Application of dehydroandrographolide in synergistic enhancement of polymyxin in the preparation of drugs against Gram-negative bacterial infections

Abstract

The present invention discloses the use of dehydroandrographolide in synergistic effect with polymyxin B in the preparation of a drug for treating Gram-negative bacterial infections. The Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Salmonella. The present invention also discloses an antibacterial combination ratio of dehydroandrographolide and the antibiotic polymyxin B, as well as an optimal ratio for treating animal infections. This invention provides a new therapeutic strategy for the clinical treatment of bacterial infectious diseases.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of dehydroandrographolide in synergizing polymyxin in the preparation of drugs for resisting Gram-negative bacterial infection. Background Art

[0002] Bacterial resistance has become a global public health issue, and curbing it is urgent. In recent years, the rapid spread of carbapenem-resistant Gram-negative bacteria in both human and animal medicine has led to a decreasing number of clinically available antibiotics. The emergence of some multidrug-resistant Gram-negative bacteria, in particular, has resulted in a near-extinction of available drugs and created a series of public health and safety concerns. In recent years, various national departments have paid considerable attention to the healthy and sustainable development of the livestock and poultry industry. my country has recently introduced a series of policies and action plans, including the "National Action Plan for Curbing Bacterial Resistance (2016-2020)" and the "National Action Plan for Curbing Animal-Source Bacterial Resistance (2017-2020)," to curb the development of bacterial resistance.

[0003] Polymyxin antibiotics include five types: polymyxin A, B, C, D, and E. Polymyxin B and E are used clinically and have strong effects against Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Polymyxin E, also known as colistin, has a CAS number of 1066-17-7 and trade names such as antimyxin, colistin, and colistin. It is primarily used clinically in the sulfate or methanesulfonate form, namely polymyxin E sulfate and polymyxin E methanesulfonate. Currently, polymyxin E is considered one of the most important antimicrobial drugs for the clinical treatment of infections such as carbapenem-resistant Escherichia coli, multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Klebsiella pneumoniae. In recent years, the emergence and spread of polymyxin resistance (MCR) plasmids carried by pathogens such as Escherichia coli has led to polymyxin resistance in some Gram-negative bacteria. These factors severely limit the clinical application of polymyxins. Therefore, the development of effective polymyxin enhancers and polymyxin compound preparations has become an important strategy for the clinical treatment of multidrug-resistant Gram-negative bacilli infections. Previous studies have reported that the combination of colistin and vancomycin has a significant synergistic antibacterial effect (Antimicrob Agents Chemother. 2010 Dec; 54(12): 5316-22.), but clinical treatment trials have confirmed that the combination of the two drugs does not have a significant advantage, but may increase the nephrotoxicity (Pharmaceutics. 2021 Jan 26; 13(2): 162). Therefore, we still need to find new and effective combination treatment strategies.

[0004] Dehydroandrographolide, CAS number 134418-28-3, molecular formula C20H28O4, molecular weight 332.43. Dehydroandrographolide is one of the main active substances in Andrographis paniculata extract, exhibiting significant anti-inflammatory and antipyretic effects. Dehydroandrographolide can inhibit the increase in capillary permeability and the development of inflammatory edema during inflammation, and can reduce inflammatory exudate, but has no significant effect on proliferative inflammation. The 2020 edition of the Chinese Pharmacopoeia has approved Andrographis paniculata capsules for therapeutic uses such as clearing heat and detoxifying, cooling blood and reducing swelling.

[0005] Although existing technologies have disclosed the combination of colistin with other antibacterial agents, most have poor drugability. The present invention not only provides in vitro synergistic experiments demonstrating the combined use of dehydroandrographolide and polymyxin, but also provides animal studies confirming the beneficial synergistic bactericidal effect. To date, no research has reported the use of dehydroandrographolide as a polymyxin potentiator to enhance the antibacterial activity of polymyxins or in combination therapy. Summary of the Invention

[0006] One object of the present invention is to provide a new medicinal use of dehydroandrographolide.

[0007] The new pharmaceutical use of dehydroandrographolide provided by the present invention is its use in preparing a synergist for antibacterial infection of polymyxin antibiotics, that is, its use in preparing a product for enhancing the antibacterial activity of polymyxin antibiotics.

