A synergistic bactericidal composition based on acacia extract and antibiotics and its application

Abstract

The application discloses a synergistic bactericidal composition, comprising the following components: a toona sinensis extract: 16-2048 mu g / mL; a macrolide antibiotic: 0.0625-4096 mu g / mL; the bacteria are staphylococcus aureus or methicillin-resistant staphylococcus aureus, and the macrolide antibiotic is erythromycin or azithromycin. The application further discloses a use of the synergistic bactericidal composition in preparation of a medicine for treating skin and soft tissue infections. The composition provided by the application can convert the traditional macrolide antibiotics such as erythromycin and azithromycin which only have a bacteriostatic effect into a preparation which has clear bactericidal activity on staphylococcus aureus and drug-resistant strains thereof. After the toona sinensis extract is combined with the antibiotic, a synergistic effect is exhibited, and the concentration of the toona sinensis extract and the antibiotic required when the toona sinensis extract and the antibiotic are used alone can be greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine application technology, specifically relating to a synergistic bactericidal composition based on Albizia bark extract and antibiotics and its application. Background Technology

[0002] Traditional Chinese medicine (TCM) is a natural drug system with multiple components and targets, showing potential in the field of antibacterial infection control. Studies have shown that some TCMs not only inhibit susceptible bacteria but also exhibit activity against drug-resistant bacteria, and can produce synergistic antibacterial effects with certain antibiotics (see CN111904995A), even helping to reverse bacterial resistance. Against this backdrop, Staphylococcus aureus (SA) and its drug-resistant strain, methicillin-resistant Staphylococcus aureus (MRSA), have become common pathogens in clinical practice, particularly prone to causing skin and soft tissue infections, posing a persistent threat to public health. Currently, macrolide antibiotics such as erythromycin and azithromycin are still used to treat SA infections, but their mechanism of action is mainly to inhibit bacterial protein synthesis, and they generally do not have the ability to directly kill bacteria. Therefore, their efficacy in treating SA and MRSA infections is limited, easily leading to prolonged illness.

[0003] Albizia bark, a traditional Chinese medicine, is known to possess various pharmacological effects, including relieving depression and calming the mind, anti-tumor activity, and anti-allergy properties. In antibacterial research, existing reports mainly focus on the inhibitory activity of Albizia leaf extract against plant pathogenic fungi, such as its inhibitory effect on *Albizia julibrissin* and *Botrytis cinerea* at higher concentrations (5 mg / ml). However, research on the inhibitory effect of Albizia bark on bacteria is relatively limited, and its antibacterial activity is generally reported to be weak. A recent study indicated that the ethanol extract of Albizia bark has antibacterial activity against *Staphylococcus aureus* and *Aspergillus niger*, and further research found that its n-butanol extract has even stronger antibacterial activity (see "Study on the Antibacterial Activity of Albizia Bark Extract against *Aspergillus niger*", Bai Xianqin et al., 2014). Nevertheless, there are currently no reports of Albizia bark having bactericidal activity against SA or MRSA, nor are there any studies on its synergistic antibacterial effects when used in combination with macrolide antibiotics such as erythromycin and azithromycin.

[0004] In summary, current research on the antibacterial activity of Albizia bark is still focused on the antibacterial aspect, and the activity is limited. There is a lack of systematic investigation into its bactericidal effect and synergistic effect with commonly used antibiotics. 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 Albizia bark extract and commonly used antibiotics work synergistically to 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] Albizia bark extract: 16 µg / mL~2048 µg / mL;

[0009] Macrolide antibiotics: 0.0625 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 Albizia bark extract and erythromycin in the synergistic bactericidal composition are respectively:

[0012] Albizia bark extract: 16 µg / mL~2048 µg / mL;

[0013] Erythromycin: 0.0625 µg / mL~1 µg / mL.

[0014] Furthermore, the concentration of the Albizia bark extract is 16 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 Albizia bark extract and azithromycin in the synergistic bactericidal composition are respectively:

[0016] Albizia bark extract: 16 µg / mL~2048 µg / mL;

[0017] Azithromycin: 0.5 µg / mL~8 µg / mL.

