Application of cepharanthine in preparation of pathogenic bacteria biofilm scavenger, anti-pathogenic bacteria drug and medical equipment coating material

Abstract

The invention relates to the field of biological medicine, in particular to application of cepharanthine in preparation of a pathogenic bacterium biofilm scavenger, an anti-pathogenic bacterium medicine and a medical equipment coating material. Staphylococcus aureus is taken as a pathogenic bacterium, the removal effect of cepharanthine on a biological membrane is detected through biological membrane formation and destruction experiments, and the curative effect of cepharanthine on infection is evaluated by constructing a staphylococcus aureus infected mouse model. The result shows that the cepharanthine inhibits the formation of the staphylococcus aureus biological membrane and destroys the formed biological membrane; animal experiments show that the cepharanthine improves the survival rate of mice infected with the staphylococcus aureus, reduces the colonial colonization number of lungs of the infected mice, and improves the elimination capability of a host to pathogenic bacteria. Therefore, the invention has important significance in the aspects of research and development of targeted biological membranes and drugs for resisting drug-resistant bacterium infection, improvement of the curative effect of antibiotics, prolonging of the service life of the drugs and the like.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to the application of cepharanthin in the preparation of pathogenic bacteria biofilm removers, anti-pathogenic bacteria drugs and medical equipment coating materials. Background Art

[0002] Bacterial biofilm (BF, also known as biofilm, bacterial biofilm) refers to a bacterial community with a certain three-dimensional structure and function that is attached to the surface of an object (such as a solid object, medical equipment, etc.) and is wrapped by bacterial extracellular macromolecules. It mainly includes extracellular polymers (composed of polysaccharides, proteins, lipids and extracellular DNA, etc.) and the bacteria wrapped in it. It is reported that more than 99% of bacteria in nature can form biofilms. The formation of biofilms has a barrier effect on antibiotics and host immune defense mechanisms, making it difficult for drugs to act on the bacterial community. Some bacteria inside the biofilm are in a static, non-dividing state (i.e., persisters), and their tolerance to antibiotics is significantly improved (10-1000 times). They can survive exposure to antibiotics and regrow under appropriate conditions, leading to persistent and repeated infections. According to statistics, about 80% of bacterial infections are caused by biofilms, such as osteomyelitis, endocarditis, gingivitis, urethritis, deep soft tissue infections, and implanted medical device infections. The antibiotics currently used in clinical practice are mainly bacterial growth inhibitors (e.g., inhibiting the synthesis of cell walls, proteins, nucleic acids, etc.), targeting planktonic bacteria in a rapidly dividing state. Most of them are ineffective against the persisters inside the biofilm and cannot effectively remove stubborn bacterial biofilms. Therefore, there is an urgent need to develop new antibacterial drugs.

[0003] At present, there are three main types of anti-biofilm compounds: biofilm inhibitors, biofilm dispersants and biofilm eradication agents (BEA). Among them, biofilm inhibitors and biofilm dispersants are usually quorum sensing inhibitors, which inhibit the formation of biofilms and destroy the formed biofilms by inhibiting or interfering with the chemical signal pathways required to maintain the existence of biofilms, respectively. They cannot directly inhibit or kill bacteria in the biofilms; while biofilm eradicators can directly kill bacteria in biofilms and remove bacterial biofilms, without the need for combination with other antibiotics, and can be used as a single drug to treat bacterial biofilm-related infections. Early research on biofilm eradicators was mainly based on an antibacterial mechanism that was independent of bacterial growth (i.e., acting on bacterial cell membranes), such as antimicrobial peptides and quaternary ammonium cations, but the former has a complex structure and the latter has high toxicity. Therefore, based on the screening and modification of natural products with antibacterial activity, the discovery of biofilm eradicators with a variety of new mechanisms can greatly promote the development of this field. Summary of the invention

[0004] The purpose of the present invention is to provide the use of cepharanthin in the preparation of pathogenic bacteria biofilm removers, anti-pathogenic bacteria drugs and medical equipment coating materials, so as to solve the problems existing in the above-mentioned prior art. The present invention is the first to discover the new use of cepharanthin in removing Staphylococcus aureus (S. aureus) biofilm and resisting bacterial infection, which makes up for the deficiencies of the existing technology and research, thereby promoting the research and development and application research of drugs against drug-resistant bacterial infections.

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

[0006] The present invention provides the use of cepharanthine in preparing a pathogenic bacteria biofilm remover, wherein the pathogenic bacteria include Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae; but are not limited to Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae, and pathogenic bacteria causing diseases including gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia and food poisoning in humans and animals are also included.