[0008] The products mentioned are human medicines, veterinary medicines, poultry medicines, etc.

[0009] Furthermore, the polymyxin antibiotic is polymyxin E (i.e., colistin) or a pharmaceutically acceptable salt thereof (such as polymyxin E sulfate (colistin sulfate), polymyxin E mesylate) or polymyxin B or a pharmaceutically acceptable salt thereof (such as polymyxin B sulfate).

[0010] Furthermore, the dehydroandrographolide is dehydroandrographolide.

[0011] Furthermore, the bacteria include bacteria containing a polymyxin resistance gene (MCR).

[0012] Furthermore, the bacteria are Gram-negative rods.

[0013] Furthermore, the Gram-negative bacilli include multidrug-resistant Gram-negative bacilli.

[0014] Furthermore, the bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Salmonella; preferably, one or more of the Escherichia coli, Klebsiella pneumoniae and Salmonella are strains with polymyxin resistance genes (MCR) or multidrug-resistant strains.

[0015] Furthermore, the bacterium is Klebsiella pneumoniae; preferably, the bacterium is Klebsiella pneumoniae with a polymyxin resistance gene (MCR) or multidrug-resistant Klebsiella pneumoniae.

[0016] Another object of the present invention is to provide an antibacterial infection composition.

[0017] The antibacterial infection composition provided by the present invention comprises dehydroandrographolide and polymyxin antibiotics.

[0018] Furthermore, the mass ratio of the polymyxin antibiotic to dehydroandrographolide is 1:40-640; specifically 1:40-160, 1:40-320, 1:160-320, 1:320-640, 1:160-640, 1:40, 1:160, 1:320, and 1:640.

[0019] In the above-mentioned antibacterial infection composition, the active ingredients of the antibacterial infection composition may be only dehydroandrographolide and the polymyxin antibiotic; it may also further include other active ingredients. The selection of other active ingredients can be determined by those skilled in the art based on the antibacterial effect.

[0020] The above composition can have a killing and / or inhibitory effect on bacteria;

[0021] The bacteria include bacteria containing a polymyxin resistance gene (MCR).

[0022] Furthermore, the bacteria are Gram-negative rods.

[0023] Furthermore, the Gram-negative bacilli include multidrug-resistant Gram-negative bacilli.

[0024] Furthermore, the bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Salmonella; preferably, one or more of the Escherichia coli, Klebsiella pneumoniae and Salmonella are strains with polymyxin resistance genes (MCR) or multidrug-resistant strains.

[0025] Furthermore, the bacterium is Klebsiella pneumoniae; preferably, the bacterium is Klebsiella pneumoniae with a polymyxin resistance gene (MCR) or multidrug-resistant Klebsiella pneumoniae.

[0026] The dosage form of the antibacterial infection composition is one of tablets, capsules, sustained-release tablets, controlled-release tablets, oral liquid, syrup, injection, dripping pills, and freeze-dried powder injection.

[0027] In addition, antibacterial products containing the above antibacterial infection composition also fall within the protection scope of the present invention.

[0028] The dosage form of the antibacterial product is one of tablets, capsules, sustained-release tablets, controlled-release tablets, oral liquid, syrup, injection dosage form, dropping pills, and freeze-dried powder injection dosage form.

[0029] The final therapeutic doses of the polymyxin antibiotic and the dehydroandrographolide are 1 mg / kg and 20 mg / kg body weight, respectively.

[0030] In order to solve the problem of drug resistance of multidrug-resistant Gram-negative bacteria and the lack of effective drugs, the present invention provides a method for enhancing the antibacterial activity of polymyxins and their combined treatment, specifically using dehydroandrographolide in combination with polymyxin, preferably polymyxin E. The two are not simply functional additions, but rather achieve a synergistic antibacterial effect. The present invention also discloses an antibacterial combination ratio of dehydroandrographolide and polymyxin E, which provides a new treatment strategy for the clinical treatment of bacterial infections, especially multidrug-resistant Gram-negative bacterial infections, specifically bacterial infections with polymyxin resistance genes (MCRs).

