Application of chlorogenic acid in inhibition of growth of carbapenem-resistant Escherichia coli

Abstract

The invention provides application of chlorogenic acid in inhibition of growth of carbapenem-resistant Escherichia coli, and belongs to the technical field of development of antibacterial materials. The invention discloses an application of chlorogenic acid in inhibiting the growth of carbapenem-resistant Escherichia coli, the chlorogenic acid can inhibit the growth of the carbapenem-resistant Escherichia coli according to the good in-vitro killing effect on the carbapenem-resistant Escherichia coli, the minimum inhibitory concentration is 5mg / mL, and the chlorogenic acid can inhibit the growth of the carbapenem-resistant Escherichia coli. The invention provides the inhibition effect of the chlorogenic acid on the carbapenem-resistant Escherichia coli, and the chlorogenic acid has a wide application value in the fields of medicines and the like, and can effectively destroy the biological membrane of the carbapenem-resistant Escherichia coli, so that the invention provides the inhibition effect of the chlorogenic acid on the carbapenem-resistant Escherichia coli.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial material development, and in particular to application of chlorogenic acid in inhibiting the growth of carbapenem-resistant Escherichia coli. Background Art

[0002] Escherichia coli is usually a normal flora in the human intestine, and it coexists with humans in a mutually beneficial symbiosis. However, under certain conditions, some strains of Escherichia coli can cause a variety of diseases. The following are diseases that may be caused by Escherichia coli: diarrhea, abdominal pain, nausea, vomiting, fever, urinary tract infection, soft tissue infection, neonatal sepsis, etc. In summary, Escherichia coli can cause a variety of diseases under certain conditions, ranging from mild gastrointestinal symptoms to severe systemic infections. With the widespread use of antibiotics in clinical practice, bacterial resistance is becoming stronger and stronger. The emergence of carbapenem-resistant Escherichia coli has brought great challenges to clinical treatment.

[0003] Traditional Chinese medicine has complex ingredients, multiple bactericidal mechanisms, is not easy to develop drug resistance, and has a certain effect in the treatment of infection. Chlorogenic acid is the main active ingredient in honeysuckle polyphenols. There are certain studies on its antioxidant, anti-cancer, antibacterial, antiviral, and anti-inflammatory effects at home and abroad. The literature (Naveed M, Hejazi V, Abbas M, Kamboh AA, Khan GJ, Shumzaid M, Ahmad F, Babazadeh D, Fang Fang X, Modarresi-Ghazani F, Wen Hua L, Xiao Hui Z. Chlorogenic acid (CGA): A pharmacological review and call for further research. Biomed Pharmacother. 2018 Jan; 97: 67-74. doi: 10.1016 / j.biopha.2017.10.064 Epub 2017 Nov 6. PMID: 29080460.) reported that chlorogenic acid (CGA, 3-CQA) is the most abundant isomer among caffeoylquinic acid isomers (3-, 4- and 5-CQA), and it is one of the most available acids among phenolic acid compounds. Chlorogenic acid is an important bioactive dietary polyphenol with multiple important therapeutic effects such as antioxidant activity, antibacterial, hepatoprotective, cardioprotective, anti-inflammatory, antipyretic, neuroprotective, anti-obesity, antiviral, antimicrobial, antihypertensive, free radical scavenger and central nervous system (CNS) stimulant. In addition, chlorogenic acid has been found to regulate lipid metabolism and glucose in genetic and health metabolism-related diseases. However, new application areas are still to be developed in the prior art. Summary of the invention

[0004] The object of the present invention is to provide the use of chlorogenic acid in inhibiting the growth of carbapenem-resistant Escherichia coli.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of chlorogenic acid in inhibiting the growth of Escherichia coli.

[0007] Preferably, the Escherichia coli is carbapenem-resistant Escherichia coli.

[0008] Preferably, the carbapenem-resistant Escherichia coli is resistant to carbapenem antibiotics;

[0009] The carbapenem antibiotics include imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline.

