CN119424431A - Application of stephanine in preparing pathogen biological film scavenger, pathogen-resistant medicine and medical equipment coating material - Google Patents
Application of stephanine in preparing pathogen biological film scavenger, pathogen-resistant medicine and medical equipment coating material Download PDFInfo
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Abstract
The invention relates to the field of biological medicine, in particular to application of cepharanthine in preparing a pathogenic bacteria biomembrane remover, an antipathogen medicine and a medical device coating material. The invention takes staphylococcus aureus as pathogenic bacteria, the cleaning effect of cepharanthine on the biological film is detected through a biological film forming and destroying experiment, and the curative effect of cepharanthine on infection is evaluated through constructing a staphylococcus aureus infection mouse model. The animal experiment shows that the stephanine promotes the survival rate of mice infected by staphylococcus aureus, reduces the colony colonization number of the lungs of the infected mice and improves the clearance capability of a host to pathogenic bacteria. Therefore, the invention has important significance in the aspects of targeting biological membranes, researching and developing medicines for resisting drug-resistant bacteria infection, improving the curative effect of antibiotics, prolonging the service life of medicines and the like.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of cepharanthine in preparing a pathogenic bacteria biomembrane remover, an antipathogen medicine and a medical device coating material.
Background
Bacterial biofilm (bacterial biofilm, BF, or bacterial film, bacterial biofilm) refers to a bacterial population which is attached to the surface of an object (such as a solid-phase object, medical equipment and facilities and the like) and is wrapped by bacterial extracellular macromolecules and has a certain three-dimensional structure and function, and mainly comprises extracellular polymers (consisting of polysaccharide, protein, lipid, extracellular DNA and the like) and wrapped bacteria and the like. More than 99% of bacteria in nature are reported to form biofilms. Biofilm formation has a barrier effect on antibiotics and host immune defense mechanisms, making it difficult for drugs to act on the flora. Part of the bacteria inside the biofilm are in a quiescent, non-dividing state (i.e. persisting bacteria), which have significantly improved resistance to antibiotics (10-1000 fold), are able to survive exposure to antibiotics and regrow under appropriate conditions, leading to persistent and recurrent infections. It is counted that about 80% of bacterial infections are caused by biofilms, such as osteomyelitis, endocarditis, gingivitis, urethritis, deep soft tissue infections, and implanted medical device infections. Antibiotics currently in clinical use are mainly bacterial growth inhibitors (e.g., inhibiting cell wall, protein, nucleic acid, etc. synthesis) against planktonic bacteria in a rapidly dividing state. Most of them are ineffective against the remaining bacteria inside the biofilm, and cannot effectively clear the recalcitrant bacterial biofilm. Therefore, development of novel antibacterial agents is urgently required.
Currently, anti-biofilm compounds are mainly 3 classes, biofilm inhibitors, biofilm dispersants and biofilm scavengers (biofilm eradication agents, BEA). The biofilm inhibitor and the biofilm dispersing agent are usually quorum sensing inhibitors, can not directly inhibit or kill bacteria in the biofilm by inhibiting or interfering with chemical signal paths required for maintaining the existence of the biofilm to inhibit the formation of the biofilm and destroy the formed biofilm respectively, and the biofilm remover can directly kill bacteria in the biofilm and remove the bacterial biofilm without being combined with other antibiotics, so that the treatment of the infection related to the bacterial biofilm can be carried out by single drug. Early biofilm remover studies were based primarily on an antibacterial mechanism (i.e., acting on the bacterial cell membrane) independent of bacterial growth, such as antibacterial peptides and quaternary ammonium cations, but the former is complex in structure and the latter is highly toxic. Therefore, based on screening and modifying natural products with antibacterial activity, the discovery of a plurality of biological film scavengers with novel mechanisms can greatly promote the development of the field.
