Application of mulberroside D in synergistic polymyxin antibacterial activity

Abstract

The application discloses application of mulberroside D in synergistic polymyxin antibacterial activity, and belongs to the technical field of medicines, and particularly relates to application of mulberroside D in synergistic polymyxin antibacterial activity. The application discloses application of mulberroside D in preparation of a product for enhancing antibacterial activity and / or bactericidal activity of an antibacterial drug. The mulberroside D can effectively synergize in-vivo and in-vitro antibacterial activity of polymyxin on multi-drug resistant bacteria, can produce synergistic antibacterial effect, and provides a new treatment strategy for clinical treatment of bacterial infectious diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of Sanggenon D in synergistic polymyxin antibacterial activity. BACKGROUND

[0002] In recent years, the generation, spread and rapid spread of multi-drug resistant bacteria, especially multi-drug resistant gram-negative bacteria, have brought great threat to livestock breeding and human health. Clinically common gram-negative pathogenic bacteria include Escherichia coli, Klebsiella pneumoniae, etc. Among them, Escherichia coli belongs to the bacterial kingdom, Proteobacteria, Gammaproteobacteria, Enterobacteriales, and Enterobacteriaceae.

[0003] Polymyxin is a group of antibiotics obtained from the culture solution of Paenibacillus polymyxa in 1947, containing polymyxin A, B, C, D, E and other components, and only polymyxin B and polymyxin E are used in clinic, and polymyxin E is also called colistin. Polymyxin is a narrow-spectrum antibiotic, which has strong antibacterial activity against some gram-negative bacilli, such as Escherichia coli, Enterobacter, Klebsiella and Pseudomonas aeruginosa, which are highly sensitive, especially Pseudomonas aeruginosa. Polymyxin B and polymyxin E have similar antibacterial spectrum and antibacterial effect in clinic. Polymyxin is considered to be the last line of treatment for clinically treating multi-drug resistant gram-negative bacterial infections. Due to the emergence of polymyxin resistance gene (MCR), the use of polymyxin in clinic has brought great challenges. The recommended clinical dose of polymyxin often cannot achieve effective therapeutic effect, and is easy to cause obvious neurotoxicity and nephrotoxicity. Therefore, it is urgent to find an effective polymyxin synergist in clinic to reduce the use of polymyxin, enhance its antibacterial activity and improve the clinical therapeutic effect. In addition, under the background of "antibiotic replacement and reduction" in the field of veterinary breeding, the use of antibacterial drug synergists can significantly reduce the use of clinical antibiotics. The discovery of high-efficiency antibacterial drug synergists from natural active substances can reduce the use of clinical antibacterial drugs, thereby reducing the probability of the emergence of drug-resistant bacteria.

[0004] Sanggenon D is one of the main active ingredients extracted from mulberry bark, and its molecular formula is C 40 H 36 O 12It has been reported in the literature that Sanggenon D has antioxidant and anti-inflammatory activities (see the literature Li X, Ren Z, Wu Z, Fu Z, Xie H, Deng L, Jiang X, Chen D. Steric Effect of Antioxidant Diels-Alder-Type Adducts: A Comparison of Sanggenon C with Sanggenon D. Molecules. 2018 Oct 11;23(10):2610.). There has been no report on Sanggenon D as an antibacterial adjuvant. SUMMARY

[0005] The technical problem to be solved by the present application is how to enhance the antibacterial activity of antibacterial drugs.

[0006] To solve the above technical problem, the present application provides an application of Sanggenon D in the preparation of a product for enhancing the antibacterial activity and / or bactericidal activity of antibacterial drugs.

[0007] In the above application, the product can be an antibacterial infection disease drug or an antibiotic synergist.

[0008] In the above application, the antibacterial drug is a drug with bactericidal activity and / or bacteriostatic activity, including various antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones and other chemical synthetic drugs, as well as some products obtained by culturing bacteria, actinomycetes, fungi and other microorganisms, or the same or similar substances manufactured by chemical semi-synthesis. The antibacterial drug can be specifically a drug containing polymyxin.

[0009] The drug containing polymyxin can be specifically polymyxin E or polymyxin B.

