阅读说明：本技术 血根碱在抑制路邓葡萄球菌生长中的应用 (Application of sanguinarine in inhibiting growth of staphylococcus lugdunensis ) 是由 钱卫东 杨敏 李鑫辰 孙照欢 刘淼 张家宁 付玉婷 王文静 于 2019-12-20 设计创作，主要内容包括：本发明公开了血根碱在抑制路邓葡萄球菌生长中的应用,根据血根碱对耐药路邓葡萄球菌具有较好的体外杀灭作用,能够抑制耐药路邓葡萄球菌的生长,且最低杀菌浓度为30μg/mL,最小抑菌浓度为15μg/mL,本发明提出了血根碱对路邓葡萄球菌的抑制作用,在医药等领域具有广泛的应用价值。(The invention discloses an application of sanguinarine in inhibiting the growth of staphylococcus lugdunensis, and provides an inhibiting effect of sanguinarine on staphylococcus lugdunensis according to the fact that sanguinarine has a good in-vitro killing effect on staphylococcus lugdunensis, the growth of staphylococcus lugdunensis can be inhibited, the minimum bactericidal concentration is 30 mu g/mL, and the minimum bacteriostatic concentration is 15 mu g/mL.)

1. Application of sanguinarine in inhibiting growth of Staphylococcus lugdunensis is provided.

2. Use according to claim 1, characterized in that: the staphylococcus lugdunensis is human staphylococcus lugdunensis.

3. Use according to claim 1, characterized in that: the drug resistance of staphylococcus lugdunensis is determined by a paper diffusion method.

4. Use according to claim 1, characterized in that: the antibiotic which is tolerated by the staphylococcus lugdunensis is selected from a plurality of penicillin, clindamycin, tetracycline, ciprofloxacin and oxacillin.

5. Use according to claim 1, characterized in that: the staphylococcus lugdunensis is selected from the group consisting of staphylococcus lugdunensis with penicillin resistance, clindamycin resistance, tetracycline resistance, ciprofloxacin resistance and oxacillin resistance.

6. Use according to claim 5, characterized in that: the minimum bactericidal concentration of sanguinarine to staphylococcus lugdunensis is 30 mug/mL, and the minimum bacteriostatic concentration is 15 mug/mL.

7. Use according to claim 1, characterized in that: the sanguinarine has an in-vitro killing effect on staphylococcus lugdunensis and can inhibit the in-vitro growth of staphylococcus lugdunensis.

8. Application of sanguinarine in preparing medicines for resisting Staphylococcus lugdunensis is provided.

9. Use according to claim 8, characterized in that: the staphylococcus lugdunensis is human staphylococcus lugdunensis.

10. Use according to claim 8, characterized in that: the staphylococcus lugdunensis is selected from staphylococcus lugdunensis which is resistant to various antibiotics in penicillin, clindamycin, tetracycline, ciprofloxacin and oxacillin.

Technical Field

The invention relates to research, development and application of a traditional Chinese medicine effective component sanguinarine as an inhibitor of staphylococcus lugdunensis, in particular to application of sanguinarine in inhibiting the growth of staphylococcus lugdunensis.

Background

Staphylococcus lugdunensis (Staphylococcus lugdunensis) is one of the more virulent coagulase-negative staphylococci, and has become an important human pathogen, which can cause a series of infectious diseases, such as endocarditis, lymphadenitis, ophthalmitis, and osteoarticular infections, but more commonly skin and soft tissue infections, which cause serious and rapid development of infectious diseases, many complications, and high mortality. According to the research of scholars, the medicine has developed drug resistance to penicillin and gradually reduced sensitivity to azithromycin, chloramphenicol and gentamicin. This is due to the emergence of drug resistant strains resistant to drugs caused by the abuse of antibiotics over a long period of time in the past, and with the increase in bacterial resistance, there is an urgent need to develop new bacteriostatic agents.