[0007] The cepharanthin described in the present invention, also known as cepharanthine, is a white solid powder that is soluble in water, benzene, chloroform, methanol, ethanol and slightly soluble in solvents such as petroleum ether.

[0008] Further preferably, the CAS registration number of the cephalaenopsis is 481-49-2, and the molecular formula is C 37 H 38 N2O6, molecular weight is 606.72, and its chemical structure is shown in Formula I:

[0009]

[0010] The present invention provides a pathogenic bacteria biofilm remover, which comprises cephalothin and pharmaceutically acceptable excipients; the pathogenic bacteria include Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae; but are not limited to Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae, and pathogenic bacteria that cause diseases including gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia and food poisoning in humans and animals are also included.

[0011] Preferably, the dosage forms of the pathogen biofilm remover include oral solution, injection, powder, tablet, capsule and granule.

[0012] The present invention provides the use of cephalanthrin in the preparation of drugs for resisting pathogenic bacteria, wherein the pathogenic bacteria include Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae; but are not limited to Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae, and pathogenic bacteria causing diseases including gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia and food poisoning in humans and animals are also included.

[0013] Preferably, the drug achieves the effect of resisting pathogens by removing the biofilm of pathogens and reducing the number of colonies colonizing the infected target organs.

[0014] The present invention provides a drug for resisting pathogenic bacteria, the drug comprises cepharanthin and pharmaceutically acceptable excipients; the pathogenic bacteria include Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae; but are not limited to Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae, and pathogenic bacteria causing diseases including gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia and food poisoning in humans and animals are also included.

[0015] The invention provides application of cephalaenopsis in preparing coating materials for medical equipment.

[0016] The invention provides a medical equipment coating material, which comprises cephalaenopsis indica and auxiliary materials.

[0017] The present invention discloses the following technical effects:

[0018] The present invention provides a natural compound with the effects of removing bacterial biofilm and resisting bacterial infection. The cephalanthrin is a dibenzylisoquinoline alkaloid mainly derived from the root or stem of the Chinese medicinal plant Cephalanthus truncatus, which is separated and extracted by solvents such as ethanol / water. At present, there are few research reports on its effect of removing bacterial biofilm. The present invention uses Staphylococcus aureus as a representative pathogen, detects the effect of cephalanthrin on biofilm removal through biofilm formation and destruction experiments, and evaluates the efficacy of cephalanthrin on infection by constructing a mouse model infected with Staphylococcus aureus. The results showed that cephalothin inhibited the formation of Staphylococcus aureus biofilm and could also destroy the formed biofilm. It can be seen that cephalothin has a dual role of intervening in the development of biofilm; at the same time, the dual role is also applicable to Escherichia coli, Pseudomonas aeruginosa, Clostridium perfringens and Klebsiella pneumoniae, which shows that cephalothin has a wide host spectrum; and cephalothin does not affect the growth of Staphylococcus aureus, so it is not easy to develop drug resistance; animal experiments show that cephalothin improves the survival rate of mice infected with Staphylococcus aureus, reduces the number of colonies colonized in the lungs of infected mice, and improves the host's ability to remove pathogens. Therefore, the present invention is of great significance for the research and development of targeted biofilms, anti-resistant bacterial infection drugs, improving the efficacy of antibiotics and extending the service life of drugs. Cephalothin can be used as a clinical antimicrobial membrane application drug preparation, as well as as a coating material for the surfaces of different objects including medical equipment, body implants and artificial organs, etc., which expands the selection and development space of drugs for treating bacterial infections, and has broad market application prospects in the future.

[0019] In addition, the application of cephalaenopsis in the field of biomedical technology also has the following advantages:

[0020] (1) Natural compounds derived from plants are specific, natural, and eco-friendly; (2) Their structure and physicochemical properties are known, their mechanism of action has been effectively elucidated, and they promote synergy with the host's immune system; (3) They do not directly kill pathogens, but mainly weaken and inhibit them, which is significantly different from the mechanism of antibiotics. They are not easy to develop drug resistance and can ensure the long-term effectiveness of such drugs during use.