Disclosure of Invention
The invention aims to provide application of stephanine in preparing pathogen biological film scavengers, antipathogenic drugs and medical equipment coating materials to solve the problems in the prior art. The invention discovers the new application of the stephanine in the aspects of removing staphylococcus aureus (golden grape bacteria) biomembrane and resisting bacterial infection for the first time, and makes up the defects of the prior art and research, thereby promoting the research and the development and the application research of medicines for resisting drug-resistant bacteria infection.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides application of stephanine (CEPHARANTHINE) in preparing a biological film scavenger of pathogenic bacteria including staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, but not limited to staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, and diseases caused by the pathogenic bacteria including gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia, food poisoning and the like of human beings and animals.
The stephanine, named stephanine, is one kind of white solid powder capable of being dissolved in water, benzene, chloroform, methanol, ethanol, petroleum ether and other solvent.
Further preferably, the cepharanthine has CAS accession number 481-49-2, molecular formula C 37H38N2O6, molecular weight 606.72, and chemical structural formula shown in formula I:
The invention provides a pathogenic bacteria biological film remover, which comprises stephanine and pharmaceutically acceptable auxiliary materials, wherein pathogenic bacteria comprise staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, but the pathogenic bacteria comprise pathogenic bacteria such as staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, and diseases caused by the pathogenic bacteria comprise gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia, food poisoning and the like of humans and animals.
Preferably, the formulation of the pathogenic bacteria biological film scavenger comprises oral liquid, injection, powder, tablet, capsule and granule.
The invention provides application of stephanine in preparing medicines for resisting pathogenic bacteria including staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, but not limited to staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, and diseases caused by the pathogenic bacteria including gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia, food poisoning and the like of human beings and animals are also included.
Preferably, the medicine reduces colony colonization number of an infected target organ by removing biological membranes of pathogenic bacteria, thereby achieving the effect of resisting the pathogenic bacteria.
The invention provides an antipathogenic medicament which comprises stephanine and pharmaceutically acceptable auxiliary materials, wherein the pathogenic bacteria comprise staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, but the antipathogenic medicament is not limited to staphylococcus aureus, escherichia coli, clostridium perfringens and klebsiella pneumoniae, and diseases caused by the pathogenic bacteria comprise human and animal gastroenteritis, pneumonia, meningitis, arthritis, enterotoxemia, food poisoning and the like.
The invention provides an application of stephanine in preparing a coating material of medical equipment.
The invention provides a medical equipment coating material, which comprises stephanine and auxiliary materials.
The invention discloses the following technical effects:
The invention provides a natural compound with the effects of removing bacterial biomembrane and resisting bacterial infection, and the stephanine is a dibenzyl isoquinoline alkaloid which is mainly derived from roots or stems of stephania japonica of the family Menispermaceae and is separated and extracted by solvents such as ethanol, water and the like. Currently, there are few reports on its removal of bacterial biofilm. The invention takes staphylococcus aureus as a pathogenic bacteria representative, the cleaning effect of cepharanthine on the biological film is detected through a biological film formation and damage experiment, and the curative effect of cepharanthine on infection is evaluated through constructing a staphylococcus aureus infection mouse model. The result shows that the stephanine inhibits the formation of staphylococcus aureus biofilm, can damage the formed biofilm, can be used for interfering with the development of the biofilm, is also suitable for escherichia coli, pseudomonas aeruginosa, clostridium perfringens and klebsiella pneumoniae, has broad-host-spectrum characteristics, does not influence the growth of staphylococcus aureus, and is not easy to generate drug resistance, and animal experiments show that the stephanine improves the survival rate of mice infected with staphylococcus aureus, reduces the colony colonization number of the mice infected with the lung and improves the capability of hosts for clearing pathogenic bacteria. Therefore, the invention has important significance in the aspects of targeting biological membranes, researching and developing medicines for resisting drug-resistant bacteria infection, improving the curative effect of antibiotics, prolonging the service life of medicines and the like. The stephanine can be used as a clinical antimicrobial film application pharmaceutical preparation, as a coating material on the surfaces of different objects including medical equipment, organism implantation equipment, artificial organs and the like, expands the selection and development space of medicines for treating bacterial infection, and has wide market application prospect in the future.
