Application of etoposide in antibacterial preparation and antibacterial preparation
Etoposide solves the problem of traditional antimicrobial resistance by downregulating bacterial biofilm gene expression, achieves effective inhibition of Streptococcus mutans, provides new antimicrobial preparations and oral care products, and reduces the prevalence of dental caries.
Patent Information
- Application Number
- CN202510938745.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-08
- Publication Date
- 2025-10-17
AI Technical Summary
The application of etoposide in the antibacterial field has long been blank. Traditional cognition believes that its mechanism of action depends on topoisomerase II unique to eukaryotic cells, while prokaryotes lack this key target, resulting in insufficient research on its antibacterial properties. Existing antibacterial agents face the problem of drug resistance and lack effective new intervention strategies.
Etoposide inhibits or destroys the formation of biofilms by downregulating the gene expression of bacterial biofilms, especially targeting Gram-positive cocci Streptococcus mutans ATCC700610. It is used to prepare antibacterial preparations, including oral care products and medical coatings, to inhibit or destroy biofilms and achieve antibacterial effects.
Etoposide significantly inhibits the formation of Streptococcus mutans biofilm, provides a new anti-infection pathway, reduces the incidence of diseases such as dental caries, and has broad application prospects and safety.
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Figure CN120789084A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of antibacterial preparations, and particularly relates to a use of etoposide in antibacterial preparations and antibacterial preparations. BACKGROUND
[0002] Etoposide is a semi-synthetic derivative of podophyllotoxin, mainly derived from the natural components of Berberidaceae plants such as American podophyllum or Tibetan podophyllum. As a classic topoisomerase II inhibitor, etoposide is mainly used in the treatment of malignant tumors, and the research on its antibacterial effect has been in a blank state for a long time. The research gap is due to the traditional cognition that the mechanism of etoposide is highly dependent on the topoisomerase II specific to eukaryotic cells, and prokaryotes lack this key target. However, with the increasingly serious problem of antibiotic resistance, it is of great scientific significance and clinical value to re-examine the antibacterial potential of existing drugs. SUMMARY
[0003] Therefore, the purpose of the present application is to provide a use of etoposide in antibacterial preparations and antibacterial preparations, which can down-regulate the gene expression of bacterial biofilm, inhibit the formation of biofilm or destroy the formed biofilm, and achieve the effect of antibacterial.
[0004] The present application solves the above technical problems through the following technical means:
[0005] In a first aspect, the present application discloses a use of etoposide in antibacterial preparations.
[0006] Further, the antibacterial preparation is a biofilm inhibitor, and etoposide can down-regulate the gene expression of bacterial biofilm, inhibit the formation of biofilm or destroy the formed biofilm.
[0007] Further, the biofilm is the biofilm of gram-positive cocci. The molecular formula of etoposide in the technical solution is C 29 H 32 O 13 , and the molecular weight is 588.557; through MIC&MBC determination test, growth curve test, bacterial biofilm determination test, bacterial live / dead staining test and qRT-PCR test, it is verified that etoposide can inhibit the growth of bacteria with biofilm, down-regulate the expression of biofilm-related genes, inhibit the formation of biofilm or destroy the formed biofilm.
[0008] Further, the biofilm is a biofilm of Streptococcus mutans ATCC700610. Streptococcus mutans ATCC700610 is a gram-positive facultative anaerobe, is a common pathogenic bacterium in the oral cavity and the main pathogen of dental caries, which can cause diseases such as enamel demineralization, dental caries and pulpitis. Its pathogenic mechanism includes: Streptococcus mutans adheres to the enamel first, secretes glycosyltransferase, catalyzes the formation of insoluble dextran in the presence of sucrose, and forms a dental biofilm; then continuously produces acid to form a cariogenic microenvironment. As the dominant cariogenic bacterium in the human oral cavity, Streptococcus mutans ATCC700610 is widely colonized in the oral cavity, can be transmitted through saliva, and has strong environmental adaptability, and is the main pathogen of dental caries worldwide. Dental caries is the most common chronic oral disease in China, and the prevalence rate presents a serious situation of "two highs and two lows", with a prevalence of 700 million, and the prevalence rate of deciduous teeth in children is over 70%, but the treatment rate and prevention awareness have been low for a long time. At present, the clinical prevention and treatment mainly depends on antibacterial agents, but the Health Oral Action Plan (2019-2025) clearly proposes to "strengthen the research on oral microecology and develop precise prevention and treatment technology". Under this guidance, new intervention strategies for Streptococcus mutans, such as targeted glycosyltransferase inhibitors, probiotic regulation, etc. have become a hot spot, aiming to break through the limitations of bacterial resistance caused by traditional antibacterial therapy, and realize safer and more effective prevention and control of dental caries.
[0009] Further, the etoposide is chemically synthesized or naturally derived.