[0018] Furthermore, the concentration of the Albizia bark extract is 16 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 Albizia bark extract and erythromycin in the synergistic bactericidal composition are respectively:

[0020] Albizia bark extract: 32 µg / mL~2048 µg / mL;

[0021] Erythromycin: 256 µg / mL~2048 µg / mL.

[0022] Furthermore, the concentration of the Albizia bark extract is 32 µg / mL, and the concentration of the erythromycin is 512 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 Albizia bark extract and azithromycin in the synergistic bactericidal composition are respectively:

[0024] Albizia bark extract: 32 µg / mL~2048 µg / mL;

[0025] Azithromycin: 1024 µg / mL~4096 µg / mL.

[0026] Furthermore, the concentration of the Albizia bark extract is 32 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 skin and soft tissue infections.

[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, into preparations that have clear bactericidal activity against Staphylococcus aureus and its drug-resistant strains, thus achieving a pharmacological leap from "inhibiting bacterial growth" to "completely killing bacteria".

[0030] 2. When Albizia bark extract is used in combination with the above-mentioned antibiotics, it exhibits a strong synergistic effect, which can significantly reduce the concentration required for Albizia bark extract and antibiotics to be effective on their own. This provides a new solution for reducing antibiotic dosage, reducing potential toxic side effects, and addressing bacterial resistance issues while improving antibacterial efficacy. 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 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 Albizia bark extract and macrolide antibiotic solution

[0038] Weigh 5 g of Albizia bark (purchased from Bozhou Pharmaceutical Market, Anhui Province) into an Erlenmeyer flask, add 100 mL of 90% ethanol, and extract with ultrasonic power of 250 W for 2 h. The extract is concentrated and dried by rotary evaporator to obtain the extract. Take 8.192 mg of the extract, dissolve it in 1 mL of methanol, filter it through a 0.45 μm microporous membrane for sterilization, and then dilute it with sterile LB medium to prepare Albizia bark preparation solution of the corresponding concentration.

[0039] Weigh out 8.192 mg of erythromycin or 8.192 mg of azithromycin standard (both purchased from Shanghai Adamas Reagent Co., Ltd., purity ≥98%), dissolve in 1 mL of ethanol, and perform serial dilution with LB medium to prepare antibiotic solutions of the required concentration.

[0040] 3. Minimum Inhibitory Concentration (MIC) Test

[0041] According to the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI), the MIC value of the drug against SA or MRSA bacteria was determined using a 96-well flat-bottom microtiter plate via serial dilution. Under aseptic conditions, 100 μL of broth culture medium was added to each well. 100 μL of Albizia bark preparation 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.

[0042] Adjust the concentration of the cultured SA or MRSA bacterial suspension to 5.0 x 10⁻⁶ using sterile LB medium. 5Add 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.

[0043] 4. Minimum Bactericidal Concentration (MBC) Test

[0044] 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.

[0045] 5. Evaluation criteria for bactericidal activity

[0046] 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.)

[0047] 6. Combined antibacterial experiment

[0048] The combined antibacterial effect of Albizia julibrissin bark extract with erythromycin or azithromycin was evaluated using the checkerboard microdilution method recommended by CLSI. Erythromycin or azithromycin and Albizia julibrissin bark extract were diluted 2-fold with LB broth medium and added to each well to achieve a maximum final concentration of 2xMIC for the diluted erythromycin or azithromycin.

[0049] Select different concentrations, adding erythromycin or azithromycin dilution to each row and Albizia bark dilution (maximum concentration 2048 μg / mL) to each column, ensuring 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 m² using sterile LB medium. 5Add 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 hours, 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 hours. The lowest concentration of erythromycin or azithromycin with no colony growth on the plate is defined as the MBC (mixed drug concentration) for this antibiotic combination therapy. An MBC (combined) / MIC (single drug) ≤ 4 is defined as bactericidal activity, while a value greater than 4 indicates bacteriostatic activity.

[0050] Example 1: MIC of the drug against SA and MRSA bacteria

[0051] In this embodiment, different concentrations of erythromycin, azithromycin, and Albizia bark extract were prepared, and their inhibitory activities on SA and MRSA were tested. The results are shown in Table 1.

