Use of carnosine in preparation of antibacterial drugs

Abstract

The application discloses application of a goose myopeptide in preparation of antibacterial drugs and belongs to the technical field of biological medicines. The antibacterial drugs include drugs against escherichia coli and drugs against klebsiella pneumoniae. The goose myopeptide has a significant inhibiting effect on escherichia coli and klebsiella pneumoniae, and has a significant enhancing effect on the anti-klebsiella pneumoniae and escherichia coli infection ability of cirrhotic mice, and reduces the mortality of the cirrhotic mice, thereby providing a new idea for preparation of antibacterial drugs and anti-interference drugs for cirrhotic patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of carnosine in the preparation of antibacterial drugs. BACKGROUND

[0002] Klebsiella pneumoniae and Escherichia coli belong to gram-negative bacteria, often infect humans, and have high morbidity and mortality, causing serious disease burden worldwide. At present, the treatment of infection of the two bacteria mainly depends on antibacterial drugs, but excessive use of antibacterial drugs can easily cause bacterial drug resistance, resulting in poorer and poorer treatment effect, and new auxiliary therapeutic drugs are urgently needed to improve the ability of patients to resist Klebsiella pneumoniae and Escherichia coli infection.

[0003] Cirrhosis is the end-stage complication caused by various chronic liver diseases, and the proportion of cirrhosis patients infected with Klebsiella pneumoniae and Escherichia coli is very high, and after infection, it can cause hepatic encephalopathy, sepsis, liver failure and other causes of patient death. At present, the treatment of cirrhosis combined with Klebsiella pneumoniae and Escherichia coli infection also mainly depends on antibacterial drugs, but it also causes increased drug resistance, and new auxiliary therapeutic drugs are urgently needed to improve the ability of cirrhosis patients to resist Klebsiella pneumoniae and Escherichia coli infection.

[0004] Carnosine is a histidine dipeptide in the animal body, mainly exists in the muscles and brains of vertebrates, is usually extracted from poultry skeletal muscle, and has significant antioxidant, immune enhancement, anti-aging, uric acid reduction, vasodilation, nerve protection and enzyme activity regulation effects, but its role in Klebsiella pneumoniae and Escherichia coli infection has not been reported. SUMMARY

[0005] The purpose of the present application is to provide the application of carnosine in the preparation of antibacterial drugs to solve the problems existing in the prior art. The present application researches and finds that carnosine has a significant inhibitory effect on Escherichia coli and Klebsiella pneumoniae, and has a significant enhancing effect on the ability of cirrhotic mice to resist Klebsiella pneumoniae and Escherichia coli infection, thereby reducing the mortality of cirrhotic mice, and providing a new idea for the preparation of antibacterial drugs and anti-interference drugs for cirrhosis patients.

[0006] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0007] The present application provides the application of carnosine in the preparation of antibacterial drugs.

[0008] Preferably, the antibacterial drug includes an anti-Escherichia coli and / or Klebsiella pneumoniae drug.

[0009] The present application also provides the application of carnosine in the preparation of anti-infection drugs for cirrhosis.

[0010] Preferably, the anti-infective drugs for cirrhosis include drugs for treating Escherichia coli and / or Klebsiella pneumoniae infection in cirrhosis.

[0011] The present invention discloses the following technical effects:

[0012] This invention has discovered that anserine can enhance the resistance of cirrhotic mice to Klebsiella pneumoniae and Escherichia coli infections, reduce bacterial load, and decrease mortality. Furthermore, anserine also inhibits the growth of Klebsiella pneumoniae and Escherichia coli in vitro, making it suitable for use in the preparation of antibacterial agents. This invention develops new functions for anserine, expands its application scope, and provides a new technical solution for the development of anti-infective drugs for patients with cirrhosis. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 To investigate the inhibitory effect of different concentrations of anserine peptide solution on the growth of Klebsiella pneumoniae;

[0015] Figure 2 The co-culture media for co-culturing Klebsiella pneumoniae with different concentrations of anserine peptide solution for 6 hours;

[0016] Figure 3 The effect of co-culturing different concentrations of anserine solution for 6 hours on the number of Klebsiella pneumoniae;

[0017] Figure 4 To investigate the inhibitory effect of different concentrations of anserine peptide solution on the growth of Escherichia coli;

[0018] Figure 5 The study investigated the effect of anserine on the survival rate of cirrhotic mice infected with Klebsiella pneumoniae. PBS+LC.I represented cirrhotic mice pretreated with PBS, while An+LC.I represented cirrhotic mice pretreated with anserine.