[0021] In summary, cephalanthrin has many advantages such as wide source, high activity, low price and green environmental protection. It can also inhibit the formation of Staphylococcus aureus biofilm and destroy the formed biofilm, improve the survival rate of mice infected with Staphylococcus aureus, reduce the number of colonies in the lungs of infected mice, and improve the host's ability to eliminate pathogens. Therefore, cephalanthrin has very important application research significance both in the extraction and preparation of antibacterial infection drugs and in the chemical synthesis of antibacterial infection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 The effect of cephalaenopsis on the biofilm of Staphylococcus aureus; the left side of the dotted line in the figure is the biofilm inhibition test, and the right side of the dotted line is the biofilm destruction test; * represents P < 0.05, ** represents P < 0.01;

[0024] Figure 2 Survival rate of mice infected with Staphylococcus aureus under different treatments; ** represents P < 0.01;

[0025] Figure 3 The number of colonies colonizing in the lungs of mice infected with Staphylococcus aureus under different treatments; ** represents P < 0.01. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Bacterial biofilm removal test

[0032] (1) Bacterial biofilm formation inhibition test: A single colony of Staphylococcus aureus was picked and placed in 2 mL of liquid culture medium for culturing at 37°C for 12 h. The colony was centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, the colony was washed once with sterile PBS, and then resuspended in TSB liquid culture medium and adjusted to OD 600nm =0.1, to obtain the adjusted bacterial culture; take 1mL of the adjusted bacterial culture, add it to a 24-well plate, then add cephalothin, so that the final concentration of cephalothin in the 24-well plate is 0μg / mL, 2μg / mL, 4μg / mL, 8μg / mL, 16μg / mL and 32μg / mL, respectively, and set up a positive control group of DMSO (without cephalothin) and a pure liquid culture medium as a negative control group, with three replicates in each group, and place the 24-well plate in a 37°C incubator for 24h. After the culture is completed, discard the supernatant, wash twice with sterile PBS, dry at 55°C for 20min, add 500μL of 0.1% crystal violet sterilized by filtration, incubate at room temperature for 45min, discard the crystal violet, and wash three times with sterile PBS. 33% acetic acid was added to each well to dissolve crystal violet. After mixing, 100 μL was added to each well of the 96-well plate. Three replicate wells were set for each group. The absorbance was measured at 570 nm on an ELISA reader. The group without cephalaenopsis was set as 100%. The biofilm formation rate was calculated based on the absorbance of crystal violet. The results are shown in Table 1. Figure 1 The results showed that at concentrations of 4 μg / mL-32 μg / mL, cephalaenopsis significantly inhibited the formation of bacterial biofilms in a concentration-dependent manner.

[0033] (2) Bacterial biofilm destruction test: A single colony of Staphylococcus aureus was picked up and placed in 2 mL of liquid culture medium for culturing at 37°C for 14 h. The colony was centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, the colony was washed once with sterile PBS, and then resuspended in TSB liquid culture medium and adjusted to OD 600nm=0.1, to obtain the adjusted bacterial culture; take 1mL of the adjusted bacterial culture, add it to a 24-well plate, place the 24-well plate in a 37°C incubator and culture it for 72h, after the bacterial biofilm appears, discard the culture medium in the well, wash it twice with sterile PBS, add fresh TSB liquid culture medium, the amount of the culture medium added is 1mL / well, then add cephalothin respectively, so that the final concentration of cephalothin in the 24-well plate is 0μg / mL, 2μg / mL, 4μg / mL, 8μg / mL, 16μg / mL and 32μg / mL, set up a positive control group of DMSO and a pure liquid culture medium as a negative control group, and repeat three times in each group. Culture for 1h, discard the supernatant, wash it twice with sterile PBS, dry it at 55°C for 20min, add 500μL of 0.1% crystal violet sterilized by filtration, incubate it at room temperature for 45min, discard the crystal violet, and wash it three times with sterile PBS. 33% acetic acid was added to each well to dissolve crystal violet. After mixing, 100 μL was added to each well of the 96-well plate. Three replicate wells were set for each group. The absorbance was measured at 570 nm on an ELISA reader. The group without cephalaenopsis was set as 100%. The biofilm formation rate was calculated based on the absorbance of crystal violet. The results are shown in Table 1. Figure 1 The results showed that: at a concentration of 4μg / mL-32μg / mL, cepharanthin significantly destroyed the formed biofilm, especially at 8μg / mL-32μg / mL; at the same time, the destructiveness of cepharanthin to the formed biofilm was concentration-dependent.

[0034] In summary, cephalothin can significantly remove the biofilm of Staphylococcus aureus.

[0035] Example 2 Protective effect of cephalaenoside on mice infected with Staphylococcus aureus

[0036] (1) Establishment of a mouse model of Staphylococcus aureus infection: 6-8 week-old female Balb / c mice (weight 16-18 g) were fed normally for 3 days and fasted for 12 h before infection. Staphylococcus aureus (5×10 6 CFU / mouse) were challenged with 5-6-year-old female Balb / c mice to establish a Staphylococcus aureus mouse infection model.