[0052] Table 1. Antibacterial activity of different drugs against SA and MRSA

[0053]

[0054] Note: - indicates no bacterial growth, + indicates bacterial growth; concentration units in Table 1 are μg / mL. The same applies below.

[0055] As shown in Table 1, the minimum inhibitory concentrations (MICs) of erythromycin, azithromycin, and Albizia julibrissin bark extract against SA bacteria were 0.5 μg / mL, 4 μg / mL, and 1024 μg / mL, respectively, and the MICs against MRSA bacteria were 1024 μg / mL, 2048 μg / mL, and 2048 μg / mL, respectively.

[0056] Example 2: MBC of the drug against SA and MRSA bacteria

[0057] In this embodiment, the MBC of erythromycin, azithromycin, and Albizia julibrissin bark extract against SA and MRSA bacteria were tested at MIC, 2×MIC, 4×MIC, and 8×MIC concentrations. The results are shown in Table 2.

[0058] Table 2. MBC of different drugs on SA and MRSA

[0059]

[0060] Note: 1x, 2x, 4x, and 8x represent 1, 2, 4, and 8 times the MIC concentration, respectively. The same applies below.

[0061] 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.

[0062] 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.

[0063] At a concentration of 4 × MIC (4096 μg / mL), *Albizia julibrissin* bark extract showed no growth of *S. aurea* (SA), indicating a mean molecular weight limit (MBC) of 4096 μg / mL and an MBC / MIC ratio of 4, demonstrating bactericidal activity against SA. Similarly, at a concentration of 2 × MIC (4096 μg / mL), *Albizia julibrissin* bark extract showed no growth of *MRSA*, indicating a mean molecular weight limit (MBC) of 4096 μg / mL and an MBC / MIC ratio of 2, demonstrating bactericidal activity against MRSA.

[0064] Example 3: Bactericidal activity of Albizia bark extract combined with erythromycin or azithromycin against SA bacteria.

[0065] In this embodiment, different concentrations of Albizia bark extract were combined with erythromycin or azithromycin to investigate their bactericidal activity against SA bacteria.

[0066] Table 3. Bactericidal activity of Albizia bark combined with erythromycin against SA

[0067]

[0068] The concentrations of Albizia bark and erythromycin in Table 3 are in μg / mL.

[0069] Table 4. Bactericidal activity of Albizia bark combined with azithromycin against SA

[0070]

[0071] The concentrations of Albizia bark and azithromycin in Table 4 are in μg / mL.

[0072] The results are shown in Tables 3 and 4. When combined with 16 μg / mL of Albizia julibrissin bark extract (only 1 / 256 of the MBC of Albizia julibrissin bark extract alone), erythromycin at a concentration of 0.125 μg / mL was effective in killing SA bacteria (MBC / MIC ≤ 4); when combined with 16 μg / mL of Albizia julibrissin bark extract (only 1 / 256 of the MBC of Albizia julibrissin bark extract alone), azithromycin at a concentration of 1 μg / mL was effective in killing SA bacteria (MBC / MIC ≤ 4). These results indicate that low concentrations (1 / 256 MBC) of Albizia julibrissin bark extract can convert erythromycin or azithromycin, which do not have SA-killing activity, into bactericidal active forms at concentrations lower than their corresponding antibacterial MICs.

[0073] Example 4: Synergistic bactericidal activity of Albizia bark extract combined with erythromycin or azithromycin against MRSA.

[0074] In this embodiment, different concentrations of Albizia julibrissin bark extract were combined with erythromycin or azithromycin to investigate its bactericidal activity against MRSA.

[0075] Table 5. Antibacterial activity of Albizia bark extract combined with erythromycin against MRSA

[0076] The concentrations of Albizia bark and erythromycin in Table 5 are in μg / mL.

[0077] Table 6. Antibacterial activity of Albizia bark extract combined with azithromycin against MRSA

[0078]

[0079] The concentrations of Albizia bark and erythromycin in Table 6 are in μg / mL.

