ampicillin sodium, pure products, powder.

[0031] 2. Chinese herbal extracts

[0032] Preparation of ginseng leaf extract: Take ginseng leaf powder, add ethanol aqueous solution containing 50% ethanol by mass according to the solid-liquid ratio of 1:10, reflux extraction, extract 3 times, each extraction for 20 minutes, filter, combine the filtrate, concentrate, and dry to obtain.

[0033] 3. Preparation of Bacteria and Drugs

[0034] 3.1 Bacterial recovery and bacterial solution preparation

[0035] Bacterial recovery: Take out the bacterial solution frozen at -80℃, dip a small amount of bacterial solution with a disposable inoculating loop, spread it on the nutrient agar plate by the three-zone plate streak method, and place it in a 37℃ incubator for 24 hours.

[0036] Preparation of bacterial solution: Pick a single colony from the nutrient agar plate with an inoculating loop and transfer it to MH broth. Cultivate at 37°C and 180 rpm with shaking until the logarithmic growth phase. Adjust the bacterial solution concentration to 2 × 10 6 CFU / mL, reserve for future use.

[0037] 3.2 Preparation of β-lactam antibiotic solution

[0038] Accurately weigh a certain amount of β-lactam antibiotic powder using an electronic analytical balance, add pure water to dissolve it, and prepare a stock solution with a concentration of 2048 μg / mL. Aliquot and store in a -20°C refrigerator for later use.

[0039] 3.3 Preparation of Chinese herbal medicine extract solution

[0040] Use an electronic analytical balance to accurately weigh 1600 mg of ginseng leaf extract into a 15 mL EP tube, add 4 mL of pure water to make a ginseng leaf extract solution with a concentration of 400 mg / mL, divide into portions, and store in a -20°C refrigerator for later use.

[0041] 4. Minimum Inhibitory Concentration (MIC) Determination

[0042] The MIC values ​​of each drug were determined by broth microdilution method according to the standard procedures of the Committee for Clinical and Laboratory Standards (CLSI). ATCC29213 was used as the quality control strain and MRS A USA 300 was used as the test strain.

[0043] Take out the stock solution of the drug, β-lactam antibiotics are 2048μg / mL, and ginseng leaf extract is 400mg / mL. First, add 100μLMH broth to each well of the 96-well pointed bottom sterile culture plate, then add 100μL of the above solution to the first well, then use a pipette to mix the solution in the first well, and draw 100μL to the second well to mix, then draw 100μL to the third well to mix, and so on, dilute to the 14th well, then draw 100μL of the solution in the 14th well and discard it, then inoculate each well with 100μL of the diluted bacterial solution, and the concentrations of the β-lactam antibiotic solutions are: 512μg / mL, 256μg / mL, 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL. L, 1μg / mL, 0.5μg / mL, 0.25μg / mL, 0.125μg / mL, 0.0625μg / mL and the concentrations of ginseng leaf extract were 100mg / mL, 50mg / mL, 25mg / mL, 12.5mg / mL, 6.25mg / mL, 3.125mg / mL, 1.5625mg / mL, 0.78125mg / mL, 0.390625mg / mL, 0.1953125mg / mL, 0.09765625mg / mL, 0.048828125mg / mL, 0.0244140625mg / mL, 0.01220703125mg / mL. Well 15 was inoculated with 100 μL of bacterial solution without adding drug solution to serve as a positive control. Well 16 was inoculated with neither drug solution nor bacterial solution to serve as a negative control. The 96-well plate was incubated at 37°C for 24 hours, and the results were recorded. The inoculated bacterial solution was divided into two groups: a quality control group and a test group, and two replicates were performed for each group for verification.

