Application of quaternary ammonium salt pyridone as broad-spectrum bactericidal enhancer of plasma microbubble discharger

Abstract

The invention discloses an application of quaternary ammonium salt pyridone as a broad-spectrum bactericidal enhancer of a plasma microbubble discharger, which comprises the following steps: dissolving quaternary ammonium salt pyridone in normal saline, and discharging in the plasma microbubble discharger for 5-15 minutes to obtain plasma microbubble discharge activated water; the obtained plasma microbubble discharge activated water and pathogenic bacteria liquid are blended in an equal amount, and sterilization is conducted; the quaternary ammonium salt pyridone is cationic pyridone or (methyl) cationic pyridone, the structural formula of the cationic pyridone is C15H27N2O, and the structural formula of the (methyl) cationic pyridone is C16H29N2O. Quaternary ammonium salt pyridone serves as a singlet oxygen carrier, the survival time of singlet oxygen is prolonged through endoperoxide loaded with singlet oxygen, and efficient broad-spectrum killing of pathogenic microorganisms is achieved on the basis that it is kept that plasma killing is not prone to generating tolerance and biological safety.

Description

Technical Field

[0001] This invention belongs to the field of broad-spectrum disinfection of pathogenic microorganisms, and relates to the application of quaternary ammonium salt pyridinone as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices. Background Technology

[0002] Bacteria, fungi, viruses, and other microorganisms are suspended in the air as aerosols, and can also deposit, attach, and accumulate on susceptible interfaces of the human body, wounds, surgical incisions, or enter the body through respiration, causing deep tissue infections and diseases. Therefore, a highly effective broad-spectrum bactericidal strategy that balances treatment safety is needed to combat various pathogens. Reactive oxygen species (ROS) play an important role in the treatment of various diseases. Investigations have revealed that singlet oxygen (ROS)... 1 O2, a key component of reactive oxygen species, often suffers from low utilization due to its short lifespan and short diffusion. 2-Hydroxypyridine can rapidly and efficiently capture oxygen through the Diels-Alder reaction. 1 O2 forms an intramolecular peroxide bond in the molecule. Moreover, this reaction is reversible. After the formation of the intramolecular peroxide bond, the reversible reaction can occur relatively quickly at body temperature (37°C), releasing oxygen. 1 O2; under in vitro light irradiation, pyridinone can rapidly capture 1 O2 is released slowly throughout the body, thus killing pathogens. 1 The process of releasing O2 does not depend on oxygen or light, and can continue for several hours. 1 O2 is widely recognized as a reliable chemical source for generating singlet oxygen. A common method for delivering singlet oxygen is photodynamic therapy, which involves photosensitizer molecules generating reactive oxygen species after exposure to light of a specific wavelength. However, this method inevitably requires the addition of photosensitizers, which is detrimental to delivery and mechanism studies, and may also introduce additional toxicity and poor stability. Therefore, achieving highly efficient, safe, and broad-spectrum therapy is of profound significance.

[0003] Plasma-activated water is a product of atmospheric pressure discharge plasma applied in the biomedical field. Due to its transient biochemical activity, the use of air discharge instead of other toxic chemicals as raw materials, and its environmental friendliness and the ability to be driven by renewable energy sources, it is considered a green and promising biochemical material. The large number of reactive oxygen ions and high-energy free radicals in the plasma oxidize and denature the proteins and nucleic acids in pathogens, leading to their death. Reactive oxygen species analysis shows that ·OH and ·OH are generated in the plasma-liquid interaction system. 1 O2、·O 2- and H2O2, while ·O 2- yes 1O2 precursors. The synergistic effect between active particles has good applications in sterilization and inhibition of viral infections. However, plasma antibacterial treatment has drawbacks such as short half-life of singlet oxygen, instability, easy quenching, inability to achieve broad-spectrum sterilization, difficulty in enrichment, and low spectral sterilization efficiency. Therefore, utilizing the reversible storage and release of singlet oxygen substances in the pyridone structure to treat deep infections provides a new strategy for improving traditional photodynamic therapy, and grafting cationic quaternary ammonium salts further enhances sterilization efficiency.