[0080] The results are shown in Tables 5 and 6. When combined with 32 μg / mL of Albizia julibrissin bark extract (1 / 128 of its MBC value against MRSA alone), erythromycin at a concentration of 512 μg / mL was effective in killing MRSA (MCB / MIC ≤ 4); and when combined with 32 μg / mL of Albizia julibrissin bark extract, azithromycin at a concentration of 2048 μg / mL was effective in killing MRSA (MCB / MIC ≤ 4). These results indicate that low concentrations (1 / 128 MBC) of Albizia julibrissin bark can transform erythromycin or azithromycin, which previously lacked MRSA-killing activity, into fungicidal agents.

[0081] In summary, this invention demonstrates through systematic in vitro antibacterial experiments that high concentrations of Albizia julibrissin bark extract not only possess certain bactericidal activity against Staphylococcus aureus (SA) and MRSA, but more importantly, when used in combination with macrolide antibiotics such as erythromycin and azithromycin, its bactericidal concentration is significantly reduced, to 1 / 256 to 1 / 128 times that when used alone. Simultaneously, it can significantly enhance the antibacterial activity of macrolide antibiotics such as erythromycin and azithromycin, transforming them from traditional "bacteriostatic" to clearly "bactericidal," and drastically reducing their effective bactericidal concentration. This synergistic bactericidal effect is significant against both sensitive and drug-resistant bacteria, providing a practical experimental basis for developing anti-infective agents based on the combined application of traditional Chinese medicine and antibiotics.

[0082] 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 containing Albizia bark extract and macrolide antibiotics, characterized in that, The bacteria is Staphylococcus aureus, the macrolide antibiotic is erythromycin, and the concentrations of Albizia bark extract and erythromycin in the synergistic bactericidal composition are respectively: Albizia bark extract: 16 µg / mL~2048 µg / mL; Erythromycin: 0.125 µg / mL~1 µg / mL.

2. The synergistic bactericidal composition according to claim 1, characterized in that, The concentration of the Albizia bark extract is 16 µg / mL, and the concentration of the erythromycin is 0.125 µg / mL.

3. A synergistic bactericidal composition containing Albizia bark extract and macrolide antibiotics, characterized in that, The bacteria is Staphylococcus aureus, the macrolide antibiotic is azithromycin, and the concentrations of Albizia bark extract and azithromycin in the synergistic bactericidal composition are respectively: Albizia bark extract: 16 µg / mL~2048 µg / mL; Azithromycin: 1 µg / mL~8 µg / mL.

4. The synergistic bactericidal composition according to claim 3, characterized in that, The concentration of the Albizia bark extract is 16 µg / mL, and the concentration of the azithromycin is 1 µg / mL.

5. A synergistic bactericidal composition containing Albizia bark extract and macrolide antibiotics, characterized in that, The bacteria are methicillin-resistant Staphylococcus aureus, the macrolide antibiotic is erythromycin, and the concentrations of Albizia bark extract and erythromycin in the synergistic bactericidal composition are respectively: Albizia bark extract: 32 µg / mL~2048 µg / mL; Erythromycin: 512 µg / mL~2048 µg / mL.

6. The synergistic bactericidal composition according to claim 5, characterized in that, The concentration of the Albizia bark extract is 32 µg / mL, and the concentration of the erythromycin is 512 µg / mL.

7. A synergistic bactericidal composition containing Albizia bark extract and macrolide antibiotics, characterized in that, The bacteria are methicillin-resistant Staphylococcus aureus, the macrolide antibiotic is azithromycin, and the concentrations of Albizia bark extract and azithromycin in the synergistic bactericidal composition are respectively: Albizia bark extract: 32 µg / mL~2048 µg / mL; Azithromycin: 2048 µg / mL~4096 µg / mL.

8. The synergistic bactericidal composition according to claim 7, characterized in that, The concentration of the Albizia bark extract is 32 µg / mL, and the concentration of the azithromycin is 2048 µg / mL.

9. Use of the synergistic bactericidal composition according to any one of claims 1 to 8 in the preparation of a medicament for treating skin and soft tissue infections.

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