[0010] In the present application, the bactericidal activity and / or bacteriostatic activity is a killing and / or inhibiting effect on at least one of the following pathogenic bacteria:

[0011] B1, gram-negative bacteria;

[0012] B2, bacteria of the phylum Proteobacteria;

[0013] B3, bacteria of the class Gammaproteobacteria;

[0014] B4, bacteria of the order Enterobacterales;

[0015] B5, bacteria of the family Enterobacteriaceae;

[0016] B6, bacteria of the genus Escherichia or bacteria of the genus Klebsiella;

[0017] B7, Escherichia coli or Klebsiella pneumoniae.

[0018] The present application provides an antibacterial composition containing mulberroside D and the antibacterial drug in the above application.

[0019] In the above antibacterial composition, the active ingredients of the antibacterial composition can be the mulberroside D and the antibacterial drug, and the active ingredients of the antibacterial composition can also contain other ingredients, and other active ingredients can be determined by those skilled in the art according to the antibacterial effect.

[0020] The above composition can have a killing and / or inhibiting effect on at least one of the following pathogenic bacteria:

[0021] B1, gram-negative bacteria;

[0022] B2, bacteria of the phylum Proteobacteria;

[0023] B3, bacteria of the class Gammaproteobacteria;

[0024] B4, bacteria of the order Enterobacterales;

[0025] B5, bacteria of the family Enterobacteriaceae;

[0026] B6, bacteria of the genus Escherichia or bacteria of the genus Klebsiella;

[0027] B7, Escherichia coli or Klebsiella pneumoniae.

[0028] The antibacterial product containing the above antibacterial composition also belongs to the protection scope of the present application.

[0029] The dosage form of the above antibacterial product can be one of tablets, creams, capsules, sustained-release tablets, controlled-release tablets, oral solutions, syrups, dripping pills, injection dosage forms, and lyophilized powder injection dosage forms.

[0030] Experiments have proved that the combination of mulberroside D and polymyxin E can reduce the MIC value of polymyxin E to standard E. coli 25922 and MCR-1 positive E. coli by 8-128 times, and the MIC value of polymyxin-resistant K. p. 1202 (ST11, KPC-2-producer, mcr-1) strain is 32 times, and the FICI value of all test strains is between 0.037 and 0.12, indicating that the combination of mulberroside D and polymyxin E has obvious synergistic antibacterial effect. At the same time, the combination of polymyxin E and mulberroside D can significantly reduce the bacterial load in the thigh muscle of mice, indicating that mulberroside D can effectively enhance the effectiveness of polymyxin in vivo, and provide a new treatment scheme for the treatment of infectious diseases caused by drug-resistant gram-negative bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1Synergistic antibacterial effect of colistin combined with Sanggenon D against different Escherichia coli and Klebsiella pneumoniae.

[0032] Figure 2 Synergistic bactericidal curve of colistin combined with Sanggenon D; Colistin (2 μg / mL); Sanggenon D (14 μg / mL); Colistin (2 μg / mL) + Sanggenon D (14 μg / mL).

[0033] Figure 3 Effect of colistin (Colistin, 10 mg / kg) or / and Sanggenon D (Sanggenon D, 20 mg / kg) alone and in combination in the treatment of drug-resistant bacterial infection model in BALB / c mice. DETAILED DESCRIPTION

[0034] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0035] In the following examples, the experimental methods are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0036] In the following examples, Sanggenon D was purchased from MCE Reagent Company, USA, with a purity of ≥99%. Sanggenon D was used to prepare a Sanggenon D stock solution with a concentration of 50 mg / mL using dimethyl sulfoxide (DMSO, solvent), which was stored in a -20°C refrigerator for later use.

[0037] In the following examples, colistin sulfate (also known as mucidin sulfate) was purchased from Aladdin Reagent Company, with a potency of ≥19000 U / mg of mucidin. Colistin sulfate was dissolved in sterile water to prepare a colistin E stock solution with a colistin sulfate content of 32 mg / mL, which was stored in a -20°C refrigerator for later use.