[0004] Only reactive oxygen species located within the cell membrane or cell interior can damage cellular components and lead to cell death. Gram-positive bacteria have a relatively dense peptidoglycan layer. Gram-negative bacteria have more complex cell walls, consisting of an outer membrane rich in lipopolysaccharides, a peptidoglycan layer, and an inner membrane. Fungal biological barriers are typically formed by a lipid bilayer encasing the cell wall. Viruses, seemingly simple in structure and lacking organelles, can enter the human or animal body and integrate their genes into host cells. Their different structures determine the varying time it takes for materials to reach effective killing sites; therefore, we need to design and synthesize cationic pyridones with different substituents to explore the relationship between singlet oxygen loading, release rate, and delivery amount. Plasma microbubble generators are characterized by low resistance, minimal irritation, and environmental safety; combining these with other technologies can effectively treat various surface or deep-seated pathogen infections. Summary of the Invention

[0005] In view of this, the present invention provides the application of quaternary ammonium salt pyridinone as a broad-spectrum bactericidal enhancer in plasma microbubble discharge devices. Specifically, the present invention provides the following technical solution:

[0006] 1. Application of quaternary ammonium pyridone as a broad-spectrum bactericidal enhancer in plasma microbubble discharge devices: Quaternary ammonium pyridone is dissolved in physiological saline and discharged in a plasma microbubble discharge device for 5-15 minutes to obtain plasma microbubble discharge activated water. The obtained plasma microbubble discharge activated water is mixed with an equal amount of pathogenic bacterial solution for sterilization. The quaternary ammonium pyridone is a cationic pyridone or a (methyl) cationic pyridone. The structural formula of the cationic pyridone is C15H27N2O, and the structural formula of the (methyl) cationic pyridone is C16H29N2O.

[0007] Furthermore, the concentration of quaternary ammonium pyridinone in physiological saline is 32-64 mM.

[0008] Furthermore, the pH of physiological saline is 8.0-8.5.

[0009] Furthermore, the preparation method of the cationic pyridone is as follows: 1) 2-hydroxypyridine is reacted with 1,4-dibromobutane and cesium carbonate to obtain bromopyridone; 2) the bromopyridone obtained in step 1) is grafted with triethylamine to obtain cationic pyridone.

[0010] Furthermore, in step 1), the molar ratio of the three reactants, 2-hydroxypyridine, 1,4-dibromobutane, and cesium carbonate, is 1:1.5:1, the reaction temperature is 25-35℃, and the reaction time is 12-24 hours.

[0011] Furthermore, in step 2), the molar ratio of bromopyridone to triethylamine is 1:1.5; the reaction temperature in step 1) is 25-35℃, and the reaction time is 12-24 hours.

[0012] Furthermore, the preparation method of the (methyl) cationic pyridone is as follows: 1) 2-hydroxy-6-methylpyridine is reacted with 1,4-dibromobutane to obtain bromopyridone; 2) the bromopyridone obtained in step 1) is grafted with triethylamine to obtain (methyl) cationic pyridone.

[0013] Furthermore, in step 1), the molar ratio of the three reactants, 2-hydroxy-6-methylpyridine, 1,4-dibromobutane, and cesium carbonate, is 1:1.5:1, the reaction temperature is 25-35℃, and the reaction time is 12-24 hours.

[0014] Furthermore, in step 2), the molar ratio of bromopyridone to triethylamine is 1:1.5; the reaction temperature in step 1) is 25-35℃, and the reaction time is 12-24 hours.

[0015] Furthermore, the pathogenic microorganisms mentioned are Staphylococcus aureus, Pseudomonas aeruginosa, or Candida albicans.

[0016] The beneficial effects of this invention are as follows: This invention utilizes the reversible storage and release of reactive oxygen species (ROS) in the pyridone structure to solve the problem of short lifetime of singlet oxygen generated by plasma microbubble discharge devices. Quaternary ammonium pyridone is used as a singlet oxygen carrier, and the survival time of singlet oxygen is extended by loading internal peroxides with singlet oxygen. After entering pathogens, singlet oxygen releases or induces the generation of high concentrations of ROS, exhibiting highly effective disinfection of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and feline calicivirus, and causing severe damage and elimination of dense, hypoxic biofilms. It achieves highly efficient and broad-spectrum disinfection of pathogenic microorganisms while retaining the advantages of plasma-induced killing, such as low tolerance and biosafety. Attached Figure Description

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0018] Figure 1 The preparation route diagram for quaternary ammonium salt pyridinones with different substituents.

