Sterilization composition containing poplatin and macrolide antibiotics and application of sterilization composition

Abstract

The invention discloses a synergistic bactericidal composition, which comprises the following components: 256g / mL to 8192 g / mL of poplar bud flavin; the content of the macrolide antibiotics is 0.125 g / mL to 4096 g / mL; the bacteria are staphylococcus aureus or methicillin-resistant staphylococcus aureus, and the macrolide antibiotics are erythromycin or azithromycin. The invention also discloses an application of the synergistic bactericidal composition in preparation of a medicine for treating infection caused by staphylococcus aureus or methicillin-resistant staphylococcus aureus. According to the composition provided by the invention, macrolide antibiotics, such as erythromycin and azithromycin, which only have a bacteriostatic effect on staphylococcus aureus and drug-resistant strains thereof traditionally can be converted into preparations with definite bactericidal activity. After the poplatin and the antibiotics are combined for use, a remarkable synergistic effect is shown, and the concentration of the antibiotics required for realizing the sterilization effect can be greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine application technology, specifically relating to a bactericidal composition of salicornin and macrolide antibiotics and its application. Background Technology

[0002] In recent years, bacterial resistance has become an increasingly serious problem, especially multidrug-resistant bacteria represented by methicillin-resistant Staphylococcus aureus (MRSA), which exhibit varying degrees of resistance to many commonly used antibiotics (such as β-lactams and macrolides), posing a major challenge to global public health. Macrolide antibiotics (such as erythromycin and azithromycin) were once widely used to treat Gram-positive bacterial infections due to their broad antibacterial spectrum and strong tissue penetration. However, with the long-term widespread use of these drugs, the resistance rate of bacteria, especially Staphylococcus aureus (SA) and its resistant strains MRSA, has increased year by year, leading to a significant decline in the efficacy of monotherapy. In some cases, these drugs only exhibit bacteriostatic rather than bactericidal effects, increasing the difficulty of treatment and the risk of recurrence.

[0003] To combat bacterial resistance, combination therapy has become an important clinical strategy. By combining drugs with different mechanisms of action, it is hoped that individual drug dosages can be reduced, side effects minimized, resistance development delayed, and the susceptibility of resistant bacteria to existing drugs may be restored. Therefore, the search for natural or synthetic compounds that can enhance the antibacterial activity of existing antibiotics, especially those with synergistic or sensitizing effects against resistant bacteria, has significant scientific research value and application prospects.

[0004] Salicin is a naturally derived flavonoid compound that has been reported to possess various biological activities, including antitumor, antioxidant, anti-inflammatory, and antibacterial effects. However, current research on the antibacterial activity of salicin largely focuses on its effects when used alone, and its inhibitory concentration against common pathogens (such as SA and MRSA) is relatively high, indicating limited antibacterial efficacy. Currently, there is a lack of systematic research on the synergistic antibacterial effects of salicin in combination with traditional antibiotics (especially macrolides). Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition in which salicornin and macrolide antibiotics synergistically kill bacteria.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a synergistic bactericidal composition comprising the following components:

[0008] Saussurein: 256 µg / mL~8192 µg / mL;

[0009] Macrolide antibiotics: 0.125 µg / mL~4096 µg / mL;

[0010] The bacteria are Staphylococcus aureus or methicillin-resistant Staphylococcus aureus, and the macrolide antibiotic is erythromycin or azithromycin.

[0011] According to a preferred embodiment of the present invention, the bacterium is Staphylococcus aureus, the macrolide antibiotic is erythromycin, and the concentrations of salinomycin and erythromycin in the synergistic bactericidal composition are respectively:

[0012] Saussurein: 256 µg / mL~8192 µg / mL;

[0013] Erythromycin: 0.125 µg / mL~1 µg / mL.

[0014] Preferably, the concentration of the flavonoid is 2048 µg / mL, and the concentration of the erythromycin is 0.125 µg / mL.

