Application of goose carnosine in preparation of antibacterial drugs

Abstract

The invention discloses application of goose carnosine in preparation of antibacterial drugs, and belongs to the technical field of biological medicines. The antibacterial medicine comprises an escherichia coli resisting medicine and a klebsiella pneumoniae resisting medicine. The research finds that the goose carnosine has a remarkable inhibition effect on escherichia coli and klebsiella pneumonia, and has a remarkable enhancement effect on the klebsiella pneumonia and escherichia coli infection resistance of mice with cirrhosis, so that the death rate of the mice with cirrhosis is reduced; a new thought is provided for preparation of antibacterial drugs and anti-interference drugs for patients with cirrhosis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of anserine in the preparation of antibacterial drugs. Background Art

[0002] Klebsiella pneumoniae and Escherichia coli are Gram-negative bacteria that often infect humans, with high morbidity and mortality, causing a serious disease burden worldwide. Currently, the treatment of these two bacterial infections mainly relies on antibiotics, but excessive use of antibiotics can easily lead to bacterial resistance, resulting in increasingly poor treatment effects. New auxiliary therapeutic drugs are urgently needed to improve patients' ability to resist Klebsiella pneumoniae and Escherichia coli infections.

[0003] Cirrhosis is a terminal complication caused by various chronic liver diseases. The incidence of Klebsiella pneumoniae and Escherichia coli infection in patients with cirrhosis is very high, and the infection can lead to hepatic encephalopathy, sepsis, liver failure, etc., leading to the patient's death. At present, the treatment of cirrhosis combined with Klebsiella pneumoniae and Escherichia coli infection also mainly relies on antibacterial drugs, but it also causes increased drug resistance. New auxiliary therapeutic drugs are urgently needed to improve the ability of patients with cirrhosis to resist Klebsiella pneumoniae and Escherichia coli infection.

[0004] Anserine is a histidine dipeptide in animals, mainly found in the muscles and brains of vertebrates. It is usually extracted from the skeletal muscle of poultry. It has significant antioxidant, immunity-enhancing, anti-aging, uric acid-lowering, vasodilation, nerve protection and enzyme activity regulation effects. However, its role in Klebsiella pneumoniae and Escherichia coli infections has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide an application of anserine in the preparation of antibacterial drugs to solve the problems existing in the above-mentioned prior art. The present invention has found that anserine has a significant inhibitory effect on Escherichia coli and Klebsiella pneumoniae, and has a significant enhancement effect on the anti-infection ability of cirrhotic mice with Klebsiella pneumoniae and Escherichia coli, reducing the mortality rate of cirrhotic mice, and providing a new idea for the preparation of antibacterial drugs and anti-interference drugs for patients with cirrhosis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides application of anserine in preparing antibacterial drugs.

[0008] Preferably, the antibacterial drugs include drugs against Escherichia coli and / or Klebsiella pneumoniae.

[0009] The invention also provides application of anserine in preparing anti-infection drugs for liver cirrhosis.

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

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

[0012] The present invention has found that anserine can enhance the ability of mice with cirrhosis to resist infection with Klebsiella pneumoniae and Escherichia coli, reduce bacterial load, and reduce mortality. In addition, anserine has the effect of inhibiting the growth of Klebsiella pneumoniae and Escherichia coli in vitro, and can be used to prepare antibacterial agents. The present invention develops new functions of anserine, expands the application range of anserine, and also provides a new technical solution for the development of anti-infective drugs for patients with cirrhosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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.

[0014] Figure 1 The figure shows the inhibitory effect of different concentrations of anserine solution on the growth of Klebsiella pneumoniae;

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

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

[0017] Figure 4 The growth inhibition effect of anserine solutions with different concentrations on Escherichia coli;

[0018] Figure 5 The effect of anserine on the survival rate of cirrhotic mice infected with Klebsiella pneumoniae, where PBS+LC.I refers to cirrhotic mice pretreated with PBS, and An+LC.I refers to cirrhotic mice pretreated with anserine;

[0019] Figure 6 The effect of anserine on the weight change of cirrhotic mice infected with Klebsiella pneumoniae, where PBS+LC.I is cirrhotic mice pretreated with PBS, and An+LC.I is cirrhotic mice pretreated with anserine;

[0020] Figure 7This is the effect of anserine on the blood bacterial load of cirrhotic mice 24 hours after infection with Klebsiella pneumoniae, wherein PBS+LC.I is the blood bacterial load of two cirrhotic mice after PBS pretreatment, and An+LC.I is the blood bacterial load of two cirrhotic mice after anserine pretreatment. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

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

[0026] Example 1

[0027] This example verifies the in vitro antibacterial effect of anserine on Klebsiella pneumoniae (KP).

[0028] 1 Experimental plan

[0029] 1.1 Preparation of anserine solution: Anserine was purchased from MCE Company with the product number HY-113354. Weigh 200 mg of anserine under light-proof conditions, add 2 mL of MH medium to dissolve, and prepare a 100 mg / mL anserine solution, which was then diluted with MH medium to 12.5 mg / mL, 25 mg / mL, and 50 mg / mL working solutions.

[0030] 1.2 Experimental method: The animals were divided into different groups according to the concentration of anserine (0 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL) and compared with 1×10 6 CFU KP (purchased from American Type Culture Collection) were co-cultured at 37°C, 220 rpm in a shaker for 24 hours. MH medium (bacteria-free) was used as the control group. The changes in bacterial concentration in each group were detected regularly using an ELISA reader. At the 6th hour of co-culture, the co-culture solution of each group was photographed and recorded. Then, an equal amount of the co-culture solution of each group was taken and diluted to 1×10 6 Then the plates were counted and photographed for 24 hours.