[0038] Pathogenic bacteria used in the following tests:

[0039] Escherichia coli ATCC 25922 (hereinafter referred to as E. coli 25922) was purchased from the China Institute of Veterinary Drug Control Culture Collection (China Institute of Veterinary Drug Control Microbial Culture Collection);

[0040] E. coli 09b19, E. coli 09b23, E. coli GZP11-15, E. coli 13h1, E. coli SC11-30, E. coli GZP08-8, E. coli 09b17, E. coli GZP11-5, E. coli EYAP10-258, E. coli GZP11-6, E. coli GZP11-9, E. coli SC11-29 (described in Lu Yang, Yingbo Shen, Junyao Jiang, Xueyang Wang, Dongyan Shao, Margaret M.C. Lam, Kathryn E. Holt, Bing Shao, Congming Wu, Jianzhong Shen, Timothy R. Walsh, Stefan Schwarz, Yang Wang & Zhangqi Shen, Distinct increase in antimicrobial resistance genes among Escherichia coli during 50 years of antimicrobial use in livestock production in China, Nature Food, 2022, 3: 197-205.); E. coli B2 described in Liu Y, Jia Y, Yang K, Tong Z, Shi J, Li R, Xiao X, Ren W, Hardeland R, Reiter RJ, Wang Z. Melatonin overcomes MCR-mediated colistin resistance in Gram-negative pathogens. Theranostics. 2020 Aug 29;10(23): 10697-10711.; K.p. 1202 (ST11, KPC-2-producer, mcr-1) described in Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, Yu LF, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu JH, Shen J.Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016 Feb; 16(2): 161-8.

[0041] The above biological materials can be obtained from the Veterinary Pharmacology and Toxicology Group of the College of Animal Medicine of China Agricultural University in China, and can only be used for repeating the experiments of the present application and cannot be used for other purposes.

[0042] The meat broth medium (MHB) used in the following examples was purchased from Beijing Land Bridge Technology Co., Ltd., and was prepared as follows: 25.0 g was weighed into 1 L of distilled water, heated and boiled until completely dissolved, 121 ℃ high pressure sterilization for 15 min, and ready for use. The MHA medium was purchased from Beijing Land Bridge Technology Co., Ltd., and was prepared as follows: 38.0 g was weighed into 1 L of distilled water, heated and boiled until completely dissolved, 121 ℃ high pressure sterilization for 15 min, and cooled to 55 ℃ for pouring flat plates.

[0043] The brain heart infusion medium (BHI) was purchased from Beijing Land Bridge Technology Co., Ltd. The preparation method is as follows: 38.5 g of the product was weighed, heated and stirred to dissolve in 1000 mL of distilled water, the pH was adjusted to 7.3, 121 ℃ high pressure sterilization for 15 minutes, and ready for use.

[0044] Example 1, synergistic antibacterial activity evaluation of morusin D and polymyxin combination

[0045] 1. Antibacterial activity of morusin D and polymyxin alone - determination of minimum inhibitory concentration (MIC)

[0046] The minimum inhibitory concentration experiment was carried out according to the standard method of American Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, CLSI), and the MIC of morusin D and polymyxin E on the strain was determined by micro-broth dilution method. The specific experimental method is as follows: a single bacterial colony was picked up in BHI broth, and the bacteria were cultured at 37℃ on a shaking table to the logarithmic growth phase. The concentration of the bacterial solution was adjusted to 0.5 McFarland turbidity by a McFarland turbidimeter, and diluted 100 times (1.0 x 10 6After the bacteria were cultured in MHB medium to 108CFUs / mL, the Morusin D and polymyxin E were diluted 10 times in MHB medium, respectively, and 100 μL of each was added to a 96-well U-shaped plate. Then, 100 μL of the diluted bacteria was added to each well. Finally, the final concentration of Morusin D in each well was 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL. The final concentrations of polymyxin B and polymyxin E were 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, and 0.015625 μg / mL. The control group contained only MHB medium and the negative and positive controls contained the bacteria to be tested. After the addition of the drug, the plates were placed in a 37°C incubator for 18 hours, and the results were observed. The minimum concentration of the drug in the sterile growth plate was the minimal inhibitory concentration (MIC).

[0047] Table 1 MIC values of Morusin D, polymyxin E, and polymyxin B for each bacteria (μg / mL)

[0048]

[0049] The results, as shown in Table 1, show that the MIC of Morusin D alone for each pathogenic bacteria was >160 μg / mL, which was the maximum concentration tested in the experiment. The MIC of polymyxin E and polymyxin B for the standard strain of Escherichia coli was 0.25 μg / mL. The MIC of polymyxin E and polymyxin B for the remaining strains was 4 μg / mL or 8 μg / mL when used alone.