[0015] According to a preferred embodiment of the present invention, the bacterium is Staphylococcus aureus, the macrolide antibiotic is azithromycin, and the concentrations of salinomycin and azithromycin in the synergistic bactericidal composition are respectively:

[0016] Saussurein: 256 µg / mL~8192 µg / mL;

[0017] Azithromycin: 1 µg / mL~8 µg / mL.

[0018] Preferably, the concentration of the flavonoid is 4096 µg / mL, and the concentration of the azithromycin is 1 µg / mL.

[0019] According to a preferred embodiment of the present invention, the bacteria is methicillin-resistant Staphylococcus aureus, the macrolide antibiotic is erythromycin, and the concentrations of salinomycin and erythromycin in the synergistic bactericidal composition are respectively:

[0020] Saussureanin extract: 256 µg / mL~8192 µg / mL;

[0021] Erythromycin: 1024 µg / mL~2048 µg / mL.

[0022] Preferably, the concentration of the flavonoid is 4096 µg / mL and the concentration of the erythromycin is 1024 µg / mL.

[0023] According to a preferred embodiment of the present invention, the bacteria is methicillin-resistant Staphylococcus aureus, the macrolide antibiotic is azithromycin, and the concentrations of salinomycin and azithromycin in the synergistic bactericidal composition are respectively:

[0024] Saussurein: 256 µg / mL~8192 µg / mL;

[0025] Azithromycin: 2048 µg / mL~4096 µg / mL.

[0026] Preferably, the concentration of the flavonoid is 4096 µg / mL and the concentration of the azithromycin is 2048 µg / mL.

[0027] A second aspect of the invention provides the use of the synergistic bactericidal composition described above in the preparation of a medicament for treating infections caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. The composition provided by the present invention can transform macrolide antibiotics such as erythromycin and azithromycin, which traditionally only have antibacterial effects against Staphylococcus aureus and its drug-resistant strains, into preparations with clear bactericidal activity, thereby achieving an improvement in pharmacological action from "inhibiting bacterial growth" to "completely killing bacteria".

[0030] 2. When used in combination with the above-mentioned antibiotics, salinomycin exhibits a significant synergistic effect, which can greatly reduce the antibiotic concentration required to achieve bactericidal effect. Thus, while improving the antibacterial efficacy against sensitive and drug-resistant bacteria, it provides a new feasible solution for reducing antibiotic dosage, reducing potential toxic side effects, and addressing the problem of bacterial resistance. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] The following lists some conventional methods used in the embodiments.

[0035] 1. Bacterial culture

[0036] Take a glycerol tube containing Staphylococcus aureus (SA, ATCC6538) or methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) and inoculate it into LB liquid medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, pH 7.0, diluted with water to 1 L) and incubate overnight at 37°C.

[0037] 2. Preparation of solutions containing salicornin and macrolide antibiotics

[0038] Weigh 131.072 mg of salicylic acid standard (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥98%), dissolve in 10 mL of ethanol, and prepare a stock solution. Weigh 32.768 mg of erythromycin standard (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥98%), dissolve in 10 mL of ethanol, and prepare a stock solution. Weigh 65.536 mg of azithromycin standard (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥98%), dissolve in 10 mL of ethanol, and prepare a stock solution. According to experimental needs, perform serial dilutions using LB medium to prepare drug solutions of the required concentrations.

[0039] 3. Minimum Inhibitory Concentration (MIC) Test

[0040] Following the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI), a 96-well flat-bottom microtiter plate was used to determine the MIC value of the drug against SA or MRSA bacteria using serial dilution techniques. Under aseptic conditions, 100 μL of LB broth was added to each well. 100 μL of aspergillin solution, erythromycin solution, or azithromycin solution was added to column 1. Using the iso-dilution method, 100 μL from the previous column was added to the next column, and this process was repeated until column 10. 100 μL from column 10 was discarded. Columns 11 and 12 were left untreated as controls. Column 11, with bacteria added but no drug added, served as a negative control, and column 12, without bacteria or drug added, served as a sterile control.