[0010] Further, the etoposide is extracted from the plant of Berberidaceae, such as American podophyllum or Tibetan podophyllum.
[0011] In the second aspect, the application further discloses an antibacterial preparation, characterized in that the antibacterial preparation comprises an etoposide component.
[0012] Further, the antibacterial preparation comprises an antibacterial drug, an oral care product, a medical coating, and a caries-preventing preparation.
[0013] Further, the antibacterial drug comprises a nanoparticle drug carrier or a local sustained-release preparation.
[0014] Further, the MIC value of the antibacterial preparation is 32 μg / mL, the MBC value of the antibacterial agent is 256 μg / mL, and the bacteria inhibited or killed by the antibacterial preparation are Streptococcus mutans ATCC700610.
[0015] In summary, the application has the following beneficial effects:
[0016] 1、The present application is confirmed by MIC and MBC determination test, growth curve test, crystal violet staining test, bacterial live / dead staining test and qRT-PCR test that etoposide can effectively down-regulate biofilm-related gene expression (such as gtfB, gtfC, gtfD, etc.), inhibit the formation of bacterial (especially Streptococcus ATCC700610) biofilm or destroy the formed biofilm, and achieve the effect of antibacterial.
[0017] 2、The etoposide of the present application can be used alone or in combination with other antibacterial ingredients for oral care products, medical coatings or caries prevention preparations, and has wide application.
[0018] 3、The present application provides a new potential lead compound and a new anti-infection approach for preventing or treating diseases caused by Streptococcus ATCC700610 infection.
[0019] BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 Figure for the determination results of the minimum bactericidal concentration of etoposide of the present application on Streptococcus ATCC700610;
[0021] Figure 2 Figure for the growth curve results of etoposide of the present application on Streptococcus ATCC700610;
[0022] Figure 3 Figure for the crystal violet staining results of etoposide of the present application on Streptococcus ATCC700610 biofilm;
[0023] Figure 4 Figure for the bacterial live / dead staining results of etoposide of the present application on Streptococcus ATCC700610;
[0024] Figure 5 Figure for the gene expression level results of Streptococcus ATCC700610 after treatment with etoposide of the present application. DETAILED DESCRIPTION
[0025] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0026] The following are examples of using etoposide to resist Streptococcus ATCC700610, including minimum inhibitory concentration, minimum bactericidal concentration test, growth curve test, bacterial biofilm determination test, bacterial live / dead staining test and qRT-PCR test.
[0027] Streptococcus mutans ATCC700610, a representative Gram-positive facultative anaerobic bacterium with biofilms, was used as a test subject to demonstrate the utility of etoposide in antibacterial preparations. Etoposide can achieve antibacterial effects by downregulating bacterial biofilm gene expression, inhibiting biofilm formation, or destroying existing biofilms. Specific examples are as follows:
[0028] Example 1
[0029] This example is a test of minimum inhibitory concentration and minimum bactericidal concentration:
[0030] Adjust the bacterial suspension overnight with BHI medium and set aside for use. Add 100 μL of the adjusted bacterial suspension to each well of a 96-well plate (the bacterial inoculum size is 1×10 6 CFUs / mL), then add the etoposide compound and mix thoroughly by pipetting to achieve final concentrations of 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, 512 μg / mL, 1024 μg / mL, 2048 μg / mL, and 4096 μg / mL. The plates were incubated anaerobically at 37°C. The bacterial growth on the 96-well plates was observed the next day. The concentration of the compound that inhibited bacterial growth with the naked eye was determined as the MIC of etoposide against S. mutans ATCC 700610. The minimum concentration at which no colonies were observed on the BHI agar plate after 24 hours of anaerobic incubation was defined as the MBC concentration.
[0031] Conclusion: If Figure 1 As shown in the figure, the MIC (minimum inhibitory concentration) value of etoposide against Streptococcus mutans ATCC700610 was 32 μg / mL, and the MBC (minimum bactericidal concentration) value was 256 μg / mL.
[0032] Example 2
[0033] This example is a growth curve test:
[0034] Streptococcus mutans ATCC700610 was co-cultured with various concentrations of etoposide overnight at 37°C for 24 hours. Groups were assigned to treat Streptococcus mutans ATCC700610 with 0 μg / mL, 4 μg / mL, 16 μg / mL, and 64 μg / mL etoposide. 0.2% chlorhexidine was used as a positive control. During the incubation period, the optical density absorbance of the bacterial samples at 600 nm was measured every hour using a microplate reader.
[0035] Conclusion: If Figure 2As shown, the addition of different concentrations of etoposide significantly inhibited the growth of S. mutans ATCC 700610.