[0010] The present invention also provides the use of chlorogenic acid in preparing a drug for inhibiting the growth of carbapenem-resistant Escherichia coli.

[0011] Preferably, the carbapenem-resistant Escherichia coli is able to tolerate multiple carbapenem antibiotics;

[0012] The carbapenem antibiotics include imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline.

[0013] Preferably, the concentration of chlorogenic acid in the drug is above 5 mg / mL.

[0014] The present invention also provides a carbapenem-resistant Escherichia coli growth inhibitor, wherein the growth inhibitor has chlorogenic acid as a main active ingredient.

[0015] Preferably, the growth inhibitor further contains other antibacterial active ingredients and / or excipients.

[0016] Preferably, the concentration of chlorogenic acid in the growth inhibitor is above 5 mg / mL.

[0017] The present invention also provides the use of chlorogenic acid in the preparation of a drug for treating or assisting in the treatment of diarrhea, abdominal pain, nausea, vomiting, fever, urinary tract infection, soft tissue infection or neonatal sepsis caused by Escherichia coli.

[0018] Beneficial effects of the present invention:

[0019] The present invention starts from the existing medicinal plant resource library to explore potential drug-resistant bacteria inhibitors. Based on the study of the effect of chlorogenic acid on carbapenem-resistant Escherichia coli, it is found that the non-antibiotic compound chlorogenic acid can effectively inhibit the growth of carbapenem-resistant Escherichia coli, which provides new ideas and sources for the research, development and application of carbapenem-resistant Escherichia coli inhibitors, and has wide application value in the fields of medicine and so on.

[0020] The present invention clarifies the inhibitory effect of chlorogenic acid on carbapenem-resistant Escherichia coli, and uses it as an antibiotic substitute to effectively alleviate or solve the drug resistance and infection problems of carbapenem-resistant Escherichia coli and reduce the mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the MIC result of chlorogenic acid against carbapenem-resistant Escherichia coli;

[0022] Figure 2 This is the imaging result of chlorogenic acid staining for the live or dead of carbapenem-resistant Escherichia coli. DETAILED DESCRIPTION

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] Example 1

[0025] 1. Antimicrobial susceptibility test of human Escherichia coli

[0026] The present invention uses multiple strains of human Escherichia coli (strain samples are taken from the Third People's Hospital of Henan Province) as starting strains, and selects drug sensitivity test strips of 7 commonly used antibiotics, including imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline, for drug sensitivity testing.

[0027] Pick the pure colonies cultured for 18 to 24 hours and evenly dissolve them in 2 to 5 mL of sterile saline, and adjust the turbidity to be equal to that of a 0.5 McFarland turbidimeter. Aseptically aspirate 50 to 200 μL of bacterial solution, evenly spread it on the plate, and place it at room temperature for 3 to 5 minutes. Use sterile tweezers to place the drug sensitivity test paper (6 mm in diameter) close to the surface of the plate as required, and three parallels for each drug sensitivity test paper. Place the inoculated plate at 36 to 37 ° C and incubate for 16 to 24 hours. Use a vernier caliper with an accuracy of 0.02 mm to measure the diameter of the inhibition zone, and use the diameter of the inhibition zone to judge the sensitivity of the drug. The inhibition results are determined according to the standards of the National Standards Management Committee of the Clinical Laboratory of the United States (CLSl2017), and the judgment criteria are shown in Table 1. The experimental results are shown in Table 2. The results show that the human Escherichia coli selected from the clinic can tolerate 8 common clinical antibiotics, so it is used as the research object for the next experiment.

[0028] Table 1 Results of the American National Standards Administration Committee for Clinical Laboratory Standardization (CLS12024)

[0029]

[0030] Table 2 MIC results of human Escherichia coli in drug sensitivity test

[0031]

[0032] Note: The unit of MIC is ug / ml, and the values ​​are the average of three parallel

[0033] 2. Inhibitory effect of chlorogenic acid on multidrug-resistant strains

[0034] In order to fully consider the safety of medication, the present invention uses chlorogenic acid, a single active ingredient, as the research object, and uses clinically collected human Escherichia coli as the experimental strain to study the drug resistance inhibition effect.