[0041] Adjust the concentration of the cultured SA or MRSA bacterial suspension to 5.0 x 10⁻⁶ using sterile LB medium. 5 Add 100 μL of CFU / mL to columns 1-11; incubate the plate at 37 ℃ for 18-24 h and observe the results visually. The minimum drug concentration corresponding to the well with no visible colony growth (i.e., no turbidity) is taken as the MIC value of the drug.

[0042] 4. Minimum Bactericidal Concentration (MBC) Test

[0043] Take 10 μL of bacterial suspension from the wells measured at MIC, 2xMIC, 4xMIC, and 8xMIC respectively, spread it evenly on LB agar plates, and incubate at 37 ℃ for 18-24 h. The lowest drug concentration at which no colonies grow on the plate is recorded as the MBC value.

[0044] 5. Evaluation criteria for bactericidal activity

[0045] Drug activity was evaluated according to the commonly used standard in the literature (MBC / MIC ratio): a ratio of MBC to MIC ≤ 4 indicates that the drug has bactericidal activity; an MBC / MIC > 4 indicates that the drug only has bacteriostatic activity. (Refer to "Understanding Bactericidal and Bacteriostatic Antibiotics", Zhang Liuli, Xu Yatong, Chen Daijie, World Clinical Drugs, 2009, 30(6): 365-369.)

[0046] 6. Combined antibacterial experiment

[0047] The combined antibacterial effect of salicin in combination with erythromycin or azithromycin was evaluated using the checkerboard microdilution method recommended by CLSI. Erythromycin or azithromycin and salicin were diluted 2-fold in LB broth and added to each well to achieve a maximum final concentration of 2xMIC for the diluted erythromycin or azithromycin.

[0048] Add a different concentration of erythromycin or azithromycin dilution to each row, and add a salicin dilution (maximum concentration 2048 μg / mL) to each column, ensuring that each well contains a unique combination of drug concentrations. Adjust the concentration of the cultured SA or MRSA bacterial suspension to 5 x 10⁻⁶ g / mL using sterile LB medium. 5 Add 100 μL of bacterial culture to each well (CFU / mL), using a bacterial culture without the drug as the positive control and a bacterial culture without bacteria as the negative control. Incubate the plate at 37 ℃ for approximately 20 h, then visually observe the results. The minimum concentration corresponding to the well with no visible bacterial growth (i.e., no turbidity) is defined as the MIC. Take 10 μL from each turbid well and spread it onto an LB agar plate. Incubate at 37 ℃ for approximately 20 h. The lowest concentration of erythromycin or azithromycin with no colony growth on the plate is defined as the MBC when the antibiotics are used in combination. An MBC (combination) / MIC (single drug) ≤ 4 is defined as bactericidal activity, while a value greater than 4 indicates bacteriostatic activity.

[0049] Example 1: MIC of the drug against SA and MRSA bacteria

[0050] In this embodiment, different concentrations of erythromycin, azithromycin, and succinate were prepared, and their inhibitory activities on SA and MRSA were tested. The results are shown in Table 1.

[0051] Table 1. Antibacterial activity of different drugs against SA and MRSA

[0052]

[0053] Note: - indicates no bacterial growth, + indicates bacterial growth; concentration units in Table 1 are μg / mL. The same applies below.

[0054] As shown in Table 1, the minimum inhibitory concentrations (MICs) of erythromycin, azithromycin, and succinate against SA bacteria were 0.5 μg / mL, 4 μg / mL, and 2048 μg / mL, respectively, and the MICs against MRSA bacteria were 1024 μg / mL, 2048 μg / mL, and 4096 μg / mL, respectively.

[0055] Example 2: MBC of the drug against SA and MRSA bacteria

[0056] In this embodiment, the MBC of erythromycin, azithromycin, and succinate against SA and MRSA bacteria were tested at MIC, 2×MIC, 4×MIC, and 8×MIC concentrations. The results are shown in Table 2.