In addition, the application of the stephanine in the technical field of biological medicine has the following advantages:
The natural compound of plant source has the characteristics of specificity, naturality, ecology, environmental protection and the like, (2) the structure and physical and chemical properties are known, the action mechanism is effectively clarified, the synergistic effect with the immune system of a host is promoted, and (3) the natural compound does not directly kill pathogenic bacteria, mainly has weakening and inhibiting effects, is obviously different from an antibiotic mechanism, is not easy to generate drug resistance, and can ensure the long-term effectiveness of the use process of the drug.
In conclusion, the stephanine has the advantages of wide sources, multiple activities, low price, environmental protection and the like, can inhibit the formation of staphylococcus aureus biological membranes and destroy the formed biological membranes, improves the survival rate of mice infected by staphylococcus aureus, reduces the colony colonization number of the lungs of infected mice, and improves the capability of a host for clearing pathogenic bacteria. Therefore, the stephanine has very important application research significance in the aspects of extraction and preparation of antibacterial infection medicaments and chemical synthesis of antibacterial infection medicaments.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 shows the effect of stephanine on Staphylococcus aureus biofilm, wherein the left side of the dotted line in the figure is a biofilm inhibition test, and the right side of the dotted line is a biofilm disruption test;
fig. 2 shows the survival rate of mice infected with staphylococcus aureus treated differently, wherein P <0.01 is represented by the following;
fig. 3 shows colony colonization numbers in lungs of mice infected with different treated staphylococcus aureus, wherein P <0.01 is represented by x.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 bacterial biofilm removal assay
(1) Bacterial biofilm formation inhibition test, namely, selecting staphylococcus aureus single colony, placing the staphylococcus aureus single colony in 2mL of liquid culture medium for culturing for 12h at 37 ℃, centrifuging for 5min at 12000rpm, discarding supernatant, washing once by using sterilized PBS, then re-suspending by using TSB liquid culture medium and adjusting to OD 600nm =0.1 to obtain an adjusted bacterial culture, taking 1mL of the adjusted bacterial culture, adding the adjusted bacterial culture into a 24-pore plate, adding stephanine respectively, enabling the final concentration of stephanine in the 24-pore plate to be 0 mug/mL, 2 mug/mL, 4 mug/mL, 8 mug/mL, 16 mug/mL and 32 mug/mL respectively, additionally arranging a positive control group and a pure liquid culture medium of DMSO (without stephanine) as a negative control group, repeating each group, and placing the 24-pore plate in a 37 ℃ incubator for standing culture for 24h. After the cultivation, the supernatant was discarded, washed twice with sterilized PBS, dried at 55℃for 20min, added with 500. Mu.L of 0.1% crystal violet sterilized by filtration, left standing at room temperature for 45min, discarded and washed three times with sterilized PBS. Adding 33% acetic acid to dissolve crystal violet in each hole, blowing and mixing, adding 100 μl of each hole of 96-well plate, arranging 3 compound holes in each group, measuring absorbance at 570nm with enzyme-labeled instrument, setting the group without adding stephanine as 100%, and calculating biofilm formation rate according to crystal violet absorbance, and the result is shown in figure 1. The results show that the stephanine obviously inhibits the formation of bacterial biofilms at the concentration of 4-32 mu g/mL and shows the trend of concentration dependence.
(2) Bacterial biofilm formation disruption test by picking single colonies of staphylococcus aureus, placing them in 2mL of liquid medium, culturing for 14h at 37 ℃, centrifuging for 5min at 12000rpm, discarding supernatant, washing once with sterilized PBS, then re-suspending with TSB liquid medium and adjusting to OD 600nm =0.1 to obtain adjusted bacterial culture, adding 1mL of adjusted bacterial culture into 24-well plate, placing 24-well plate in 37 ℃ incubator, standing and culturing for 72h, discarding the in-well medium after appearance of bacterial biofilm, sterilizing PBS, washing twice, adding fresh TSB liquid medium with the addition amount of 1 mL/well, adding cepharanthine into the 24-well plate respectively, so that the final concentration of cepharanthine in the 24-well plate is 0 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL, 16 μg/mL and 32 μg/mL respectively, setting positive control group and pure liquid medium of DMSO as negative control group, and repeating three times each group. Culturing for 1h, discarding supernatant, washing twice with sterilized PBS, drying at 55deg.C for 20min, adding 500 μL of 0.1% crystal violet sterilized by filtration, standing at room temperature for 45min, discarding crystal violet, and washing three times with sterilized PBS. Adding 33% acetic acid to dissolve crystal violet in each hole, blowing and mixing, adding 100 μl of each hole of 96-well plate, arranging 3 compound holes in each group, measuring absorbance at 570nm with enzyme-labeled instrument, setting the group without adding stephanine as 100%, and calculating biofilm formation rate according to crystal violet absorbance, and the result is shown in figure 1. The results show that the stephanine significantly damages the formed biological film, especially 8-32 mug/mL, at the concentration of 4-32 mug/mL, and the stephanine is destructive to the formed biological film and is concentration dependent.
