Application of phenazine-1-carboxylic acid in preparation of medicine for inhibiting methicillin-resistant staphylococcus aureus

Abstract

The invention discloses application of phenazine-1-carboxylic acid in preparation of a medicine for inhibiting methicillin-resistant staphylococcus aureus, and belongs to the technical field of biological medicine. The phenazine-1-carboxylic acid has a good antagonistic effect, shows good antibacterial activity to various gram-positive bacteria and fungi, has good temperature stability and illumination stability, has good safety to various animals, and can be applied to preparation of drugs for treating various animals. The phenazine-1-carboxylic acid has a good treatment effect on greater wax moth larvae and mice infected with methicillin-resistant staphylococcus aureus. The phenazine-1-carboxylic acid disclosed by the invention is expected to enrich the existing antibacterial drug library at present, and provides an important basis for treating infection of methicillin-resistant staphylococcus aureus.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of phenazine-1-carboxylic acid in preparing a drug for inhibiting methicillin-resistant Staphylococcus aureus. Background Art

[0002] With the irrational use of antibiotics in clinic, microbial resistance to antibiotics is becoming more and more serious. The development speed of microbial resistance has exceeded the development speed of new antimicrobial drugs, which brings severe challenges to the clinical treatment of bacterial infectious diseases. According to statistics from the World Health Organization, at least 700,000 people die from multidrug-resistant pathogens every year. Methicillin-resistant Staphylococcus aureus (MRSA) refers to Staphylococcus aureus strains resistant to isoxacillin, methicillin and other isoxacillin, also known as "super bacteria". It spreads quickly and has strong adaptability. Since it was first discovered by Jevons in the UK in 1961, the infection has spread almost all over the world. The spread of MRSA infection poses a major threat to public health. It not only increases the complexity of infection and the difficulty of treatment, but also may be widely spread among different populations through various channels, leading to serious health consequences and economic burden. Therefore, it is crucial to strengthen preventive measures and control strategies.

[0003] Phenazine-1-carboxylic acid (PCA) is a compound with a unique chemical structure. It was first isolated from the metabolites of Bacillus bacteria in 1930. PCA and its derivatives have attracted widespread attention due to their broad-spectrum antibacterial, antifungal, antiviral and other biological activities. Studies have found that PCA can effectively inhibit a variety of plant pathogenic fungi, such as rice sheath blight and wheat fusarium, with an inhibition rate of about 90-100%. The antibacterial mechanism of PCA is mainly achieved by interfering with the growth and reproduction of pathogens. Phenazine-1-carboxylic acid and its derivatives have significant antifungal activity against all plant pathogenic fungi, so they have important application prospects in the field of pesticides. However, the antibacterial effect on MRSA or other bacteria has not been fully explored. PCA is expected to become a new direction for the development and application of drugs against MRSA infection or bacterial infection. Summary of the invention

[0004] The purpose of the present invention is to provide an application of phenazine-1-carboxylic acid in the preparation of a drug for inhibiting methicillin-resistant Staphylococcus aureus, so as to solve the problems existing in the above-mentioned prior art. Phenazine-1-carboxylic acid has antibacterial activity against a variety of Gram-positive bacteria and animal pathogenic fungi, especially has a significant inhibitory effect on methicillin-resistant Staphylococcus aureus, and is expected to enrich the current antibacterial drug library and provide new hope for the effective treatment of methicillin-resistant Staphylococcus aureus infection.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of phenazine-1-carboxylic acid in any of the following:

[0007] (1) Application in the preparation of drugs for inhibiting bacteria;

[0008] (2) Application in the preparation of drugs for treating bacterial infections.

[0009] Optionally, the bacteria include Gram-positive bacteria.

[0010] Preferably, the Gram-positive bacteria include Methicillin-resistant Staphylococcus aureus, Aeromonas veronii and Bacillus subtilis.

[0011] The present invention also provides the use of phenazine-1-carboxylic acid in any of the following:

[0012] (1) Application in the preparation of drugs for inhibiting Candida albicans;

[0013] (2) Application in the preparation of drugs for treating diseases caused by Candida albicans infection.

[0014] The invention also provides the use of phenazine-1-carboxylic acid in preparing a medicine for improving the survival rate of bacterial infection receptors.

[0015] Optionally, the bacteria include Gram-positive bacteria.

[0016] Preferably, the Gram-positive bacteria include methicillin-resistant Staphylococcus aureus, Aeromonas veronii and Bacillus subtilis.

[0017] Optionally, the recipient includes animals and insects.

[0018] The present invention also provides an anti-Gram-positive bacteria and / or fungus drug, which comprises phenazine-1-carboxylic acid and pharmaceutically acceptable adjuvants and carriers.

[0019] Optionally, the Gram-positive bacteria include methicillin-resistant Staphylococcus aureus, Aeromonas veronii and Bacillus subtilis, and the fungi include Candida albicans.