[0031] 1.3 Statistical methods: Data are expressed as mean ± standard error. Statistical differences were analyzed by one-way analysis of variance (ANOVA) (when more than two groups were compared) using GraphPad Prism 8 medical graphics analysis software. P values ​​< 0.05 were considered statistically significant, and the smaller the p value, the more significant the difference. * in the figure represents p < 0.05.

[0032] 2 Experimental results

[0033] 2.1 The inhibitory effect of anserine on the growth of KP: Figure 1 As shown in the figure, compared with the MH medium group (without bacteria), the bacterial concentration of 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, but compared with 0 mg / mL, the addition of anserine significantly inhibited bacterial growth, and the inhibitory effect was most obvious when the concentration of anserine was 100 mg / mL. The co-culture solution at the 6th hour of co-culture is as follows Figure 2 As shown, compared with the MH medium group (without bacteria), the turbidity of the bacterial solution in the groups containing different concentrations of anserine decreased with the increase of anserine concentration, and the turbidity of the bacterial solution was the lowest when the concentration was 100 mg / mL.

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

[0035] Example 2

[0036] This example verifies the in vitro antibacterial effect of anserine on Escherichia coli (E. coli).

[0037] 1 Experimental plan

[0038] 1.1 Preparation of anserine solution: Anserine was purchased from MCE Company with the product number HY-113354. Weigh 200 mg of anserine under light-proof conditions, add 2 mL of MH medium to dissolve, and prepare a 100 mg / mL anserine solution, which was then diluted with MH medium to 12.5 mg / mL, 25 mg / mL, and 50 mg / mL working solutions.

[0039] 1.2 Experimental method: The animals were divided into different groups according to the concentration of anserine (0 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL) and compared with 1×10 6 CFU E. coli (purchased from American Type Culture Collection) were co-cultured at 37°C, 220 rpm in a shaker for 24 hours, MH medium was used as the control group, and the changes in bacterial concentration in each group were regularly detected using an enzyme marker.

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

[0041] 2 Experimental results

[0042] The inhibitory effect of anserine on the growth of E.coli: Figure 4 As shown, compared with the MH medium group (without bacteria), the bacterial concentration of 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, but compared with 0 mg / mL, the addition of anserine significantly inhibited bacterial growth, and the inhibitory effect was most obvious when the concentration of anserine was 100 mg / mL.

[0043] Example 3

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

[0045] 1 Experimental plan

[0046] 1.1 Preparation of anserine solution: Anserine was purchased from MCE Company with the product number HY-113354. Under light-proof 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 mixed thoroughly using a vortexer for 30 s. Then, anserine powder was dissolved by ultrasound for 5 min to prepare a 10 mg / mL anserine solution.

[0047] 1.2 Experimental subjects: Male C57BL / 6J mice. The mice were housed in separate cages, with free access to food and water, and were kept in an environment with a 12-hour light and dark cycle.

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

[0049] 1.4 Model pretreatment and administration: After 7 days of stopping oral gavage treatment of the cirrhotic mice established in 1.3, they were randomly divided into two groups: a control group and anserine treatment group. The mice in the anserine treatment group were intraperitoneally injected with anserine solution (100 mg / Kg) once a day for 7 days; the mice in the control group were intraperitoneally injected with PBS solution, the injection amount was the same as that of the anserine treatment group, once a day for 7 days.

[0050] 1.5 Establishment of liver cirrhosis infection model: After the pretreatment administration in 1.4, the mice in the control group and anserine treatment group were injected with a single dose of 2×10 5 CFU KP (purchased from American Type Culture Collection) was processed, and then blood was collected from the orbital vein to detect the blood bacterial load. The status of the mice was closely observed, and the weight changes and survival rate of the mice were recorded.

[0051] 1.6 General description: The animal experiments involved in this example comply with relevant ethical regulations and are conducted in accordance with the protocol approved by the Animal Ethics Committee of Central South University. C57BL / 6J mice are recognized as an ideal animal model. This experimental protocol uses as few animals as possible to obtain reliable experimental results. In order to reduce the pain of experimental animals, this experiment uses intraperitoneal injection and tail vein injection for drug administration, and the experimental process complies with the humanitarian standards for ending animal life.

[0052] 1.7 Statistical analysis: Data are expressed as mean ± standard error. Survival comparison was performed using the Kaplan-Meier test. P values ​​< 0.05 were considered statistically significant, and 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 cirrhotic mice after infection: Figure 5As shown, after infection of cirrhotic mice, all mice pretreated with PBS died within 25 days, while the survival rate of mice pretreated with anserine for 7 days was 100% within 25 days, which was statistically different from the control group (P<0.05).

[0055] 2.2 Effect of anserine on body weight changes in cirrhotic mice after infection: Figure 6 As shown, it can be seen that the body weight of mice pretreated with PBS gradually decreased after infection. In contrast, the body weight of mice pretreated with anserine did not change significantly after infection.

[0056] 2.3 Effect of anserine on blood bacterial load in cirrhotic mice 24 hours after infection: Figure 7 As shown, it can be seen that the blood bacterial load of mice in the anserine-treated group was not obvious, on the contrary, the blood bacterial load of mice in the control group increased significantly.

[0057] The above results suggest that anserine has a significant protective effect on cirrhotic mice against KP infection.

[0058] 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 anserine in the preparation of antibacterial drugs.

2. The use according to claim 1, characterized in that: The antibacterial drugs include drugs against Escherichia coli and / or Klebsiella pneumoniae.

3. Application of anserine in the preparation of anti-infective drugs for liver cirrhosis.

4. The use according to claim 3, characterized in that: The liver cirrhosis anti-infection drugs include liver cirrhosis anti-Escherichia coli and / or liver cirrhosis anti-Klebsiella pneumoniae infection drugs.

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