[0031] The present invention demonstrates the synergistic antibacterial activity of dehydroandrographolide with polymyxins through a checkerboard minimum inhibitory concentration test and in vitro bactericidal curves. Unlike existing methods of combining polymyxins with antibiotics, the present invention provides a mouse model of drug-resistant bacterial infection, demonstrating at the animal level that dehydroandrographolide can effectively enhance the effectiveness of polymyxins and their in vivo efficacy, further supporting further clinical application. The present invention demonstrates that dehydroandrographolide can restore the sensitivity of polymyxin-resistant bacteria and further evaluates the in vitro and in vivo effectiveness of their combined use. This approach contributes to the development of a new class of compound preparations, improving therapeutic efficacy and overcoming the problem of multidrug-resistant Gram-negative infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the time-kill curve of dehydroandrographolide combined with polymyxin E against Escherichia coli E. coli B2 in Example 3 of the present invention.

[0033] Figure 2 This is the time-kill curve of dehydroandrographolide combined with polymyxin E against Escherichia coli ATCC25922 in Example 3 of the present invention.

[0034] Figure 3The results show the therapeutic effects of polymyxin E and dehydroandrographolide alone and in combination on diarrhea caused by multidrug-resistant bacteria in mice in Example 4 of the present invention. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0036] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0037] The dehydroandrographolide used in the following examples has a CAS number of 134418-28-3, a molecular formula of C20H28O4, a molecular weight of 332.43, and was purchased from Aladdin Reagent Company with a purity of ≥98%.

[0038] Colistin sulfate was purchased from Aladdin Reagents, catalog number C114323, with an activity of >19000 U / mg.

[0039] Polymyxin B sulfate was purchased from Aladdin Reagent Company with a potency greater than or equal to 6000 U / mg.

[0040] Weigh a certain amount of colistin sulfate or polymyxin B sulfate to a stock solution concentration of 16 mg / mL. Prepare dehydroandrographolide using DMSO to a stock solution concentration of 40 mg / mL. Store in a -20°C freezer.

[0041] The standard strain of Escherichia coli was Escherichia coli ATCC 25922 (hereinafter referred to as E. coli 25922), which was purchased from the China Veterinary Supervision Institute’s Culture Collection Center (China Veterinary Microbial Culture Collection Administration Center).

[0042] Escherichia coli B2 (E. coli B2) carries a colistin resistance gene 1 (MCR-1) plasmid and a New Delhi metallo-β-lactamase 5 (NDM-5) plasmid. Detailed information has been reported in the following references: Song M, Liu Y, Huang X, Ding S, Wang Y, Shen J, Zhu KA. Broad-spectrum antibiotic adjuvant reverses multidrug-resistant Gram-negative pathogens. Nat Microbiol. 2020 Aug; 5(8): 1040-1050.

[0043] The specific information of Klebsiella pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1) has been described in the following literature: 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. This strain carries the MCR-1 plasmid and is resistant to colistin.

[0044] The standard strain of Pseudomonas aeruginosa ATCC15692 (ie, Pseudomonas aeruginosa PA01) was purchased from the American Type Culture Collection (ATCC).

[0045] Salmonella SH16SF0764, isolated from clinical samples and carrying the MCR-1 plasmid, was provided by the National Center for Veterinary Drug Safety Evaluation, China Agricultural University.

[0046] The standard strain of Acinetobacter baumannii ATCC 19606 was purchased from the American Type Culture Collection (ATCC).

[0047] Escherichia coli E.coli09b19, E.coli13h1, and E.coli GZP080-8 are described in the following literature: Lu Yang, Yingbo Shen, Junyao Jiang, Xueyang Wang, Dongyan Shao, Margaret MCLam, 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; all carry the MCR-1 plasmid and are resistant to polymyxins.

[0048] Bacterial culture media were used in the following experiments:

[0049] MHB broth culture medium was purchased from Beijing Luqiao Technology Co., Ltd. and prepared as follows: weigh 25.0 g into 1 L of distilled water, heat and boil until completely dissolved, sterilize under high pressure at 121°C for 15 min, and set aside.

[0050] MHA medium was purchased from Beijing Luqiao Technology Co., Ltd. and prepared as follows: weigh 38.0 g and dissolve in 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121°C under high pressure for 15 min, cool to 55°C and pour into a plate for later use.