[0057] Table 2. MBC of different drugs on SA and MRSA

[0058]

[0059] Note: 1x, 2x, 4x, and 8x represent 1, 2, 4, and 8 times the MIC concentration, respectively, and the same applies below.

[0060] As shown in Table 2, at a concentration of 8 × MIC (4 μg / mL), erythromycin still caused SA bacteria to grow, i.e., MBC > 4 μg / mL, and its MBC / MIC ratio > 4, indicating that erythromycin has antibacterial activity against SA bacteria, rather than bactericidal activity. Similarly, at a concentration of 8 × MIC (8096 μg / mL), erythromycin still caused MRSA bacteria to grow, i.e., MBC > 8096 μg / mL, and its MBC / MIC ratio > 4, indicating that erythromycin has antibacterial activity against MRSA bacteria, rather than bactericidal activity.

[0061] At a concentration of 8 × MIC (32 μg / mL), azithromycin still caused SA bacteria to grow, indicating that MBC > 32 μg / mL and its MBC / MIC ratio > 4, suggesting that azithromycin exhibits antibacterial activity rather than bactericidal activity against SA bacteria. Similarly, at a concentration of 8 × MIC (16192 μg / mL), azithromycin still caused MRSA bacteria to grow, indicating that MBC > 16192 μg / mL and its MBC / MIC ratio > 4, suggesting that azithromycin exhibits antibacterial activity rather than bactericidal activity against MRSA bacteria.

[0062] At a concentration of 8 × MIC (16384 μg / mL), flavonoids inhibited the growth of SA bacteria (i.e., the mean squared mass index (MBC) was 16384 μg / mL), but the MBC / MIC ratio was greater than 4, indicating that flavonoids exhibited antibacterial activity rather than bactericidal activity against SA bacteria. At a concentration of 8 × MIC (32768 μg / mL), flavonoids still inhibited the growth of MRSA bacteria (i.e., the MBC was greater than 32768 μg / mL), and the MBC / MIC ratio was greater than 4, indicating that flavonoids exhibited antibacterial activity rather than bactericidal activity against MRSA bacteria.

[0063] Example 3: Bactericidal activity of salinomycin combined with erythromycin or azithromycin against SA bacteria.

[0064] In this embodiment, different concentrations of salinomycin were combined with erythromycin or azithromycin to investigate its bactericidal activity against SA bacteria.

[0065] Table 3. Bactericidal activity of salinomycin combined with erythromycin against SA

[0066]

[0067] The concentration units in Table 3 are μg / mL.

[0068] Table 4. Bactericidal activity of salinomycin combined with azithromycin against SA

[0069]

[0070] The concentration units in Table 4 are μg / mL.

[0071] The results are shown in Tables 3 and 4. When combined with 256 μg / mL of salinomycin (only 1 / 64 of the MBC of salinomycin alone), erythromycin at a concentration of 0.5 μg / mL was effective in killing SA bacteria (erythromycin MBC / MIC ≤ 4); when combined with 256 μg / mL of salinomycin, azithromycin at a concentration of 4 μg / mL was effective in killing SA bacteria (azithromycin MBC / MIC ≤ 4). These results indicate that low concentrations (1 / 64 MBC) of salinomycin can transform erythromycin or azithromycin, which previously lacked SA-killing activity, into bacteria with bactericidal activity.

[0072] Example 4: Synergistic bactericidal activity of salinomycin combined with erythromycin or azithromycin against MRSA.

[0073] In this embodiment, different concentrations of salinomycin were combined with erythromycin or azithromycin to investigate its bactericidal activity against MRSA.

[0074] Table 5. Bactericidal activity of salinomycin combined with erythromycin against MRSA

[0075]

[0076] Table 6. Bactericidal activity of salinomycin combined with azithromycin against MRSA

[0077]

[0078] The concentration units in Tables 5 and 6 are μg / mL.

