Application of chlorogenic acid in preparation of carbapenem-resistant klebsiella pneumoniae product

Abstract

The invention discloses application of chlorogenic acid in preparation of a product for resisting carbapenem-resistant klebsiella pneumoniae, and belongs to the technical field of medicines. It is verified that chlorogenic acid has a good in-vitro killing effect on carbapenem-resistant klebsiella pneumonia, growth of the carbapenem-resistant klebsiella pneumonia can be inhibited, the minimum inhibitory concentration is 20 mg / mL, and a biological membrane of the carbapenem-resistant klebsiella pneumonia can be effectively destroyed. The invention provides the inhibition effect of chlorogenic acid on carbapenem-resistant klebsiella pneumoniae, and the chlorogenic acid has wide application value in the fields of medicines and the like.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of chlorogenic acid in the preparation of products for resisting carbapenem-resistant Klebsiella pneumoniae. Background Art

[0002] Klebsiella pneumoniae is a common nosocomial pathogen and the most important bacterium in the genus Klebsiella of the family Enterobacteriaceae. Klebsiella pneumoniae can cause typical primary pneumonia and various extrapulmonary infections, such as infant enteritis, meningitis, nosocomial urinary tract infections in adults, as well as traumatic infections and bacteremia. With the widespread use of antibiotics in clinical practice, bacterial resistance is becoming stronger. The emergence of carbapenem-resistant Klebsiella pneumoniae 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): Apharmacological 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, including 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, there are few reports on chlorogenic acid inhibiting the growth of carbapenem-resistant Klebsiella pneumoniae. Summary of the invention

[0004] The present invention aims at solving the drug resistance problem currently faced by clinical medicine, and aims to provide application of chlorogenic acid in preparing a product for resisting carbapenem-resistant Klebsiella pneumoniae.

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

[0006] The present invention provides an application of chlorogenic acid in preparing a product for resisting carbapenem-resistant Klebsiella pneumoniae.

[0007] Furthermore, the product is a drug for use against carbapenem-resistant Klebsiella pneumoniae.

[0008] Furthermore, the carbapenem-resistant Klebsiella pneumoniae is human Klebsiella pneumoniae.

[0009] Furthermore, the drug resistance of the Klebsiella pneumoniae is determined by a disk diffusion method.

[0010] The antibiotics to which the carbapenem-resistant Klebsiella pneumoniae is resistant are selected from one or more of imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline.

[0011] Furthermore, the minimum inhibitory concentration of chlorogenic acid against carbapenem-resistant Klebsiella pneumoniae is 20 mg / ml.

[0012] Furthermore, the drug also contains one or more pharmaceutically acceptable carriers.

[0013] In addition, the present invention also provides a use of a composition containing chlorogenic acid in the preparation of a drug for resisting carbapenem-resistant Klebsiella pneumoniae.

[0014] Furthermore, the composition containing chlorogenic acid is a pharmaceutical composition.

[0015] Furthermore, the pharmaceutical composition contains one or more pharmaceutically acceptable carriers.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 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 Klebsiella pneumoniae, it is found that the non-antibiotic compound chlorogenic acid can effectively inhibit the growth of carbapenem-resistant Klebsiella pneumoniae, which provides new ideas and sources for the research, development and application of carbapenem-resistant Klebsiella pneumoniae inhibitors, and has wide application value in the fields of medicine and so on.

[0018] Furthermore, the present invention clarifies the inhibitory effect of chlorogenic acid on carbapenem-resistant Klebsiella pneumoniae. Using it as an antibiotic substitute can effectively alleviate or solve the drug resistance and infection problems of carbapenem-resistant Klebsiella pneumoniae and reduce the mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the MIC result of chlorogenic acid against carbapenem-resistant Klebsiella pneumoniae in Example 1;

[0020] Figure 2 The live and dead staining imaging results of chlorogenic acid on carbapenem-resistant Klebsiella pneumoniae in Example 1; DETAILED DESCRIPTION

[0021] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0022] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0023] Example 1

[0024] 1. Antimicrobial susceptibility test of human Klebsiella pneumoniae

[0025] The present invention uses multiple strains of human Klebsiella pneumoniae (strain samples were taken from the Third People's Hospital of Henan Province) as the starting strains, and selects 7 commonly used antibiotics including imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline for drug sensitivity test. The antibacterial results are determined according to the standards of the National Standardization Management Committee of Clinical Laboratory of the United States (CLS12017), and the judgment criteria are shown in Table 1. The experimental results are shown in Table 2.

[0026] Table 1 Results of the American National Standards Administration Committee for Clinical Laboratory Standardization (CLS12024)

[0027]

[0028] Table 2 MIC results of drug susceptibility test of human Klebsiella pneumoniae

[0029]

[0030] Note: The unit of MIC is ug / ml, and the values ​​are the average of three parallel assays.