[0020] The present invention discloses the following technical effects:

[0021] The present invention finds that phenazine-1-carboxylic acid has antibacterial activity against a variety of Gram-positive bacteria and animal-derived fungal pathogens, and especially shows good antibacterial activity and stability against MRSA; phenazine-1-carboxylic acid has good safety for a variety of animals, and especially has good therapeutic effects on mice and greater wax moth infected with MRSA, wherein 20 mg / kg of phenazine-1-carboxylic acid can increase the survival rates of greater wax moth larvae and mice infected with MRSA by 70% and 50%, respectively, and has the potential to become an anti-MRSA drug.

[0022] The experiments of the present invention prove that phenazine-1-carboxylic acid is expected to enrich the existing antibacterial drug library and provide new hope for the effective treatment of MRSA infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 is the bactericidal curve of phenazine-1-carboxylic acid;

[0025] Figure 2 It is the result of hemolytic activity assay;

[0026] Figure 3 This is the result of the survival rate test of the larvae of G. mellonella;

[0027] Figure 4 The results of the mouse survival rate determination;

[0028] Figure 5 The results of the bacterial load determination of each organ of mice;

[0029] Figure 6 Results of histopathological analysis of various organs of mice. Scale bar: 1:400. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The chemical formula of phenazine-1-carboxylic acid is C 13 H8N2O2, CAS number: 2538-68-3, the structural formula is as follows:

[0036]

[0037] The phenazine-1-carboxylic acid used in the present invention is purchased from conventional commercial channels.

[0038] Example 1 Stability Analysis of Antibacterial Substance Phenazine-1-Carboxylic Acid

[0039] 1. Temperature stability

[0040] In a sterile environment, prepare 5 sterilized 1.5mL centrifuge tubes, inject 100μL of the antibacterial substance phenazine-1-carboxylic acid into each of the 5 sterile centrifuge tubes, then heat the 4 centrifuge tubes containing the antibacterial substance in a water bath at 40℃, 60℃, 80℃, and 100℃ for 30 minutes, and place the remaining antibacterial substance centrifuge tube at room temperature for 30 minutes. MRSA is used as an indicator bacteria, and the antibacterial activity of the antibacterial substance in each tube is verified by the agar diffusion method.

[0041] 2. Light stability

[0042] In two sterilized EP tubes, the same volume of antimicrobial solution was injected respectively, and one of the samples was placed under a UV lamp, 30 cm away from the light source. After 60 minutes, the sample was taken out for testing, and the other group was placed under sunlight for 60 minutes. Using MRSA as the indicator bacteria, the antimicrobial activity of the antimicrobial substances in each tube was verified by the agar diffusion method.

[0043] The stability evaluation was verified by the agar diffusion method. The experimental results showed that phenazine-1-carboxylic acid had good temperature stability and light stability. The results are shown in Table 1.

[0044] Table 1 Stability analysis

[0045]

[0046] Example 2 Determination of the antibacterial effect of phenazine-1-carboxylic acid on different strains

[0047] This embodiment is to determine the minimum inhibitory concentration of the antibacterial substance phenazine-1-carboxylic acid against different drug-resistant bacteria. The MIC of the antibacterial substance phenazine-1-carboxylic acid against Escherichia coli B2, Pseudomonas aeruginosa ATCC9027, Escherichia coli K88, Acinetobacter baumannii, Escherichia coli ATCC 25922, Salmonella, Shigella Castellani, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Aeromonas veronii, Enterococcus faecalis, Bacillus subtilis and Candida albicans was determined. The above indicator bacteria are all stored in the Pathogenic Microbiology Laboratory of the College of Life Sciences of Jilin Agricultural University. The public can obtain them from the applicant and only use them for repeated implementation of the experiment of the present invention.

[0048] The MIC determination method is as follows:

[0049] (1) Prepare the antibacterial substance phenazine-1-carboxylic acid at a concentration of 1024 μg / mL.

[0050] (2) Inoculate the indicator bacteria mentioned above into MH broth medium and culture in a shaking incubator at 37°C until OD 600 The bacterial concentration was adjusted to 0.5 using MH medium. 5 CFU / mL.

[0051] (3) Add 100 μL of MH broth medium to wells 1 to 12 of a sterile 96-well plate, add 100 μL of phenazine-1-carboxylic acid to well 1, pipette and mix well from well 1 and pipette 100 μL to well 2, dilute to well 10 in sequence as described above, and then pipette 100 μL of the diluted indicator bacteria in Table 2 and add them to wells 1 to 10 in sequence. Add only 200 μL of MH broth medium to well 11 as a negative control, and add 100 μL of MH medium and 100 μL of pathogenic bacteria solution to well 12 as a positive control.