[0079] The results are shown in Tables 5 and 6. When combined with 256 μg / mL of salinomycin, erythromycin at a concentration of 2048 μg / mL was effective in killing MRSA (erythromycin MBC / MIC ≤ 4); similarly, when combined with 256 μg / mL of salinomycin, azithromycin at a concentration of 4096 μg / mL was effective in killing MRSA (azithromycin MBC / MIC ≤ 4). These results indicate that low concentrations of salinomycin can transform erythromycin or azithromycin, which previously lacked MRSA-killing activity, into fungicidal agents.

[0080] In summary, this invention systematically evaluated the in vitro antibacterial effects of salicin combined with erythromycin or azithromycin against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) using the microbroth dilution method and the checkerboard microdilution method. The results showed that in the combined system, low concentrations of salicin could transform the antibacterial activity of erythromycin and azithromycin against the above-mentioned strains from bacteriostatic to bactericidal, with the evaluation criterion being a ratio of the combined MBC (micro-cell cross-section) of the antibiotics to their single MIC ≤ 4. For multidrug-resistant MRSA strains, this combined strategy can significantly reduce the concentration required for macrolide antibiotics to achieve bactericidal effects. Those skilled in the art will readily understand that, based on the above synergistic bactericidal effect, compositions containing salicin and macrolide antibiotics can be used to prepare bactericidal drugs for treating infections caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0081] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A synergistic bactericidal composition, characterized in that, Includes the following components: Saussurein: 256 µg / mL~8192 µg / mL; Macrolide antibiotics: 0.125 µg / mL~4096 µg / mL; The bacteria are Staphylococcus aureus or methicillin-resistant Staphylococcus aureus, and the macrolide antibiotic is erythromycin or azithromycin.

2. The synergistic bactericidal composition according to claim 1, characterized in that, The bacteria is Staphylococcus aureus, the macrolide antibiotic is erythromycin, and the concentrations of salinomycin and erythromycin in the synergistic bactericidal composition are respectively: Saussurein: 256 µg / mL~8192 µg / mL; Erythromycin: 0.125 µg / mL~1 µg / mL.

3. The synergistic bactericidal composition according to claim 2, characterized in that, The concentration of the flavonoid is 2048 µg / mL, and the concentration of the erythromycin is 0.125 µg / mL.

4. The synergistic bactericidal composition according to claim 1, characterized in that, The bacteria is Staphylococcus aureus, the macrolide antibiotic is azithromycin, and the concentrations of salinomycin and azithromycin in the synergistic bactericidal composition are respectively: Saussurein: 256 µg / mL~8192 µg / mL; Azithromycin: 1 µg / mL~8 µg / mL.

5. The synergistic bactericidal composition according to claim 4, characterized in that, The concentration of the flavonoid is 4096 µg / mL, and the concentration of the azithromycin is 1 µg / mL.

6. The synergistic bactericidal composition according to claim 1, characterized in that, The bacteria are methicillin-resistant Staphylococcus aureus, the macrolide antibiotic is erythromycin, and the concentrations of salinomycin and erythromycin in the synergistic bactericidal composition are respectively: Saussureanin extract: 256 µg / mL~8192 µg / mL; Erythromycin: 1024 µg / mL~2048 µg / mL.

7. The synergistic bactericidal composition according to claim 6, characterized in that, The concentration of the flavonoid is 4096 µg / mL, and the concentration of the erythromycin is 1024 µg / mL.

8. The synergistic bactericidal composition according to claim 1, characterized in that, The bacteria are methicillin-resistant Staphylococcus aureus, the macrolide antibiotic is azithromycin, and the concentrations of salinomycin and azithromycin in the synergistic bactericidal composition are respectively: Saussurein: 256 µg / mL~8192 µg / mL; Azithromycin: 2048 µg / mL~4096 µg / mL.

9. The synergistic bactericidal composition according to claim 8, characterized in that, The concentration of the salicin was 4096 µg / mL, and the concentration of the azithromycin was 2048 µg / mL.

10. Use of the synergistic bactericidal composition according to any one of claims 1 to 9 in the preparation of a medicament for treating infections caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

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