[0044] Reading the MIC results: Observe bacterial growth at the bottom of the 96-well pointed-bottom plate. If the culture medium in the wells becomes turbid or precipitates form, it indicates bacterial growth and is recorded as "+." If the culture medium is clear and transparent with no visible bacteria, it indicates no bacterial growth and is recorded as "-." The minimum inhibitory concentration (MIC) is the lowest drug concentration in the wells where no obvious bacterial growth is observed. The result is considered valid only if bacterial growth is observed in the positive control wells and the negative control wells remain clear.

[0045] The MIC results of β-lactam antibiotics and ginseng leaf extract against the control bacteria and MRSA are shown in Tables 1 to 5. The MIC results show that the MICs of ceftiofur, penicillin, amoxicillin, and ampicillin against the control bacteria were 2 μg / mL, 2 μg / mL, 2 μg / mL, and 1 μg / mL, respectively, which were within the control range. The MICs of ceftiofur, penicillin, amoxicillin, and ampicillin against the test bacteria were 64 μg / mL, 128 μg / mL, 256 μg / mL, and 256 μg / mL, respectively. The MICs of ginseng leaf extract against the control bacteria and the test bacteria were 25 mg / mL and 50 mg / mL, respectively.

[0046] Table 1 MIC test results of ceftiofur

[0047]

[0048]

[0049] Table 2 MIC test results of penicillin

[0050] serial number 1 2 3 4 5 6 7 8 Concentration (μg / mL) 512 256 128 64 32 16 8 4 Quality control bacteria - - - - - - - - MRSA - - - + + + + + serial number 9 10 11 12 13 14 15 16 Concentration (μg / mL) 2 1 0.5 0.25 0.125 0.0625 PC NC Quality control bacteria - + + + + + + - MRSA + + + + + + + -

[0051] Table 3 MIC test results of amoxicillin

[0052] serial number 1 2 3 4 5 6 7 8 Concentration (μg / mL) 512 256 128 64 32 16 8 4 Quality control bacteria - - - - - - - - MRSA - - + + + + + + serial number 9 10 11 12 13 14 15 16 Concentration (μg / mL) 2 1 0.5 0.25 0.125 0.0625 PC NC Quality control bacteria - + + + + + + - MRSA + + + + + + + -

[0053] Table 4 MIC test results of ampicillin

[0054] serial number 1 2 3 4 5 6 7 8 Concentration (μg / mL) 512 256 128 64 32 16 8 4 Quality control bacteria - - - - - - - - MRSA - - + + + + + + serial number 9 10 11 12 13 14 15 16 Concentration (μg / mL) 2 1 0.5 0.25 0.125 0.0625 PC NC Quality control bacteria - - + + + + + - MRSA + + + + + + + -

[0055] Table 5 MIC test results of ginseng leaf extract

[0056]

[0057]

[0058] 5. Combined Antibacterial Index Determination

[0059] In order to determine the antibacterial effect of ginseng leaf extract and β-lactam antibiotics against MRSA, the microdilution checkerboard method was used to determine the graded inhibitory concentration index (FICI). MRSA was used as the test strain and the MRSA solution was diluted to 2×10 6 CFU / mL for later use.

[0060] Based on the MIC results of ginseng leaf extract and β-lactam antibiotics against MRSA and previous preliminary experiments, the components were combined in a 96-well sterile microtiter plate in a 6×8 pattern. The two combined components were diluted separately, with ginseng leaf extract diluted vertically and antibiotics diluted horizontally.

[0061] 50 μL of MHB medium was added to a 96-well sterile microplate. After 50 μL of ginseng leaf extract was added to the first horizontal column, an eight-channel pipette was used to dilute the extract in a gradient manner in the vertical direction by row. After mixing, 50 μL of MHB medium was pipetted into the next column and mixed evenly. After diluting to the seventh column in this way, 50 μL of the mixture was aspirated and discarded. The concentrations of each well from top to bottom were 6.25 mg / mL, 3.125 mg / mL, 1.5625 mg / mL, 0.78125 mg / mL, 0.390625 mg / mL, 0.1953125 mg / mL, 0.09765625 mg / mL, and 0 mg / mL. Antibiotics were added to each column according to their concentration from high to low, 50 μL per well, and 50 μL of MHB broth was added to the 8th column. The concentrations of antibiotics from left to right were 1 MIC, 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, 1 / 16 MIC, and 0 μg / mL. Finally, add 100 μL of the diluted bacterial solution to each well. Incubate at 37°C for 18–24 hours, observing and recording the results. Calculate FICI using the following formula:

[0062] FICI=MIC 甲药联用 / MIC 甲药单用 +MIC 乙药联用 / MIC 乙药单用

[0063] FICI interpretation criteria: when FICI ≤ 0.5, it is a synergistic effect; when 0.5 < FICI ≤ 1, it is an additive effect; when 1 < FICI ≤ 2, it is an irrelevant effect; when FICI > 2, it is an antagonistic effect.

[0064] The antibacterial effect of ginseng leaf extract combined with β-lactam antibiotics was investigated using a microdilution checkerboard assay. The bacterial growth observed in each well of the checkerboard was tabulated, with a "-" indicating no bacterial growth and a "+" indicating bacterial growth.

[0065] The results in Table 6 show that the MIC of ginseng leaf extract when used alone is 50 mg / mL, and the MIC when used in combination is 0.78125 mg / mL;

[0066] The MIC of ceftiofur when used alone was 64 μg / mL, and the MIC of ceftiofur when used in combination was 4 μg / mL;

[0067] Therefore, FICI = 0.78125 / 50 + 4 / 64 = 0.078125 < 0.5, and it is determined that the two have a synergistic effect.

[0068] Table 6 Anti-MRSA effect of ceftiofur combined with ginseng leaf extract

[0069] concentration 64 32 16 8 4 0 (ceftiofur μg / mL) 6.25 - - - - - + 3.125 - - - - - + 1.5625 - - - - - + 0.78125 - - - - - + 0.390625 - - - - + + 0.1953125 - + + + + + 0.09765625 - + + + + + 0(Ginseng leaf mg / mL) - + + + + +

[0070] The results in Table 7 show that the MIC of ginseng leaf extract when used alone is 50 mg / mL, and the MIC when used in combination is 6.25 mg / mL;

[0071] The MIC of penicillin when used alone was 128 μg / mL, and the MIC of penicillin when used in combination was 16 μg / mL;

[0072] Therefore, FICI=6.25 / 50+16 / 128=0.25<0.5, and it is determined that the two have a synergistic effect.

[0073] Table 7 Anti-MRSA effect of penicillin combined with ginseng leaf extract

[0074] concentration 128 64 32 16 8 0 (penicillin μg / mL) 6.25 - - - - + + 3.125 - - - + + + 1.5625 - - + + + + 0.78125 - + + + + + 0.390625 - + + + + + 0.1953125 - + + + + + 0.09765625 - + + + + + 0(Ginseng leaf mg / mL) - + + + + +

[0075] The results in Table 8 show that the MIC of ginseng leaf extract when used alone is 50 mg / mL, and the MIC when used in combination is 1.5625 mg / mL;

[0076] The MIC of amoxicillin was 256 μg / mL when used alone and 32 μg / mL when used in combination;

[0077] Therefore, FICI=1.5625 / 50+32 / 256=0.15625<0.5, and it is determined that the two have a synergistic effect.

[0078] Table 8 Anti-MRSA effect of amoxicillin combined with ginseng leaf extract

[0079] concentration 256 128 64 32 16 0 (amoxicillin μg / mL) 6.25 - - - - + + 3.125 - - - - + + 1.5625 - - - - + + 0.78125 - - - + + + 0.390625 - - + + + + 0.1953125 - + + + + + 0.09765625 - + + + + + 0(Ginseng leaf mg / mL) - + + + + +

[0080] The results in Table 9 show that the MIC of ginseng leaf extract when used alone is 50 mg / mL, and the MIC when used in combination is 1.5625 mg / mL;

[0081] The MIC of ampicillin when used alone was 256 μg / mL, and the MIC of ampicillin when used in combination was 32 μg / mL;

[0082] Therefore, FICI=1.5625 / 50+32 / 256=0.15625<0.5, and it is determined that the two have a synergistic effect.