[0031] The results showed that the human Klebsiella pneumoniae selected from clinical sources could tolerate 8 common clinical antibiotics, so it was selected as the research subject for the next experiment.

[0032] 2. Inhibitory effect of chlorogenic acid on multidrug-resistant strains

[0033] In order to fully consider the safety of medication, the present invention uses chlorogenic acid, a single active ingredient, as the research object, and clinically collected human Klebsiella pneumoniae as the experimental strain to study the drug resistance inhibition effect.

[0034] MIC test: The bacterial solution was prepared using LB liquid culture medium, and the bacterial solution concentration was 10 6 CFU / mL; Use 10% DMSO solution to dissolve the chlorogenic acid powder, then use LB liquid culture medium to dilute the sample mother solution 1 (chlorogenic acid sigma) to 80 mg / mL (2×), and dilute the sample mother solution 2 (chlorogenic acid MCE) to 100 mg / mL (2×), and use the two-fold dilution method to dilute the concentration step by step. The 10% DMSO solution is also diluted step by step by the two-fold dilution method. After the treatment, in the experimental group, 100 μL of bacterial solution was added to the well plate, and 100 μL of the diluted chlorogenic acid solution was added in turn; in addition to the experimental group, control group 1 (bacterial solution + DMSO) and control group 2 (DMSO) were set up. In the control group, control group 1 added 100 μL of bacterial solution and 100 μL of DMSO solution diluted in sequence to the well plate, and control group 2 only added 200 μL of DMSO solution diluted in sequence to the well plate; after addition, the well plate was placed at 37°C for 24 hours, and the lowest concentration at which the culture medium was clear and no bacterial growth was observed by naked eye was determined as the minimum inhibitory concentration of the drug, i.e., the MIC value. Each group was set up for 3 parallels. Take pictures and record. The experimental results are shown in Figure 1 .

[0035] According to the MIC value, live and dead staining imaging was performed. Add cell slides to the 24-well plate and take 1 mL (10 6 CFU / mL) bacterial solution was added to a 24-well plate and placed in a 37°C constant temperature incubator for 48 hours, with fresh LB liquid culture medium replaced every 24 hours. Gently rinse the unattached bacteria with sterile PBS solution, and then add the MIC concentration of drugs to the 24-well plate according to the grouping, and incubate in a 37°C constant temperature incubator for 24 hours. After incubation, gently rinse 3 times with PBS. Take 200μL of the staining working solution SYTO-9 (5μM) and PI (2μg / mL) respectively and add them to the sample surface, and incubate at 37°C in the dark for 15 minutes. After staining is completed, discard the staining solution and take a picture at 400 times. The experimental results are as follows Figure 2 .

[0036] Depend on Figure 1 and Figure 2It can be seen that chlorogenic acid has a good inhibitory effect on Klebsiella pneumoniae resistant to imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline, and its MIC is 20 mg / mL. The results show that chlorogenic acid has a good inhibitory effect on carbapenem-resistant Klebsiella pneumoniae, effectively destroys the biofilm of carbapenem-resistant Klebsiella pneumoniae, and can be used to inhibit the growth of carbapenem-resistant Klebsiella pneumoniae.

[0037] According to the above experimental results, 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 Klebsiella pneumoniae, which can effectively alleviate or solve the infection problem of carbapenem-resistant Klebsiella pneumoniae and reduce the mortality rate. It provides a scientific basis for the study of antibacterial agents for clinically isolated Klebsiella pneumoniae and provides new ideas and sources for the development of drugs and antibiotic alternatives to inhibit carbapenem-resistant Klebsiella pneumoniae.

[0038] 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. Application of chlorogenic acid in the preparation of products against carbapenem-resistant Klebsiella pneumoniae.

2. The use according to claim 1, characterized in that The product is a drug against carbapenem-resistant Klebsiella pneumoniae.

3. The use according to claim 1, characterized in that The carbapenem-resistant Klebsiella pneumoniae is human Klebsiella pneumoniae.

4. The use according to claim 1, characterized in that The drug resistance of Klebsiella pneumoniae was determined by disk diffusion method.

5. The use according to claim 1, characterized in that The antibiotics to which the carbapenem-resistant Klebsiella pneumoniae is resistant are selected from one or more of imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, levofloxacin and tetracycline.

6. The use according to claim 1, characterized in that The minimum inhibitory concentration of chlorogenic acid against carbapenem-resistant Klebsiella pneumoniae is 20 mg / ml.

7. The use according to claim 1, characterized in that The drug further contains one or more pharmaceutically acceptable carriers.

8. Use of a composition containing chlorogenic acid in the preparation of a drug for use against carbapenem-resistant Klebsiella pneumoniae.

9. The use according to claim 8, characterized in that The chlorogenic acid-containing composition is a pharmaceutical composition.

10. The use according to claim 9, characterized in that The pharmaceutical composition contains one or more pharmaceutically acceptable carriers.

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