Taken together, cepharanthine can significantly clear the biofilm of staphylococcus aureus.
EXAMPLE 2 protective Effect of Cepharanthine on mice infected with Staphylococcus aureus
(1) Establishing a staphylococcus aureus infection mouse model, namely, 6-8 week old female Balb/c mice (weight is 16-18 g), feeding for 3 days in normal adaptation, fasted for 12 hours before infection, and using staphylococcus aureus (5X 10 6 CFU/mouse) to attack the female Balb/c mice by a nasal drip mode to establish a staphylococcus aureus mouse infection model.
(2) Survival test the adaptively raised mice were randomly divided into healthy control groups, infected control groups (staphylococcus aureus infection + physiological saline treatment) and stephanine groups (staphylococcus aureus infection + stephanine treatment), 10 each. The mice of the cepharanthine group are subcutaneously injected into the neck of the mice after the bacteria attack at a dose of 10mg/kg of cepharanthine solution for 1 time/d for 4 days, wherein the mice infected with the control group and the healthy control group are normally isolated and fed without any treatment, and the mental state and death of the mice of the different treatment groups are counted for 10 days continuously, and the result is shown in fig. 2. The results show that no death occurs in the healthy control group 10d, the survival rate is 100.00%, the death rates of the infected control group 3d and 5d are respectively 60.00% and 100.00%, the survival rate of the stephanine group 5d is 80.00%, and the protection rate is obviously higher than that of the infected control group. Thus, the stephanine can obviously improve the survival rate of mice infected by staphylococcus aureus.
EXAMPLE 3 detection of colony colonization number of target organ
The model was constructed according to the staphylococcus aureus mouse infection model establishment method in example 2, adaptively bred mice were randomly divided into healthy control groups, infected control groups (staphylococcus aureus infection+physiological saline treatment) and stephanine groups (staphylococcus aureus infection+stephanine treatment), 10 mice per group were challenged with staphylococcus aureus (1×10 6 CFU/mice) by nasal drip to establish a mouse sublethal infection model, and the treatment methods of the groups after the challenging were the same as in example 2. Mice were sacrificed at day 5 post-infection cervical dislocation, liver tissues of each group of mice were weighed and ground to prepare tissue homogenates, which were spread on LB agar plates after PBS-fold dilution, and colony counts were performed after incubation at 37 ℃ for 12h, and the results are shown in fig. 3. The results show that the colony colonization number of the target organ (lung) of the mice is significantly reduced after the cepharanthine treatment compared with the infection control group. Thus, the stephanine can obviously reduce colony colonization number of staphylococcus aureus in target organs of infected mice.
In conclusion, the invention uses staphylococcus aureus as a pathogen representative, the effect of the stephanine on clearing the biological film is detected through a biological film forming and destroying experiment, and the curative effect of the stephanine on infection is evaluated through constructing a staphylococcus aureus infection mouse model. As a result, it was found that cepharanthine inhibits the formation of Staphylococcus aureus biofilm and also damages the formed biofilm, and that this scavenging effect is equally applicable to Escherichia coli, pseudomonas aeruginosa, clostridium perfringens and Klebsiella pneumoniae. Animal experiments show that the stephanine improves the survival rate of mice infected by staphylococcus aureus, reduces colony colonization number of lungs of infected mice, and improves the capability of a host for removing pathogenic bacteria. Therefore, the stephanine can be used as a biological film scavenger for pathogenic bacteria, an antipathogen drug and a coating material for medical equipment.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
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