[0051] Brain heart infusion medium (BHI) was purchased from Beijing Luqiao Technology Co., Ltd. and was prepared as follows: 38.5 g of the product was weighed and dissolved in 1000 mL of distilled water with heating and stirring, the pH was adjusted to 7.3, and the medium was sterilized by autoclaving at 121°C for 15 minutes and set aside.

[0052] BHI solid culture medium was purchased from Beijing Road & Bridge Technology Co., Ltd. and was prepared as follows: 50.0 g of the product was weighed and dissolved in 1000 mL of distilled water. The mixture was heated and boiled until completely dissolved. The medium was sterilized by autoclaving at 121°C for 20 min and cooled to 55°C before being poured into a plate for later use.

[0053] Example 1. Evaluation of Minimum Inhibitory Concentrations of Dehydroandrographolide, Polymyxin B, and Polymyxin E

[0054] (1) Determination of the minimum inhibitory concentration (MIC) of antibacterial activity of dehydroandrographolide, polymyxin B, and polymyxin E alone

[0055] The minimum inhibitory concentration (MIC) of different strains to dehydroandrographolide, polymyxin B and polymyxin E was detected according to the standard method of the Clinical and Laboratory Standards Institute (CLSI). The specific operation is as follows: pick a single bacterial colony in BHI broth, culture the bacteria in a shaking incubator at 37°C until the logarithmic growth phase, and adjust the concentration of the bacterial solution to 0.5 McFarland turbidity using a McFarland turbidimeter for use. Use MHB medium to dilute dehydroandrographolide, polymyxin B and polymyxin E in multiple concentration gradients. Take 100 μL of bacterial solution (the final concentration of the bacterial solution in each well is 1.0x10 6 CFUs / mL) were added to a 96-well U-shaped plate, and then different drugs were added to each well. The final concentrations of dehydroandrographolide were 500μg / mL, 250μg / mL, 125μg / mL, and 62.5μg / mL, respectively; the final concentrations of polymyxin B and polymyxin E were set at 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, 0.125μg / mL, and 0.0625μg / mL. The negative control group contained only MHB medium; the positive control group contained 100μL of the test bacteria solution, and 1μL of DMSO was added to each well. After drug addition, the culture plates were placed in a 37°C incubator for 18 hours and then observed. The drug concentration in the wells that were clear to the naked eye in the 96-well plate was the minimum inhibitory concentration (MIC). The test results are shown in Table 1.

[0056] Table 1 MIC values ​​of dehydroandrographolide, polymyxin E, and polymyxin B against various bacteria (μg / mL)

[0057]

[0058] As shown in Table 1, dehydroandrographolide has an inhibitory effect on the tested strains E. coli 25922, E. coli B2, ATCC15692, K. pneumoniae1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1), E. coli 09b19, E. coli13h1, E. coli GZP080-8, ATCC 19606, Salmonella The antibacterial activity against Gram-negative bacteria such as SH16SF0764 was greater than the maximum concentration measured, i.e., >500 μg / mL; the MICs of polymyxin E against the above-mentioned Gram-negative bacteria were 0.25 μg / mL, 8 μg / mL, 1 μg / mL, 8 μg / mL, 4 μg / mL, 8 μg / mL, 8 μg / mL, 0.5 μg / mL, and 8 μg / mL, respectively; the MICs of polymyxin B against the above-mentioned Gram-negative bacteria were 0.25 μg / mL, 8 μg / mL, 1 μg / mL, 8 μg / mL, 4 μg / mL, 4 μg / mL, 4 μg / mL, 0.5 μg / mL, and 8 μg / mL, respectively.