[0083] Table 9 Anti-MRSA effect of ampicillin combined with ginseng leaf extract

[0084] concentration 256 128 64 32 16 0 (ampicillin μg / mL) 6.25 - - - - + + 3.125 - - - - + + 1.5625 - - - - + + 0.78125 - - - + + + 0.390625 - - + + + + 0.1953125 - + + + + + 0.09765625 - + + + + + 0(Ginseng leaf mg / mL) - + + + + +

[0085] 6. Growth Curve Determination

[0086] The microdilution checkerboard assay results show that ceftiofur combined with ginseng leaf extract has the lowest FICI, indicating the best combined efficacy. Ceftiofur is a third-generation β-lactam antibiotic with a broad antimicrobial spectrum and long-lasting efficacy. Restoring ceftiofur's sensitivity to MRSA and reinstating it as a first-line antibiotic has important practical significance. Therefore, the present invention employed a growth curve assay to further evaluate the synergistic anti-MRSA activity of ginseng leaf extract combined with ceftiofur.

[0087] Preparation of bacterial solution: MRSA was used as the test strain and the MRSA bacterial solution was diluted to 2×10 6 CFU / mL for later use.

[0088] Drug preparation: Based on the results of the MIC test and microdilution checkerboard method, ceftiofur and ginseng leaf extract were diluted with MH broth. Ceftiofur: 4μg / mL, 8μg / mL, 16μg / mL; ginseng leaf extract: 0.39mg / mL, 0.58mg / mL, 0.78mg / mL. The experiment was divided into the following groups: control (CON) group, MRSA group, 0.39mg / mL ginseng leaf extract group, 0.58mg / mL ginseng leaf extract group, 0.78mg / mL ginseng leaf extract group, 4μg / mL ceftiofur group, 8μg / mL ceftiofur group, 16μg / mL ceftiofur group, 0.39mg / mL ginseng leaf extract + 4μg / mL ceftiofur group, 0.39mg / mL ginseng leaf extract + 8μg / mL ceftiofur group, 0.39mg / mL ginseng leaf extract +16μg / mL ceftiofur group, 0.58mg / mL ginseng leaf extract + 4μg / mL ceftiofur group, 0.58mg / mL ginseng leaf extract + 8μg / mL ceftiofur group, 0.58mg / mL ginseng leaf extract + 16μg / mL ceftiofur group, 0.78mg / mL ginseng leaf extract + 4μg / mL ceftiofur group, 0.78mg / mL ginseng leaf extract + 8μg / mL ceftiofur group, 0.78mg / mL ginseng leaf extract + 16μg / mL ceftiofur group.

[0089] Growth curve drawing: The above antimicrobial drug combination was set up in triplicate for each group, and each group was added to a sterile 96-well plate (100 μL / well). 100 μL of MRSA bacterial suspension was then added and placed in a 37°C microplate reader for incubation. Within 0 to 16 hours, the OD value of the bacterial solution was measured at a wavelength of 600 nm on the microplate reader every hour, and the obtained OD600 value was plotted against the corresponding time to form a bacterial growth curve. The results are shown in Figure 1 、 Figure 2 and Figure 3 .

[0090] Figure 1The results of the effect of 0.39 mg / mL ginseng leaf extract combined with ceftiofur on the growth of MRSA; GLE: ginseng leaf extract, CF: ceftiofur. Figure 2 The results of the effect of 0.58 mg / mL ginseng leaf extract combined with ceftiofur on the growth of MRSA; GLE: ginseng leaf extract, CF: ceftiofur. Figure 3 The results of the effect of 0.78 mg / mL ginseng leaf extract combined with ceftiofur on the growth of MRSA; GLE: ginseng leaf extract, CF: ceftiofur.