[0037] (2) Survival rate test: The above adaptively fed mice were randomly divided into a healthy control group, an infection control group (Staphylococcus aureus infection + saline treatment) and a cephalothin group (Staphylococcus aureus infection + cephalothin treatment), with 10 mice in each group. After infection, the mice in the cephalothin group were subcutaneously injected with a 10 mg / kg cephalothin solution in the neck, once a day, for 4 consecutive days. The mice in the infection control group and the healthy control group were isolated and fed normally without any treatment. The mental state and death of the mice in the different treatment groups were counted for 10 consecutive days. The results are shown in the figure. Figure 2The results showed that there was no death in the healthy control group within 10 days, and the survival rate was 100.00%; the mortality rates of the infected control group on the 3rd and 5th days were 60.00% and 100.00% respectively; the survival rate of the cephalothin group on the 5th day was 80.00%, and the protection rate was significantly higher than that of the infected control group. It can be seen that cephalothin can significantly improve the survival rate of mice infected with Staphylococcus aureus.

[0038] Example 3 Detection of colony count in target organs

[0039] The model was constructed according to the method for establishing the mouse infection model of Staphylococcus aureus in Example 2, and the adaptively fed mice were randomly divided into a healthy control group, an infection control group (Staphylococcus aureus infection + saline treatment) and a cephalothin group (Staphylococcus aureus infection + cephalothin treatment), with 10 mice in each group. Staphylococcus aureus (1×10 6 CFU / mouse) was used to establish a sublethal infection model in mice. After infection, the treatment methods of each group were the same as those in Example 2. On the 5th day after infection, the mice were killed by cervical dislocation. The liver tissues of mice in each group were taken, weighed and ground to prepare tissue homogenates, and then diluted in PBS in multiple ratios and applied to LB agar culture plates. After incubation at 37°C for 12 hours, the colonies were counted. The results are shown in the figure. Figure 3 As shown. The results showed that compared with the infected control group, the number of colonies in the target organ (lung) of mice was significantly reduced after treatment with cephalothin. It can be seen that cephalothin can significantly reduce the number of colonies of Staphylococcus aureus in the target organs of infected mice.

[0040] In summary, the present invention uses Staphylococcus aureus as a representative of pathogens, detects the removal effect of cephalothin on biofilms through biofilm formation and destruction experiments, and evaluates the efficacy of cephalothin on infection by constructing a mouse model infected with Staphylococcus aureus. The results showed that cephalothin inhibited the formation of Staphylococcus aureus biofilms and could also destroy the formed biofilms. This removal effect is also applicable to Escherichia coli, Pseudomonas aeruginosa, Clostridium perfringens and Klebsiella pneumoniae. Animal experiments have shown that cephalothin increased the survival rate of mice infected with Staphylococcus aureus; reduced the number of colonies colonized in the lungs of infected mice, and improved the host's ability to remove pathogens. It can be seen that cephalothin can be used as a pathogen biofilm remover, an anti-pathogen drug, and a coating material for medical equipment.

[0041] 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. The use of cepharanthin in the preparation of a pathogenic bacteria biofilm remover, characterized in that: The pathogenic bacteria include Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae.

2. A pathogenic bacteria biofilm remover, characterized in that: The pathogenic bacteria biofilm remover comprises cephalothin and pharmaceutically acceptable auxiliary materials; the pathogenic bacteria comprise Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae.

3. The pathogenic bacteria biofilm remover according to claim 2, characterized in that: The dosage forms of the pathogen biofilm remover include oral liquid, injection, powder, tablet, capsule and granule.

4. The use of cepharanthin in the preparation of drugs against pathogenic bacteria, characterized in that: The pathogenic bacteria include Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae.

5. The use according to claim 4, characterized in that: The drug achieves the effect of resisting pathogens by removing the biofilm of pathogens and reducing the number of colonies colonizing the infected target organs.

6. A drug for resisting pathogenic bacteria, characterized in that: The medicine comprises cepharanthin and pharmaceutically acceptable auxiliary materials; the pathogenic bacteria comprise Staphylococcus aureus, Escherichia coli, Clostridium perfringens and Klebsiella pneumoniae.

7. Application of cephalaenopsis in the preparation of coating materials for medical devices.

8. A medical device coating material, characterized in that: The medical device coating material comprises cephalaenopsis indica and auxiliary materials.

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