Chemical components in Chinese herbal medicine plants are used as a new bacteriostatic agent development object, and the bacteriostatic agent has the advantages of little toxic and side effect, wide sources and lower treatment cost in the aspect of treating diseases. Sanguinarine (C) ₂₀H ₁₄NO ₄) The benzophenanthridine isoquinoline alkaloid is separated from papaveraceae plants such as celandine, macleaya cordata and the like, has various pharmacological activities such as insecticidal, anti-tumor, antibacterial, anti-inflammatory, liver function improvement, immunity enhancement and the like, currently, sanguinarine is reported to have an inhibiting effect on candida albicans, pseudomonas aeruginosa, escherichia coli, staphylococcus aureus and the like, however, staphylococcus lugdunensis has obvious difference with pathogenic bacteria causing intestinal tract and respiratory tract infection in an infection way and a treatment scheme, and the sanguinarine is also rarely reported in the aspect of inhibiting the growth of the staphylococcus lugdunensis.

The effective active ingredient sanguinarine is extracted from natural medicinal plant macleaya cordata in literature (Liu Zhu Ying, Wang Xiang Ming, Tang Liang Ming, sanguinarine action mechanism and application effect thereof in animal production [ J ]. Hunan feed, 2019,168(01):41-45.), and the pharmacological action and the application status of the sanguinarine are researched. The result shows that the minimum inhibitory concentration of the sanguinarine to escherichia coli is 640 mug/mL, the minimum inhibitory concentration to the tetranectococcus cereus is 80 mug/mL, the minimum inhibitory concentration to staphylococcus aureus and bacillus subtilis is less than 40 mug/mL, and in addition, the antibacterial activity of the sanguinarine to the tetranectococcus cereus and bacillus subtilis is stronger compared with that of penicillin.

Disclosure of Invention

Aiming at the problem of drug resistance in current clinical medicine, the invention aims to provide the application of sanguinarine in inhibiting the growth of staphylococcus lugdunensis.

In order to achieve the purpose, the invention adopts the technical scheme that:

the drug resistance of staphylococcus lugdunensis to antibiotics is determined by a paper diffusion method, and the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of sanguinarine to staphylococcus lugdunensis are determined by a trace double dilution method. The results show that: sanguinarine has good bacteriostatic action on drug-resistant staphylococcus lugdunensis, and can be used for drug-resistant inhibition of the growth of staphylococcus lugdunensis.

Preferably, the staphylococcus lugdunensis is human staphylococcus lugdunensis resistant to various antibiotics in penicillin, clindamycin, tetracycline, ciprofloxacin and oxacillin.

Preferably, the staphylococcus lugdunensis is selected from the group consisting of penicillin-resistant, clindamycin, tetracycline, ciprofloxacin and oxacillin-resistant staphylococcus lugdunensis.

Preferably, the minimum bactericidal concentration of the sanguinarine is 30 mug/mL, and the minimum bacteriostatic concentration is 15 mug/mL.

The invention has the beneficial effects that:

the invention starts from the existing medicinal plant resource library, excavates a potential drug-resistant bacteria inhibitor, and finds that the non-antibiotic compound sanguinarine can effectively inhibit the growth of staphylococcus lugdunensis based on the research on the effect of sanguinarine on the drug-resistant staphylococcus lugdunensis, thereby providing a new thought and source for the development and application of the staphylococcus lugdunensis inhibitor, and having wide application value in the fields of medicine and the like.

Furthermore, the invention defines the inhibition effect of sanguinarine on the drug-resistant staphylococcus lugdunensis, and the sanguinarine is used as an antibiotic substitute, so that the drug-resistant and infection problems of staphylococcus lugdunensis can be effectively relieved or solved, and the fatality rate is reduced.

Detailed Description

The present invention will be described in further detail with reference to examples. The examples are only for explaining the present invention and do not limit the scope of protection of the present invention.

1. Drug sensitivity experiment of human staphylococcus lugdunensis

The invention takes a plurality of strains of human staphylococcus lugdunensis (strain samples are taken from Ningbo city disease prevention and control center) as starting strains, and selects drug sensitive test paper sheets of 6 common antibiotics of penicillin, clindamycin, tetracycline, ciprofloxacin, vancomycin and oxacillin to carry out drug sensitive tests.