[0059] Example 2: Antibacterial activity test of combined application of dehydroandrographolide and antibacterial drug polymyxin

[0060] Determination of the combined use index (FICI) of dehydroandrographolide and the antimicrobial drug polymyxin against pathogens: The FICI values ​​of dehydroandrographolide combined with antimicrobial drugs against E. coli 25922, E. coli B2, ATCC15692, K. pneumoniae1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1), E. coli09b19, E. coli 13h1, E. coli GZP080-8, ATCC 19606, and Salmonella SH16SF0764 were determined using the checkerboard method. The specific procedures are as follows:

[0061] The antimicrobial drugs polymyxin E (as drug A) and dehydroandrographolide (as drug B) were diluted into 8-11 concentrations using MHB broth medium at a maximum concentration of 2 MIC. 50 μL of MHB broth containing different concentrations of the two drugs was added along the horizontal and vertical axes of the 96-well microplate, and then 50 μL of various pathogenic bacteria were added to each well to make the final bacterial count of each well 1×10 6 CFU were cultured at 37°C for 18-24 hours, and the results were observed. The MICs of the two drugs when used alone and in combination were recorded, and the FICI values ​​(fractional inhibitory concentration index) were calculated according to the following formula.

[0062] FICI = MIC of drug A combined / MIC of drug A alone + MIC of drug B combined / MIC of drug B alone

[0063] Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no effect; FICI > 2, antagonistic effect. Specifically, the FICI of dehydroandrographolide and colistin = MIC (colistin combination) / MIC (colistin alone) + MIC (dehydroandrographolide combination) / MIC (dehydroandrographolide alone). The experimental results are shown in Table 2.

[0064] Table 2 FICI values ​​of combined use of dehydroandrographolide and antimicrobial drugs against Escherichia coli standard strains E. coli 25922, E. coli B2, ATCC 15692, K. pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1), E. coli 09b19, E. coli 13h1, E. coli GZP08-8, ATCC 19606, and Salmonella SH16SF0764

[0065]

[0066] As shown in Table 2, the in vitro MIC assay results show that the MIC of dehydroandrographolide alone against the above-mentioned Gram-negative bacteria is greater than the maximum tested concentration, i.e., >500 μg / mL. The combination of dehydroandrographolide and polymyxin E can enhance the antibacterial activity of polymyxin E:

[0067] When polymyxin E and dehydroandrographolide were used together against the standard Escherichia coli strain ATCC25922, the two achieved maximum synergy at concentrations of 40 μg / mL of dehydroandrographolide and 0.0625 μg / mL of polymyxin E, with an FICI of 0.33, indicating a synergistic effect. This indicates that the optimal synergistic effect was achieved at a mass ratio of 1:640 between polymyxin E and dehydroandrographolide.

[0068] When polymyxin E and dehydroandrographolide were used together against K. pneumoniae 1202 (ST11, KPC-2-producer) and pHNSHP45 (mcr-1), the two achieved maximum synergy at concentrations of 20 μg / mL and 0.5 μg / mL, with an FICI of 0.1025, indicating a synergistic effect. This indicates that a mass ratio of 1:40 between polymyxin E and dehydroandrographolide resulted in the highest synergistic effect.

[0069] When polymyxin E and dehydroandrographolide were used together against ATCC15692 strains, the two achieved maximum synergy at concentrations of 40 μg / mL dehydroandrographolide and 0.25 μg / mL, with an FICI of 0.33, indicating a synergistic effect. This indicates that the optimal synergistic effect was achieved at a mass ratio of 1:160 between polymyxin E and dehydroandrographolide.

[0070] When polymyxin E and dehydroandrographolide were used together against clinical E. coli B2 strains, the two achieved maximum synergy at concentrations of 20 μg / mL of dehydroandrographolide and 0.5 μg / mL of polymyxin E, with an FICI of 0.1025, indicating a synergistic effect. This indicates that the optimal synergistic effect was achieved at a mass ratio of 1:40 between polymyxin E and dehydroandrographolide.

[0071] When polymyxin E and dehydroandrographolide were used together against the clinical E. coli 09b19 strain, the two achieved maximum synergy at concentrations of 40 μg / mL of dehydroandrographolide and 0.25 μg / mL of polymyxin E, with an FICI of 0.205, indicating a synergistic effect. This indicates that the optimal synergistic effect was achieved at a mass ratio of 1:160 between polymyxin E and dehydroandrographolide.

[0072] When polymyxin E and dehydroandrographolide were used together against the clinical E. coli 13h1 strain, the two achieved maximum synergy at concentrations of 40 μg / mL of dehydroandrographolide and 0.25 μg / mL of polymyxin E, with an FICI of 0.142, indicating a synergistic effect. This indicates that the optimal synergistic effect was achieved at a mass ratio of 1:160 between polymyxin E and dehydroandrographolide.