[0091] The growth curves show that both ginseng leaf extract and ceftiofur exhibited a certain degree of antibacterial activity when used alone, and this activity increased with increasing concentrations of the extract and ceftiofur. Furthermore, the combined use of ginseng leaf extract and ceftiofur demonstrated a synergistic antibacterial effect. The combination of 0.78 mg / mL ginseng leaf extract and 4, 8, and 16 μg / mL ceftiofur completely inhibited MRSA proliferation, with this activity persisting for up to 16 hours.

[0092] 7. Discussion of Results

[0093] Microdilution checkerboard assays revealed that ginseng leaf extract exhibited a synergistic effect when combined with β-lactam antibiotics (ceftiofur, penicillin, amoxicillin, and ampicillin), significantly enhancing each other's in vitro antibacterial activity against MRSA compared to either agent alone. Furthermore, the combination of ceftiofur and ginseng leaf extract exhibited the lowest FICI, indicating the best combined effect. Growth curve experiments further demonstrated that the combination of ginseng leaf extract and ceftiofur exhibited a significant synergistic antibacterial effect. The combination of 0.78 mg / mL ginseng leaf extract with 4, 8, and 16 μg / mL ceftiofur completely inhibited MRSA proliferation for up to 16 hours.

[0094] Combined with the results of microdilution checkerboard method and growth curve experiment, when ginseng leaf extract and ceftiofur were used together, the ratio relationship was within a certain range and had a good synergistic effect.

[0095] In other words, the present invention explored the optimal ratio of ginseng leaf extract and ceftiofur in combination, through extensive experiments, and obtained a good antibacterial formula. The ginseng leaf extract screened in this experiment was able to inhibit the growth of MRSA in combination with ceftiofur, which provides a direction for further research on the mechanism of action of combined Chinese and Western medicine against bacteria and also has reference significance for exploring the in vitro antibacterial effects of Chinese herbal extracts against other drug-resistant bacteria.

[0096] in conclusion:

[0097] While studying the combined use of a Chinese medicine extract and ceftiofur, the present invention unexpectedly discovered that when a ginseng leaf extract was used in combination with ceftiofur, it significantly inhibited MRSA in vitro, with the effect being superior to either drug used alone. Therefore, the combined use of ceftiofur and the ginseng leaf extract exhibited a significant synergistic effect. In the antibacterial composition of the present invention, the combined use of ginseng leaf extract and ceftiofur exhibited synergistic and toxicity-reducing advantages. This significantly reduced the relative amount of each substance in the composition, thereby minimizing potential adverse reactions and side effects, improving medication safety, and increasing patient compliance, thereby ensuring efficacy.

[0098] Comparative Example

[0099] During the screening process for Chinese herbal extracts, the antibacterial effects of various herbal extracts combined with ceftiofur were compared. The results showed that not every combination had a strong antibacterial effect or synergistic effect. In some cases, even if the Chinese herbal extract itself had antibacterial effects, when combined with ceftiofur, it did not show effective inhibitory or synergistic effects against MRSA.

[0100] Following the same experimental method as in Example 1 of the present invention, the combination of Terminalia chebula extract, Elsholtzia ciliata extract, and Eucommia ulmoides leaf extract with ceftiofur did not demonstrate a synergistic effect. The microdilution checkerboard assay results are shown in Tables 10 to 12, with FICI values ​​of 1.03125, 1.125, and 1.0625, respectively, indicating a lack of synergistic activity.