[0050] 2. Antibacterial activity of Morusin D and polymyxin E alone and in combination

[0051] FICI determination of the combination of Morusin D and polymyxin for drug-resistant bacteria: The FICI values of the combination of Morusin D and polymyxin E for the standard strain of gram-negative drug-resistant bacteria Escherichia coli ATCC25922 and MCR-1-producing Escherichia coli and Klebsiella pneumoniae were determined. The method (checkerboard method) and results are as follows:

[0052] Antibacterial drug polymyxin E (as a drug A) and mulberrocytonin D (as a drug B) were diluted with MHB broth medium in 8 concentrations with 2 MIC as the highest concentration of each, and 50 μL of the medium containing different concentrations of the two drugs was added along the horizontal and vertical axes of the 96-well microplate, respectively, and then 100 μL of each pathogenic bacteria solution was added to make the final pathogenic bacteria content 1×10 6 CFU / well, 37°C constant temperature incubation for 18-24 h, and the results were observed. The MIC of each drug was recorded when used alone and in combination, and the FIC value (partial inhibitory concentration index) was calculated according to the following formula.

[0053] FICI = MIC 甲药联合 / MIC 甲药单用 + MIC 乙药联合 / MIC 乙药单用

[0054] Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no effect; FICI > 2, antagonistic effect. That is: the FICI of mulberrocytonin D and polymyxin is MIC (polymyxin E combination) / MIC (polymyxin E alone) + MIC (mulberrocytonin D combination) / MIC (mulberrocytonin D alone).

[0055] Table 2 FICI value of mulberrocytonin D combined with antibacterial drugs on standard strain ATCC 25922 of Escherichia coli, MCR-1 producing E. coli and Klebsiella pneumoniae K.p1202

[0056]

[0057]

[0058]

[0059]

[0060] The test results are shown in Table 2.

[0061] The MIC of polymyxin E (as a drug A) and mulberrocytonin D (as a drug B) used in combination on ATCC 25922 was 0.875 μg / mL and 0.03125 μg / mL, respectively, and the synergistic index FICI was 0.068.

[0062] The MIC of polymyxin E (as a drug A) and mulberrocytonin D (as a drug B) used in combination on E. coli GZP11-5 was 3.5 μg / mL and 0.125 μg / mL, respectively, and the synergistic index FICI was 0.053.

[0063] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli B2 were 3.5 μg / mL, 0.061 μg / mL, respectively, and the synergistic index FICI was 0.037.

[0064] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli 09b17 were 3.5 μg / mL, 0.125 μg / mL, respectively, and the synergistic index FICI was 0.053.

[0065] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli EYAP10-258 were 3.5 μg / mL, 0.063 μg / mL, respectively, and the synergistic index FICI was 0.038.

[0066] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli GZP11-9 were 1.75 μg / mL, 0.25 μg / mL, respectively, and the synergistic index FICI was 0.073.

[0067] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli GZP11-15 were 3.5 μg / mL, 0.5 μg / mL, respectively, and the synergistic index FICI was 0.084.

[0068] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli GZP11-6 were 3.5 μg / mL, 0.125 μg / mL, respectively, and the synergistic index FICI was 0.053.

[0069] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli 09b19 were 1.75 μg / mL, 0.125 μg / mL, respectively, and the synergistic index FICI was 0.053.

[0070] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against K. pneumoniae K.p1202 (ST11, KPC-2-producer, mcr-1) were 1.75 μg / mL, 0.125 μg / mL, respectively, and the synergistic index FICI was 0.042.

[0071] The MICs of polymyxin E (as the partner drug) and morusin D used in combination against E. coli SC11-29 were 14 μg / mL, 0.125 μg / mL, respectively, and the synergistic index FICI was 0.12.

[0072] The MIC of colistin (as the first drug) and Sanggenon D combined use against E. coli 13h1 was 3.5 μg / mL, 0.25 μg / mL respectively, and the synergistic index FICI was 0.084.

[0073] The MIC of colistin (as the first drug) and Sanggenon D combined use against E. coli SC11-30 was 7 μg / mL, 0.125 μg / mL respectively, and the synergistic index FICI was 0.059.