[0035] MIC test: Each group was set up for 3 parallel tests, and control group 1 (LB liquid culture medium + bacterial solution) and control group 2 (LB liquid culture medium) were set up. Sample stock solution 1 (chlorogenic acid sigma) was diluted to 80 mg / mL (2×) and sample stock solution 2 (chlorogenic acid MCE) was diluted to 100 mg / mL (2×) using LB liquid culture medium. The samples were diluted by two-fold dilution method, and then cultured at 37°C for 24 hours. The samples were photographed and the MIC values ​​were recorded. The experimental results are shown in Figure 1 .

[0036] According to its MIC value, live and dead staining imaging was performed. Add cell slides to the 24-well plate, take 1mL (106CFU / mL) of bacterial solution and add it to the 24-well plate, place it in a 37°C constant temperature incubator for 48 hours, and replace fresh culture medium every 24 hours. Gently rinse the unattached bacteria with sterile PBS, and then add drugs with MIC concentrations corresponding to different strains to the 24-well plate according to the grouping, and incubate it in a 37°C constant temperature incubator for 24 hours. After the incubation, rinse gently with PBS 3 times. SYTO-9 (5μM) and PI (2μg / mL), take 200μL of staining working solution respectively and add it to the sample surface, incubate it in the dark at 37°C for 15 minutes. After the staining is completed, discard the staining solution and shoot at 400 times. The experimental results are as follows Figure 2 .

[0037] Depend on Figure 1 and Figure 2It can be seen that chlorogenic acid has a good inhibitory effect on Escherichia coli resistant to imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline, and its MIC is 5 mg / mL. The results show that chlorogenic acid has a good inhibitory effect on carbapenem-resistant Escherichia coli, effectively destroys the biofilm of carbapenem-resistant Escherichia coli, and can be used to inhibit the growth of carbapenem-resistant Escherichia coli.

[0038] It can be seen from the above embodiments that, combined with the characteristics of Chinese herbal medicine such as wide sources, few adverse reactions and not easy to develop drug resistance, the active single ingredient chlorogenic acid can be obtained to directly inhibit the main clinical carbapenem-resistant Escherichia coli, which can effectively alleviate or solve the infection problem of carbapenem-resistant Escherichia coli, reduce the mortality rate, provide a scientific basis for the study of antibacterial agents for clinically isolated Escherichia coli, and provide new ideas and sources for the development of drugs and antibiotic substitutes for inhibiting carbapenem-resistant Escherichia coli.

[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of chlorogenic acid in inhibiting the growth of Escherichia coli.

2. The use according to claim 1, characterized in that: The Escherichia coli is carbapenem-resistant Escherichia coli.

3. The use according to claim 2, characterized in that: The carbapenem-resistant Escherichia coli is resistant to carbapenem antibiotics; The carbapenem antibiotics include imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline.

4. The use of chlorogenic acid in the preparation of drugs for inhibiting the growth of carbapenem-resistant Escherichia coli.

5. The use according to claim 4, characterized in that: The carbapenem-resistant Escherichia coli can tolerate multiple carbapenem antibiotics; The carbapenem antibiotics include imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline.

6. The use according to claim 4, characterized in that: The concentration of chlorogenic acid in the drug is above 5 mg / mL.

7. A carbapenem-resistant Escherichia coli growth inhibitor, characterized in that: The growth inhibitor has chlorogenic acid as a main active ingredient.

8. The growth inhibitor according to claim 7, characterized in that The growth inhibitor may also contain other antibacterial active ingredients and / or auxiliary materials.

9. The growth inhibitor according to claim 7, characterized in that The concentration of chlorogenic acid in the growth inhibitor is above 5 mg / mL.

10. Use of chlorogenic acid in the preparation of drugs for treating or assisting in the treatment of diarrhea, abdominal pain, nausea, vomiting, fever, urinary tract infection, soft tissue infection or neonatal sepsis caused by Escherichia coli.

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