[0073] When polymyxin E and dehydroandrographolide were used together against the clinical E. coli GZP08-8 strain, the two achieved maximum synergy at concentrations of 20 μg / mL and 0.5 μg / mL, with an FICI of 0.1025, indicating a synergistic effect. This indicates that a mass ratio of 1:40 between polymyxin E and dehydroandrographolide resulted in the best synergistic effect.

[0074] When polymyxin E and dehydroandrographolide were used together against ATCC 19606 strain, the two exhibited maximum synergy at concentrations of 20 μg / mL dehydroandrographolide and 0.0625 μg / mL, with an FICI of 0.165, indicating a synergistic effect. This indicates that the optimal synergistic effect was achieved at a mass ratio of 1:320 between polymyxin E and dehydroandrographolide.

[0075] When polymyxin E and dehydroandrographolide were used together against the clinical Salmonella SH16SF0764 strain, the two achieved maximum synergy at a dehydroandrographolide concentration of 20 μg / mL and a polymyxin E concentration of 1 μg / mL, with an FICI of 0.165, indicating a synergistic effect. This indicates that a mass ratio of polymyxin E to dehydroandrographolide of 1:20 provides the best synergistic effect.

[0076] In summary, dehydroandrographolide significantly enhanced the antibacterial activity of polymyxin E when added at 20-40 μg / mL.

[0077] Example 3: Synergistic bactericidal curve of polymyxin combined with dehydroandrographolide

[0078] 2.1 Test materials

[0079] Dehydroandrographolide and polymyxin E were purchased as in Example 1.

[0080] 2.2 In vitro sterilization curve test

[0081] After EcoliB2 and ATCC25922 were cultured in BHI broth for 6 hours, equal volumes of DMSO (final concentration 0.1%), polymyxin E, dehydroandrographolide, and a mixture of polymyxin E and dehydroandrographolide were added.

[0082] (1) For EcoliB2 strain, the groups and dosages are as follows:

[0083] Control group: 0.1% DMSO;

[0084] Polymyxin E group: the final concentration of polymyxin E sulfate was 4 μg / mL;

[0085] Dehydroandrographolide group: the final concentration of dehydroandrographolide was 40 μg / mL;

[0086] Dehydroandrographolide and polymyxin E combined group: the final concentration of dehydroandrographolide was 40 μg / mL, and the final concentration of polymyxin E sulfate was 4 μg / mL.

[0087] Then, 100 μL of bacterial solution was spread on an MHA agar culture plate with a diameter of 10 cm at 1 h, 3 h, 6 h, 12 h and 24 h, and the colonies were counted after overnight culture.

[0088] (2) For Escherichia coli ATCC25922, the groups and dosages are as follows:

[0089] Control group: 0.1% DMSO;

[0090] Polymyxin E group: the final concentration of polymyxin E sulfate was 0.25 μg / mL;

[0091] Dehydroandrographolide group: the final concentration of dehydroandrographolide was 40 μg / mL;

[0092] Dehydroandrographolide and polymyxin E combined group: the final concentration of dehydroandrographolide was 40 μg / mL, and the final concentration of polymyxin E sulfate was 0.25 μg / mL.

[0093] Then, 100 μL of bacterial solution was spread on an MHA agar culture plate with a diameter of 10 cm at 1 h, 3 h, 6 h, 12 h and 24 h, and the colonies were counted after overnight culture.

[0094] 2.3 Test results

[0095] like Figure 1 As shown in Figure 2, for E. coli B2 strain, at 24h, compared with the control group, the treatment with polymyxin E and dehydroandrographolide alone had no significant effect on the bacterial colony count. When dehydroandrographolide and polymyxin E were treated together, the colony count was 2.41Log at 3h. 10 CFU / mL, until the 24th hour, the colony count was 1.32Log 10 CFU / mL.