[0019] Figure 6 The study investigated the effect of anserine on body weight changes in cirrhotic mice after infection with Klebsiella pneumoniae. PBS+LC.I represented cirrhotic mice pretreated with PBS, while An+LC.I represented cirrhotic mice pretreated with anserine.

[0020] Figure 7The effect of anserine on blood bacterial load in cirrhotic mice 24 hours after infection with Klebsiella pneumoniae was investigated. PBS+LC.I represents the blood bacterial load of two cirrhotic mice pretreated with PBS, and An+LC.I represents the blood bacterial load of two cirrhotic mice pretreated with anserine. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1

[0027] This embodiment verifies the in vitro antibacterial effect of goose muscle peptide on Klebsiella pneumoniae (KP).

[0028] 1 Experimental Design

[0029] 1.1 Preparation of Anserine Solution: Anserine was purchased from MCE, catalog number HY-113354. 200 mg of anserine was weighed under light-protected conditions and dissolved in 2 mL of MH medium to prepare a 100 mg / mL anserine solution. This solution was then diluted with MH medium to produce working solutions of 12.5 mg / mL, 25 mg / mL, and 50 mg / mL.

[0030] 1.2 Experimental Methods: Based on the concentration of anserine, participants were divided into different groups (0 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL) and compared with 1×10⁻⁶ ppm. 6 CFU KP (purchased from American Type Culture Collection) was co-cultured at 37°C and 220 rpm on a shaker for 24 hours, with MH medium (sterile-free) as the control group. Changes in bacterial concentration in each group were monitored periodically using a microplate reader. At the 6th hour of co-culture, the co-culture media of each group were photographed and recorded. Then, equal volumes of each co-culture media were diluted 1×10⁻⁶. 6 Then, the tablets were counted, and photos were taken of the tablets over 24 hours.

[0031] 1.3 Statistical Methods: Data are expressed as mean ± standard error. Statistical differences were analyzed using one-way ANOVA (when comparing more than two groups) with GraphPad Prism 8 medical graphing and analysis software. A p-value < 0.05 was considered statistically significant; the smaller the p-value, the more significant the difference. * in the figure represents p < 0.05.

[0032] 2 Experimental Results

[0033] 2.1 Growth inhibitory effect of anserine on KP: such as Figure 1 As shown, compared with the MH medium group (without bacteria), the bacterial concentration in the groups containing different concentrations of anserine (0 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL) increased with time. However, compared with 0 mg / mL, the addition of anserine significantly inhibited bacterial growth, and the inhibitory effect was most pronounced at an anserine concentration of 100 mg / mL. The co-culture medium at 6 hours of co-culturing is shown in the figure. Figure 2 As shown, compared with the MH culture medium group (without bacteria), the turbidity of the bacterial solutions containing different concentrations of anserine decreased with the increase of anserine concentration, and the turbidity of the bacterial solutions was lowest at 100 mg / mL.

[0034] 2.2 Antibacterial effect of anserine on KP: such as Figure 3 As shown, the number of bacteria decreased with increasing concentration of anserine peptide after co-culturing with KP at different concentrations.

[0035] Example 2

[0036] This embodiment verifies the in vitro antibacterial effect of goose muscle peptide on Escherichia coli (E. coli).

[0037] 1 Experimental Design

[0038] 1.1 Preparation of Anserine Solution: Anserine was purchased from MCE, catalog number HY-113354. 200 mg of anserine was weighed under light-protected conditions and dissolved in 2 mL of MH medium to prepare a 100 mg / mL anserine solution. This solution was then diluted with MH medium to produce working solutions of 12.5 mg / mL, 25 mg / mL, and 50 mg / mL.

[0039] 1.2 Experimental Methods: Based on the concentration of anserine, participants were divided into different groups (0 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL) and compared with 1×10⁻⁶ ppm. 6 CFU E. coli (purchased from American Type Culture Collection) was co-cultured in a shaker at 37°C and 220 rpm for 24 hours, with MH medium as the control group. The changes in bacterial concentration in each group were detected at regular intervals using an ELISA reader.

[0040] 1.3 Statistical methods: Data are expressed as mean ± standard error.