[0036] Example 3,
[0037] In this example, a bacterial biofilm assay was performed:
[0038] S. mutans ATCC 700610 was diluted into a 96-well plate with BHIS medium to a concentration of 2 x 10 6 CFUs / mL. Different concentrations of etoposide were added to each well, with a final concentration of 0, 4, 16, and 64 μg / mL. After 12 h of anaerobic incubation at 37 °C, the 96-well plate was removed, and the excess medium was removed. The loosely adherent planktonic bacteria were removed by washing three times with 200 μL of PBS. The biofilm in the wells was fixed with 4% (wt / vol) paraformaldehyde for 15 min. Then, 0.1% (wt / vol) crystal violet-ethanol solution was added to each well for 30 min. After removing the solution, the wells were washed three times with 200 μL of PBS to remove the excess dye. Anhydrous ethanol was added to dissolve the dye, and an equal volume of the solution from each well was transferred to a new 96-well plate. The absorbance was measured at 575 nm using a microplate reader.
[0039] Conclusion: As shown in Figure 3, the addition of different concentrations of etoposide significantly inhibited the formation of S. mutans ATCC 700610 biofilm. Figure 3
[0040] Example 4,
[0041] In this example, a bacterial live / dead staining assay was performed:
[0042] The bacterial biofilm was cultured in a 96-well plate as previously described, and a gradient concentration of etoposide was set (64 μg / mL, 16 μg / mL, 4 μg / mL, and 0 μg / mL). After 12 h of growth, the bacterial solution was discarded, and the surface-floating bacteria were rinsed with sterile PBS solution. After completion, the liquid was discarded, and the plate was dried. The biofilm was observed using an inverted fluorescence microscope. DMAO & PI were used to stain the biofilm.
[0043] Conclusion: As shown in Figure 4, the resulting images reflect the green (live bacteria) and red (dead bacteria) fluorescence intensity. Without the addition of etoposide, the biofilm was evenly distributed and relatively dense in structure, completely covering the surface. After treatment with etoposide, the biofilm was highly dispersed, and the biofilm structure was significantly loose. The results indicate that etoposide can reduce the surface area covered by the biofilm, leading to a significant reduction in biofilm biomass. Figure 4
[0044] Example 5,
[0045] This example is a qRT-PCR experiment:
[0046] S. mutans ATCC 700610 was cultured in BHIS and etoposide was added to a final concentration of 64 μg / mL and incubated anaerobically at 37°C for 12 h. The bacteria were collected by centrifugation (4°C, 4000 rpm) and then treated with 100 μL of lysozyme (30 mg / mL) for 30 min. Total RNA was extracted and the purified RNA was dissolved in 80 μL of RNase-Free water. The bacterial RNA was then reverse transcribed to generate cDNA. qRT-PCR was performed using a 96-well plate. After obtaining the data, the relative gene expression was calculated using the 2 -ΔΔCt Method to calculate relative gene expression.
[0047] Conclusion: As shown in Table 1, the expression of gtfB, gtfC, gtfD, gbpC, LuxS, and srtA genes were significantly inhibited when S. mutans ATCC 700610 was treated with 64 μg / mL of etoposide. Figure 5
[0048] The above examples are only used to illustrate the technical solutions of the present application and not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and all such modifications or replacements should be included in the scope of the claims of the present application. The technical, shape, and structure parts not described in detail in the present application are well-known technologies.
Claims
1. Use of etoposide in antibacterial preparations.
2. The use of etoposide in antibacterial preparations according to claim 1, characterized in that: The antibacterial preparation is a biofilm inhibitor. Etoposide inhibits the formation of biofilm or destroys the formed biofilm by down-regulating the gene expression of bacterial biofilm.
3. The use of etoposide in antibacterial preparations according to claim 2, characterized in that: The biofilm is a biofilm of Gram-positive cocci.
4. The use of etoposide in antibacterial preparations according to claim 3, characterized in that: The biofilm is a biofilm of Streptococcus mutans ATCC700610.
5. The use of etoposide in antibacterial preparations according to claim 4, characterized in that: The etoposide is chemically synthesized or derived from natural sources.
6. The use of etoposide in antibacterial preparations according to claim 5, characterized in that: The etoposide is extracted from the Berberidaceae plants Podophyllum americanum or Podophyllum tibetum.
7. An antibacterial preparation, characterized in that The antibacterial preparation includes an etoposide component.
8. The antibacterial preparation according to claim 7, characterized in that The antibacterial preparations include antibacterial drugs, oral care products, medical coatings, and anti-caries preparations.
9. The antibacterial preparation according to claim 8, characterized in that The antibacterial drug includes nanoparticle-loaded drug or local sustained-release preparation.
10. The antibacterial preparation according to claim 9, characterized in that The MIC value of the antibacterial preparation is 32 μg / mL, the MBC value of the antibacterial preparation is 256 μg / mL, and the bacterium inhibited or killed by the antibacterial preparation is Streptococcus mutans ATCC700610.