[0096] like Figure 2 As shown, for ATCC25922 strain, compared with the control group, at 24h, the colony count in the polymyxin E-treated group was reduced to 5.82Log 10 CFU / mL, dehydroandrographolide had no significant effect on the number of bacterial colonies. When dehydroandrographolide and polymyxin E were treated together, the number of colonies decreased to 3.6Log at 1h, 3h, 6h, 12h, and 24h. 10 CFU / mL, 2.4Log 10 CFU / mL, 1.8Log 10 CFU / mL, 1.2Log 10 CFU / mL, 0.3Log 10 CFU / mL.

[0097] Example 4: Therapeutic Effect of Polymyxin E Combined with Dehydroandrographolide on Bacterial Clearance Rate in Wound Infection in Mice

[0098] Male BALB / c mice, weighing 18–20 g, were purchased from Beijing Weitonglihua Co., Ltd. Mice were housed in the experimental animal room of the National Center for Veterinary Drug Safety, College of Veterinary Medicine, China Agricultural University, at room temperature (25 ± 2°C, relative humidity 50 ± 10%), with a standard light schedule of 12 h day:12 h night, and fed a standard mouse maintenance pellet diet. Mice were acclimated to the animal room for 1 week before formal experiments. All animal experiments were approved by the Animal Ethics Committee of China Agricultural University.

[0099] Fifty-six mice were randomly divided into seven groups, each with eight mice, including a blank control group (normal saline), an infection model group (normal saline injection), a polymyxin E monotherapy group (1 mg / kg intraperitoneal injection), a dehydroandrographolide 20 mg / kg monotherapy group (20 mg / kg dehydroandrographolide intraperitoneal injection), and a polymyxin E combined with dehydroandrographolide treatment group (1 mg / kg colistin sulfate + 20 mg / kg dehydroandrographolide intraperitoneal injection). The inoculum size of each mouse in the infection model and each treatment group was 2 × 10 9 After infection with CFU of Escherichia coli E.coli B2, treatment was performed once at 2 hours and 24 hours after infection, and the diarrhea index was detected 3 hours after the last treatment. The number of loose stools, total number of stools, and loose stool size of each group of mice were counted, and the loose stool rate, loose stool grade, and diarrhea index were calculated. Loose stool grade: The degree of loose stool is divided into 4 levels based on the size of the stain area formed by loose stool contamination of filter paper. Diameter <1cm is grade 1, 1~<2cm is grade 2, 2~<3cm is grade 3, and ≥3cm is grade 4. If the stain is a relatively regular circle, its diameter is measured. If it is an irregular shape, the average of the longest and approximate circle diameters is measured. The calculation formula is as follows:

[0100] Loose stool rate = number of loose stools / total number of stools × 100%;

[0101] Average loose stool grade = sum of loose stool grades / number of loose stools;

[0102] Diarrhea index = loose stool rate × average loose stool grade.

[0103] The test results are as follows:

[0104] like Figure 3 As shown, compared with the control group (non-infected group), after E. coli B2 infection, the diarrhea index of mice increased to 0.93. The diarrhea index of the polymyxin E 1mg / kg treatment group did not change significantly, while the diarrhea index of the dehydroandrographolide 20mg / kg treatment group decreased to 0.59. The diarrhea index of the polymyxin E combined with dehydroandrographolide treatment group was significantly reduced to 0.18. This shows that the combined treatment of polymyxin E and dehydroandrographolide has a significant synergistic effect on the diarrhea caused by E. coli infection.

[0105] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. An antibacterial infection composition, the active ingredients of which are dehydroandrographolide and polymyxin antibiotics; The polymyxin antibiotic is polymyxin E or a pharmaceutically acceptable salt thereof, polymyxin B or a pharmaceutically acceptable salt thereof; The mass ratio of the polymyxin antibiotic to dehydroandrographolide is 1:40-640; The bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Salmonella.

2. The antibacterial infection composition according to claim 1, characterized in that: The bacteria include bacteria containing polymyxin resistance genes.

3. An antibacterial product comprising the antibacterial infection composition according to claim 1 or 2.

4. The antibacterial product according to claim 3, characterized in that: The dosage form of the antibacterial product is one of tablets, capsules, oral liquid, syrup, injection, dripping pills and freeze-dried powder injection.

5. The antibacterial product according to claim 4, characterized in that: The tablet is a sustained-release tablet or a controlled-release tablet.

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