[0074] The MIC of colistin (as the first drug) and Sanggenon D combined use against E. coli GZP08-8 was 5 μg / mL, 0.25 μg / mL respectively, and the synergistic index FICI was 0.063.

[0075] The MIC of colistin (as the first drug) and Sanggenon D combined use against E. coli GZP08-9 was 3.5 μg / mL, 0.25 μg / mL respectively, and the synergistic index FICI was 0.084.

[0076] In summary, the MIC of colistin combined with Sanggenon D against standard E. coli 25922 and MCR-1 positive E. coli was reduced by 8-128 times, and the MIC against MCR-1 positive K. pneumoniae K.p.1202 (ST11, KPC-2-producer, mcr-1) strain was reduced by 32 times. The FICI values of all tested strains were between 0.037 and 0.12, indicating that the combination of Sanggenon D and colistin had a significant synergistic antibacterial effect.

[0077] Example 2, Synergistic bactericidal curve of colistin combined with Sanggenon D

[0078] Four treatments were set up for parallel experiments, namely the control group (Control), the colistin group (Colistin), the Sanggenon D group (Sanggenon D), and the colistin + Sanggenon D group (Colistin + Sanggenon D). The four treatments were the same in operation except for the different drugs added.

[0079] 2.1 Control group (Control): First, E. coli B2 was cultured in BHI broth medium for 6 hours, then an equal volume of DMSO solution (solvent was BHI broth medium) was added to make the content of DMSO in the culture system 0.1%. Then 100 μL of bacterial solution was taken at 4h, 8h, 12h and 24h and spread on MHA medium, and after overnight culture, colony counting was performed.

[0080] 2.2 Polymyxin E group (Colistin): First, E. coli B2 was cultured in BHI broth medium for 6 hours, then an equal volume of polymyxin E solution (a liquid obtained by diluting the polymyxin E stock solution with BHI broth medium) was added to make the content of polymyxin E in the culture system 2 μg / mL. Then 100 μL of bacterial solution was taken at 4 h, 8 h, 12 h and 24 h respectively and coated on MHA medium, and after overnight culture, colony counting was performed.

[0081] 2.3 Sanggenon D group: First, E. coli B2 was cultured in BHI broth medium for 5 hours, then an equal volume of sanggenon D solution (a liquid obtained by diluting the sanggenon D stock solution with BHI broth medium) was added to make the content of sanggenon D in the culture system 14 μg / mL. Then 100 μL of bacterial solution was taken at 4 h, 8 h, 12 h and 24 h respectively and coated on MHA medium, and after overnight culture, colony counting was performed.

[0082] 2.4 Group and Polymyxin E + Sanggenon D group (Colistin + Sanggenon D): First, E. coli B2 was cultured in BHI broth medium for 5 hours, then an equal volume of sanggenon D and polymyxin E mixed solution (a liquid obtained by mixing the liquid obtained by diluting the sanggenon D stock solution with BHI broth medium and the liquid obtained by diluting the polymyxin E stock solution with BHI broth medium in proportion) was added to make the content of sanggenon D in the culture system 14 μg / mL and the content of polymyxin E 2 μg / mL. Then 100 μL of bacterial solution was taken at 4 h, 8 h, 12 h and 24 h respectively and coated on MHA medium, and after overnight culture, colony counting was performed.

[0083] The results are shown in Table 1. Figure 2 Compared with polymyxin E or sanggenon D alone, the combination of polymyxin E and sanggenon D significantly reduced the number of bacteria, and the number of bacteria was reduced to 4 Log 10 CFU / mL after 24 h of combination.

[0084] Example 3, Evaluation of the therapeutic effect of polymyxin E and sanggenon D alone and in combination in a drug-resistant bacterial infection model in BALB / c mice

[0085] 3.1 Experimental grouping and dosing

[0086] To avoid the influence of mouse autoimmunity on the experiment, all mice were given cyclophosphamide twice, 100 mg / kg intraperitoneally, 4 days and 1 day before infection, to cause neutropenia and immunodeficiency in mice. 24 6-8 week old BALB / c female mice (body weight 20 g ± 2 g) were randomly divided into model control group PBS buffer (i.e. non-treatment group), polymyxin E treatment group, Sanggenon D treatment group and Sanggenon D + polymyxin E combined treatment group, 6 in each group.