And selecting and culturing the pure colonies for 18-24 h, uniformly dissolving the pure colonies in 2-5 mL of sterile TSB liquid culture medium, and adjusting the turbidity of the pure colonies to be equal to that of a 0.5 McLeod turbiditube. And (3) sucking 50-200 mu L of bacterial liquid in a sterile environment, uniformly coating the bacterial liquid on a flat plate, and standing at room temperature for 3-5 min. The drug sensitive test paper pieces (diameter is 6mm) are tightly attached to the surface of the flat plate by using sterile forceps according to requirements, and each drug sensitive test paper piece is made into three parallel pieces. And standing the inoculated plate in an incubator at the temperature of 36-37 ℃ for incubation for 16-24 h. The diameter of the bacteriostatic circle is measured by a vernier caliper with the precision of 0.02mm, the sensitivity of the medicine is judged according to the diameter of the bacteriostatic circle, the bacteriostatic result is judged according to the standard of the national standard administration committee of the clinical laboratory (CLSl2017), and the judgment standard is shown in Table 1. The experimental results are shown in table 2, and the results show that the 3# human staphylococcus lugdunensis has strong tolerance to 5 clinically common antibiotics, namely penicillin, clindamycin, tetracycline, ciprofloxacin and oxacillin, and belongs to multiple drug-resistant strains, so that the strain is used as a research object of the next experiment.

TABLE 1 results of the national Committee for standardization management of the clinical laboratory (CLSl2017) standards

TABLE 2 diameter results of zone of inhibition in drug susceptibility experiments with Staphylococcus hernalis

Note: the unit of the diameter of the inhibition zone is mm, and the numerical values are the average values of three parallels

2. Inhibition effect of sanguinarine on drug-resistant staphylococcus lugdunensis

In order to fully consider the medication safety, the invention takes sanguinarine as a single active ingredient as a research object, takes ATCC700328 as a reference strain (the reference strain is highly sensitive to gentamicin, vancomycin, linezolid and moxifloxacin), and takes 3# humanized staphylococcus lugdunensis as an experimental strain to research the drug resistance inhibition effect. Selecting overnight single colony, inoculating to 3mL TSB liquid culture medium, adjusting turbidity to 0.5 McLeod turbiditube, and measuring OD ₆₀₀The value is obtained. The sanguinarine with the concentration of 10mg/mL is prepared by dimethyl sulfoxide, the liquid medicine, the bacterial liquid and the TSB liquid culture medium are added into a 96-hole culture plate for overnight culture by using a test tube double dilution method, three liquid medicine groups with different concentrations are respectively parallel, and the reliability of experimental data is ensured.

The Minimum Inhibitory Concentration (MIC) of sanguinarine to Staphylococcus lugdunensis is measured by a microplate reader, a culture solution with the concentration being the concentration of the MIC diluted by the former drug is transferred to a sterile TSA solid culture medium for continuous culture for 24h, whether a single colony is generated or not is observed, if not, the concentration is the Minimum Bactericidal Concentration (MBC) of sanguinarine to Staphylococcus lugdunensis, and the experimental result is shown in Table 3.

TABLE 3 inhibition of drug-resistant Staphylococcus lugdunensis by sanguinarine

The results in Table 3 show that sanguinarine has good inhibition effect on penicillin, clindamycin, tetracycline, ciprofloxacin and oxacillin-resistant Staphylococcus lugdunensis, the MIC is 15 mug/mL, and the MBC is 30 mug/mL. The result of comparison with the reference strain ATCC700328 shows that sanguinarine not only has bacteriostatic action on the staphylococcus lugdunensis reference strain ATCC700328, but also has bacteriostatic action on the drug-resistant staphylococcus lugdunensis (such as 3# strain) in the invention.

According to the experimental results, by combining the characteristics of wide sources of Chinese herbal medicines, less adverse reactions, difficult generation of drug resistance and the like, the sanguinarine serving as an effective monomer ingredient of the Chinese herbal medicines can directly play an inhibiting role for clinically main drug-resistant staphylococcus lugdunensis, so that the infection problem of the drug-resistant staphylococcus lugdunensis is effectively relieved or solved, the death rate is reduced, a scientific basis is provided for researching clinically separated bacteriostats of the staphylococcus lugdunensis, and a new thought and source are provided for developing drugs and antibiotic substitutes for inhibiting the drug-resistant staphylococcus lugdunensis.