[0041] 2 Experimental Results

[0042] The growth-inhibiting effect of anserine on E. coli: such as Figure 4 As shown, compared with the MH culture medium group (without bacteria), the bacterial concentration of different concentrations of anserine (0 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL) increased over time. However, compared with 0 mg / mL, the addition of anserine significantly inhibited bacterial growth, and the inhibitory effect was most obvious when the anserine concentration was 100 mg / mL.

[0043] Example 3

[0044] This embodiment verifies the effect of angiosin (An) on the ability of cirrhotic mice to resist Klebsiella pneumoniae (KP) infection.

[0045] 1 Experimental Design

[0046] 1.1 Preparation of Anserine Solution: Anserine was purchased from MCE Company, catalog number HY-113354. Under light-protected conditions, 20 mg of anserine powder was weighed and placed in a sterile 5 mL EP tube, 2 mL of PBS buffer was added, and the mixture was vortexed for 30 s to mix thoroughly. Then, the anserine powder was dissolved by sonication for 5 min to prepare a 10 mg / mL anserine solution.

[0047] 1.2 Experimental subjects: Male C57BL / 6J mice. Mice were housed separately in cages with free access to food and water, and kept in environments with 12 hours of light and 12 hours of darkness.

[0048] 1.3 Establishment of a mouse model of liver cirrhosis: Mice were treated with 20% CCl4 (corn oil: CCl4 = 4:1) by gavage at a frequency of 3 times / week for 15 weeks.

[0049] 1.4 Pretreatment of the model: Seven days after stopping the gavage treatment in the cirrhotic mice established in 1.3, they were randomly divided into two groups: a control group and a goose muscle peptide treatment group. Mice in the goose muscle peptide treatment group were injected intraperitoneally with goose muscle peptide solution (100 mg / Kg) once a day for 7 days; mice in the control group were injected intraperitoneally with PBS solution at the same dose as the goose muscle peptide treatment group once a day for 7 days.

[0050] 1.5 Establishment of a liver cirrhosis infection model: Mice in the control group and the anserine-treated group, which had undergone pretreatment in 1.4, were given a single tail vein injection of 2×10⁻⁶ mg / L of the drug 24 hours after the last administration. 5 CFU KP (purchased from American Type Culture Collection) was used for treatment, followed by blood sampling from the orbital vein to detect blood bacterial load. The mice were closely observed, and changes in body weight and survival rate were recorded.

[0051] 1.6 General Notes: The animal experiments involved in this embodiment comply with relevant ethical regulations and are conducted in accordance with the protocols approved by the Animal Ethics Committee of Central South University. The C57BL / 6J mouse is a recognized ideal animal model, and this experimental protocol uses the minimum number of animals possible to obtain reliable experimental results. To minimize the suffering of the experimental animals, intraperitoneal and tail vein injections were used for drug administration, and the experimental process complied with humanitarian standards for the termination of animal life.

[0052] 1.7 Statistical Analysis: Data are expressed as mean ± standard error. The Kaplan-Meier test was used for survival comparisons. A p-value < 0.05 was considered statistically significant; the smaller the p-value, the more significant the difference. * in the figure represents p < 0.05.

[0053] 2 Experimental Results

[0054] 2.1 Effect of anserine on the survival rate of infected mice with liver cirrhosis: such as Figure 5As shown, after infection, all mice with liver cirrhosis who were pretreated with PBS died within 25 days, while mice pretreated with anserine for 7 days had a 100% survival rate within 25 days, which was statistically different from the control group (P < 0.05).

[0055] 2.2 Effects of goose muscle peptide on body weight changes in infected mice: such as Figure 6 As shown, mice pretreated with PBS gradually lost weight after infection, while mice pretreated with anserine showed no significant change in weight after infection.

[0056] 2.3 Effect of anserine on blood bacterial load 24 hours after infection in cirrhotic mice: Figure 7 As shown, the bacterial load in the blood of mice treated with goose muscle peptides was not significant, while the bacterial load in the blood of mice in the control group was significantly increased.

[0057] The above results suggest that goose muscle peptide has a significant protective effect against KP infection in cirrhotic mice.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of anserine in the preparation of antibacterial drugs, characterized in that, The antibacterial drug is an antibacterial drug against Escherichia coli and / or Klebsiella pneumoniae.

2. The application of anserine in the preparation of anti-infective drugs for liver cirrhosis, characterized in that, The aforementioned anti-infective drug for liver cirrhosis is a drug for treating Klebsiella pneumoniae infection in patients with liver cirrhosis.

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