[0087] 3.2 Bacterial culture

[0088] E. coli B2 was inoculated into BHI broth medium and cultured at 37°C to the logarithmic phase, the bacterial cells were collected by centrifugation, resuspended with PBS buffer, vortexed and mixed, and then diluted with PBS buffer to 1 × 10 7 CFU / mL to obtain an E. coli suspension for use, and the amount of E. coli injected into each mouse was 1 × 10 6 CFU.

[0089] 3.3 Construction of mouse infection model

[0090] Control group (Control): each mouse was injected with 0.1 mL of E. coli suspension into the right thigh muscle of the abdominal cavity; 1 hour later, the mouse was injected with 200 μL of sterile normal saline into the abdominal cavity;

[0091] Polymyxin E (colistin) treatment group: each mouse was injected with 0.1 mL of E. coli suspension into the right thigh muscle of the abdominal cavity; 1 hour later, the mouse was injected with 200 μL of polymyxin E liquid (obtained by diluting polymyxin E stock solution with sterile normal saline) into the abdominal cavity, so that the dose of polymyxin E per mouse was 10 mg / kg body weight;

[0092] Sanggenon D treatment group: each mouse was injected with 0.1 mL of E. coli suspension into the right thigh muscle of the abdominal cavity; 1 hour later, the mouse was injected with 200 μL of Sanggenon D liquid (obtained by diluting Sanggenon D stock solution with sterile normal saline, so that the dose of Sanggenon D per mouse was 20 mg / kg body weight) into the abdominal cavity;

[0093] In the combined treatment group of sanggenon D and polymyxin E (Colistin + Sanggenon D), each mouse received an intramuscular injection of 0.1 mL of Escherichia coli suspension in the right thigh. One hour later, the mice were injected intraperitoneally with a mixture of sanggenon D and 200 μL of polymyxin E (the mixture of sanggenon D stock solution diluted with sterile saline and polymyxin E stock solution diluted with sterile saline in a specific ratio). The dosage of sanggenon D was 20 mg / kg body weight per mouse, and the dosage of polymyxin E was 10 mg / kg body weight per mouse.

[0094] Twenty-four hours after infection, all mice were euthanized, and the muscle from the right thigh of each mouse was removed and placed in 3 mL of PBS buffer. The muscle was then lysed using a low-temperature tissue homogenizer, and 100 μL of the lysate was plated for colony counting.

[0095] The results are as follows Figure 3 As shown, the bacterial load in the thigh muscles of mice treated with the negative control PBS buffer, polymyxin E, and morula ketone D alone was comparable, with no significant difference. This indicates that the three drugs alone are not effective in treating E. coli B2 infections. Conversely, the combined treatment with polymyxin E and morula ketone D significantly reduced the bacterial load in the thigh muscles of mice. These results demonstrate that morula ketone D can effectively enhance the efficacy of polymyxins in vivo, providing a new treatment option for infectious diseases caused by drug-resistant Gram-negative bacteria.

[0096] Table 3. Evaluation of the efficacy of each treatment group in the E. coli B2 infection model (n=10)

[0097] Group Total bacteria (Log 10 CFU / mL) Control group (sterile saline) 7.1 Polymyxin E treatment group 6.75 Morin D treatment group 6.86 Morin D + polymyxin E combined treatment group 4.23

[0098] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An antibacterial composition, characterized in that: The antibacterial composition is a combination of mulberry root ketone D and an antibacterial drug, wherein the antibacterial drug is polymyxin E; the mass ratio of mulberry root ketone D to polymyxin E in the composition is 0.875-56:

1.

2. The composition according to claim 1, characterized in that: The composition has a killing and / or inhibitory effect on Escherichia coli or Klebsiella pneumoniae.

3. An antibacterial product comprising the antibacterial composition as described in claim 1 or 2.

4. The antibacterial product according to claim 3, characterized in that: The product dosage form is one of the following: capsules, sustained-release tablets, controlled-release tablets, oral liquids, drop pills, injections, or lyophilized powder injections.

5. The antibacterial product according to claim 3 or 4, characterized in that: The antibacterial product has a killing and / or inhibitory effect on Escherichia coli or Klebsiella pneumoniae.

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