[0052] (4) Place the 96-well plate in a 37°C incubator and culture for 12-14 hours (the time varies depending on the logarithmic growth phase of the indicator bacteria). Use an ELISA reader to measure the OD 600 The results are shown in Table 2.

[0053] Table 2 MIC determination of antibacterial substance phenazine-1-carboxylic acid against various pathogens

[0054]

[0055] Example 3 Time-kill curve of phenazine-1-carboxylic acid against MRSA

[0056] 1. Preparation of MRSA bacterial suspension

[0057] Take MRSA out of the -80℃ freezer, streak it on the LB agar plate, and then place it in a 37℃ incubator for 12h. Pick a single colony and inoculate it in MH broth medium, and culture it on a shaker at 37℃ until its optical density (OD 600nm ) reached 0.5, a 100-fold dilution was performed to make the colony forming unit 10 6 CFU / mL of bacterial solution.

[0058] 2. Sterilization curve determination

[0059] 10 6 The bacterial suspension of CFU / mL and PCA with concentrations of 0.5×MIC, 1×MIC, 2×MIC, 4×MIC and 8×MIC were added to a 96-well microplate and then placed in a shaker at 37°C. The absorbance of the bacterial suspension was detected by an ELISA reader every 2 hours.

[0060] The results are as follows Figure 1 As shown in the figure, compared with the PBS group, the number of MRSA after antimicrobial treatment decreased significantly with the extension of the action time. Under 1×MIC treatment, the bacteria decreased by about 2 orders of magnitude within 24 hours, and under 2×MIC treatment, MRSA growth was quickly inhibited within 4 hours. Under 4×MIC and 8×MIC treatment, MRSA almost stopped growing.

[0061] Example 4 Safety Assessment of Phenazine-1-Carboxylic Acid

[0062] 1. Hemolytic activity assay

[0063] Collect fresh blood from healthy sheep, put it into a pre-prepared centrifuge tube (containing 0.2% sodium heparin), centrifuge at 8500rpm / min for 15min, wash 3 times with sterile PBS, and prepare a 1% red blood cell suspension. Prepare 11 centrifuge tubes, add 100μL of blood cells and 100μL of different concentrations of antibacterial substances (1μg / mL, 2μg / mL, 4μg / mL, 8μg / mL, 16μg / mL, 32μg / mL, 64μg / mL, 128μg / mL, 256μg / mL, 512μg / mL) to the 1st to 9th centrifuge tubes, and then gently shake and mix. Add 100μL of red blood cells and 100μL of phosphate buffer (PBS) to the 10th centrifuge tube as a negative control. Add 100μL of red blood cells and 100μL of TritonX-100 with a final concentration of 0.2% to the 11th centrifuge tube as a positive control. Place these centrifuge tubes in a 37°C incubator for 1 hour, centrifuge at 3000 rpm / min for 8 minutes, and then transfer 150 μL of supernatant to a sterile 96-well plate. Use an ELISA reader to read the absorbance at 576 nm. Set up three biological replicates for each treatment. Reference formula for hemolytic activity calculation:

[0064] Hemolysis rate (%) = (OD sample -OD blank ) / (OD Triton-100 -OD blank )×100

[0065] The results are as follows Figure 2 As shown, after treatment with different concentrations of antibacterial substances (0.5-512 μg / mL), the hemolysis rate of red blood cells was less than 5%, indicating that the compound had almost no hemolytic activity.

[0066] 2. Acute toxicity test

[0067] Mouse breeding and mouse experiments were carried out in full accordance with the animal use requirements of Jilin Agricultural University. Ten mice, 5 males and 5 females, were acclimated to the breeding for one week, and then intraperitoneally injected with 20 mg / kg of the antibacterial substance phenazine-1-carboxylic acid dilution, and the mice were bled after 48 hours of eye removal. The blood collected by the mice was tested for blood indicators using a blood analyzer, and the remaining mice were observed for survival within 7 days. The results are shown in Table 3. All indicators of the blood tests of the mice were normal, and there was no death of the mice by observation.

[0068] Table 3 Detection of blood indexes in mice

[0069]

[0070]

[0071] Example 5: Experiment on protection of wax moth larvae

[0072] Fifty wax moth larvae were kept overnight at room temperature and randomly divided into four groups, with 10 larvae in each group. The larvae in groups one, two and three were injected with 10 μL of MRSA solution (1×10 7 CFU / mL), and 1 hour later, the first and second groups of wax moth larvae were injected with 10mg / kg and 20mg / kg of phenazine-1-carboxylic acid into the right ventral foot for treatment. The third group of wax moth larvae were treated with 10μL sterile PBS as a negative control group. The fourth group of wax moth larvae were only given 10μL PBS as a blank control group. The survival of wax moth larvae was observed every day for 72 hours and the survival of wax moth larvae was recorded.