[0101] The results in Table 10 show that the MIC of Eucommia leaf extract when used alone is 0.048 mg / mL, and the MIC when used in combination is 0.0015 mg / mL;

[0102] The MIC of ceftiofur when used alone was 64 μg / mL, and the MIC of ceftiofur when used in combination was 64 μg / mL;

[0103] Therefore, FICI = 0.0015 / 0.048 + 64 / 64 = 1.03125 > 1, which is determined to be an irrelevant effect.

[0104] Table 10 Anti-MRSA effect of ceftiofur combined with Terminalia chebula extract

[0105] concentration 64 32 16 8 4 0 (ceftiofur μg / mL) 0.096 + - - - - - 0.048 + + + + + - 0.024 + + + + + + 0.012 - + + + + + 0.006 - + + + + + 0.003 - + + + + + 0.0015 - + + + + + 0 (Terminalia chebula mg / mL) - + + + + +

[0106] The results in Table 11 show that the MIC of Elsholtzia ciliata extract when used alone is 0.78125 mg / mL, and the MIC of Elsholtzia ciliata extract when used in combination is 0.09765625 mg / mL;

[0107] The MIC of ceftiofur when used alone was 64 μg / mL, and the MIC of ceftiofur when used in combination was 64 μg / mL;

[0108] Therefore, FICI = 0.09765625 / 0.78125 + 64 / 64 = 1.125 > 1, which is determined to be an irrelevant effect.

[0109] Table 11 Anti-MRSA effect of ceftiofur combined with Elsholtzia ciliata extract

[0110] concentration 64 32 16 8 4 0 (ceftiofur μg / mL) 1.5625 + + + + + - 0.78125 + + + + + - 0.390625 + + + + + + 0.1953125 + + + + + + 0.09765625 - + + + + + 0 (Elsholtzia mg / mL) - + + + + +

[0111] The results in Table 12 show that the MIC of Eucommia leaf extract when used alone is 6.25 mg / mL, and the MIC when used in combination is 6.25 mg / mL;

[0112] The MIC of ceftiofur when used alone was 64 μg / mL, and the MIC of ceftiofur when used in combination was 4 μg / mL;

[0113] Therefore, FICI=6.25 / 6.25+4 / 64=1.0625>1, which is determined to be an irrelevant effect.

[0114] Table 12 Anti-MRSA effect of ceftiofur combined with Eucommia ulmoides leaf extract

[0115] concentration 64 32 16 8 4 0 (ceftiofur μg / mL) 6.25 - - - - - - 3.125 - + + + + + 1.5625 - + + + + + 0.78125 - + + + + + 0.390625 - + + + + + 0.1953125 - + + + + + 0.09765625 - + + + + + 0(Eucommia ulmoides leaves mg / mL) - + + + + +

[0116] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An antibacterial composition containing ginseng leaf extract, characterized in that: The antibacterial composition comprises a β-lactam antibiotic and a ginseng leaf extract obtained by extracting with a 50% ethanol aqueous solution; the β-lactam antibiotic comprises one or more of ceftiofur, penicillin, amoxicillin and ampicillin.

2. The antibacterial composition according to claim 1, characterized in that The β-lactam antibiotic is ceftiofur.

3. The antibacterial composition according to claim 1, characterized in that The preparation method of the ginseng leaf alcohol extract comprises the following steps: mixing ginseng leaves with 50% ethanol aqueous solution, refluxing extraction, filtering the filtrate, concentrating, and drying to obtain the ginseng leaf extract.

4. The antibacterial composition according to claim 1, characterized in that The mass ratio of the β-lactam antibiotics to the ginseng leaf extract is (4-256): (97-6250).

5. The antibacterial composition according to claim 1, characterized in that When the β-lactam antibiotic is ceftiofur, the mass ratio of ceftiofur to ginseng leaf extract is (4-64): (97-6250).

6. Use of the antibacterial composition according to any one of claims 1 to 5 in the preparation of a drug against Staphylococcus aureus.

7. The use according to claim 6, characterized in that The Staphylococcus aureus includes drug-resistant Staphylococcus aureus.

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