[0073] like Figure 3 As shown, compared with the control group, the survival rate of the wax moth larvae within 72 hours was 50% after treatment with 10 mg / kg of phenazine-1-carboxylic acid, and the survival rate of the wax moth larvae within 72 hours was 70% after treatment with 20 mg / kg of the antibacterial substance phenazine-1-carboxylic acid.

[0074] Example 6 Mouse protection experiment

[0075] Thirty 4-week-old Kunming mice (half male and half female) were randomly divided into three groups, with 10 mice in each group.

[0076] Group 1: Bacteria group (2×10 9 CFU / mL) + phenazine-1-carboxylic acid (10 mg / kg, 100 μL);

[0077] Group 2: Bacteria group (2×10 9 CFU / mL) + phenazine-1-carboxylic acid (20 mg / kg, 100 μL);

[0078] Group 3: Bacteria group (2×10 9 CFU / mL) + sterile PBS buffer (100 μL);

[0079] The mice in the first, second and third groups were intraperitoneally injected with 500 μL of MRSA (2×10 9CFU / mL), 1h later, the first and second groups were treated with 10mg / kg and 20mg / kg of phenazine-1-carboxylic acid, respectively, and the third group was treated with 100μL of sterile PBS buffer as a negative control group. The mice were observed for 48h and the survival of the mice was recorded. After 48h, the mice were dissected, the lung changes were examined and photographed. Then the heart, liver, spleen, lungs and kidneys were taken, and some organs were washed with sterile saline, ground and diluted in multiples for colony counting. The other part was rinsed with sterile saline, fixed with paraformaldehyde, and the samples were sent to Sevier Biotechnology Co., Ltd. for HE staining.

[0080] After 48 hours, the survival of mice in each group was as follows Figure 4 As shown, compared with the third group, the survival rate of mice within 2 days was 40% after treatment with 10 mg / kg of phenazine-1-carboxylic acid, and the survival rate of mice within 2 days was 50% after treatment with 20 mg / kg of the antibacterial substance phenazine-1-carboxylic acid.

[0081] The bacterial load of each organ of mice is as follows Figure 5 As shown, compared with the third group of mice, the bacterial load in the organs of mice treated with phenazine-1-carboxylic acid was significantly reduced. Except for the kidneys, which showed no significant changes, the bacterial content in each organ could be reduced by about 2-4 orders of magnitude. The experimental results show that different doses of phenazine-1-carboxylic acid can significantly reduce the bacterial load in the organs of mice infected with MRSA, and have a good therapeutic effect on mice infected with MRSA.

[0082] Organ histopathological analysis Figure 6 As shown in the figure, the liver of mice infected with MRSA was severely vacuolated and had obvious inflammatory infiltration. The heart, spleen, lungs and kidneys showed hemorrhages, punctate necrosis, dark red cytoplasm, interstitial hemorrhage, granular chromatin and other histopathological changes. After treatment with phenazine-1-carboxylic acid, the pathological changes in various tissues and organs of mice infected with MRSA were alleviated.

[0083] The above results indicate that phenazine-1-carboxylic acid can effectively alleviate the pathological lesions of mouse organs caused by MRSA infection in mice, and has a good improvement effect on the damage of mouse tissues and organs.

[0084] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of phenazine-1-carboxylic acid in any of the following: (1) Application in the preparation of drugs for inhibiting bacteria; (2) Application in the preparation of drugs for treating bacterial infections.

2. The use according to claim 1, characterized in that The bacteria include Gram-positive bacteria.

3. The use according to claim 2, characterized in that The Gram-positive bacteria include Methicillin-resistant Staphylococcus aureus, Aeromonas veronii and Bacillus subtilis.

4. Use of phenazine-1-carboxylic acid in any of the following: (1) Application in the preparation of drugs for inhibiting Candida albicans; (2) Application in the preparation of drugs for treating diseases caused by Candida albicans infection.

5. Application of phenazine-1-carboxylic acid in the preparation of drugs for improving the survival rate of bacterial infection receptors.

6. The use according to claim 5, characterized in that The bacteria include Gram-positive bacteria.

7. The use according to claim 6, characterized in that The Gram-positive bacteria include methicillin-resistant Staphylococcus aureus, Aeromonas veronii and Bacillus subtilis.

8. The use according to claim 5, characterized in that The recipients include animals and insects.

9. An anti-Gram-positive bacteria and / or fungus drug, characterized in that: The anti-Gram-positive bacteria and / or fungus drug comprises phenazine-1-carboxylic acid and pharmaceutically acceptable adjuvants and carriers.

10. The drug according to claim 9, characterized in that The Gram-positive bacteria include methicillin-resistant Staphylococcus aureus, Aeromonas veronii and Bacillus subtilis, and the fungi include Candida albicans.

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