[0019] Figure 2 This is the NMR spectrum of a cationic pyridinone.

[0020] Figure 3 The NMR spectrum of (methyl) cationic pyridone is shown.

[0021] Figure 4 The image shows the singlet oxygen ESR diagrams for two quaternary ammonium pyridinone salts.

[0022] Figure 5 This is a diagram showing the singlet oxygen release of two quaternary ammonium salt pyridinones.

[0023] Figure 6 The image shows the results of endothelial damage in Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans after different treatment groups.

[0024] Figure 7 The results of ROS levels in Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans after different treatment groups were statistically analyzed.

[0025] Figure 8 The image shows the killing effect of quaternary ammonium salt pyridinone on Staphylococcus aureus and Pseudomonas aeruginosa.

[0026] Figure 9 The results show the killing effect of quaternary ammonium salt pyridinone on Candida albicans.

[0027] Figure 10 Statistical graph showing the killing effect of (methyl) cationic pyridone on feline calicivirus.

[0028] Figure 11 This is a statistical chart showing the damage caused by (methyl) cationic pyridone to biomembranes. Detailed Implementation

[0029] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0030] Figure 1 This paper presents the preparation routes for quaternary ammonium pyridones with different substituents. First, bromopyridones are prepared via a substitution reaction, followed by the introduction of triethylamine to obtain quaternary ammonium pyridones. Quaternary ammonium pyridones can enhance the broad-spectrum bactericidal performance of plasma microbubble discharge devices.

[0031] Example 1: Preparation of cationic pyridone

[0032] (1) Synthesis of 2-bromopyridone.

[0033] 282.6 mg of 2-hydroxypyridine and 537.4 μL of 1,4-dibromobutane (1:1.5) were dissolved in 15 mL of tetrahydrofuran, and then 1955 mg of cesium carbonate was added. The mixture was stirred at 35 °C for 24 h (the reaction can be scaled up three times). After the reaction was complete, all the product was collected and centrifuged at 3000 rpm for 5 min to separate the catalyst cesium carbonate. Then, tetrahydrofuran was removed by rotary evaporation to obtain the crude product. The crude product was separated by column chromatography using an eluent (methanol:dichloromethane = 1:20) to obtain 2-bromopyridone.

[0034] (2) Synthesis of cationic pyridinone.

[0035] Take 100 mg of 2-bromopyridone from step 1) and dissolve it in 4 mL of acetonitrile with 150 mg of triethylamine. Stir at 84 °C for 12 h. After the reaction is complete, remove the product and then remove the solvent by rotary evaporation. Quaternized pyridone is obtained as a red oil. Dissolve the product in a small amount of methanol, wash with isopropyl ether, remove the isopropyl ether, and wash with n-hexane. This washing process can be repeated several times. After washing, vacuum is applied to obtain cationic pyridone.

[0036] Characterization of cationic pyridinones. The products of this example were characterized by nuclear magnetic resonance (NMR). All products were analyzed by 1H NMR spectra obtained in CDCl3 using an NMR spectrometer.

[0037] Figure 2 The image shows the NMR spectrum of a cationic pyridinone. The 1H NMR spectrum of the 2-bromopyridinone obtained in step 1) is shown below. Figure 2 (Left). δ 7.30 (1H), 6.46 (1H), 6.18 (1H), 3.98 (2H), 3.45 (2H), 1.94 (4H). The first four peaks are characteristic peaks generated by the corresponding hydrogen atoms on the pyridone, and the last three peaks are characteristic peaks generated by the hydrogen atoms on the carbon chain of 1,4-dibromobutane. From the peak area integration of the NMR spectrum, we can obtain: a:b:c:d:e:f = 2:1:1:2:2:4. This proves that 1,4-dibromobutane replaced the hydrogen atom on the nitrogen atom of pyridone, generating 2-bromopyridone. The 1H NMR spectrum of the final product, cationic pyridone, is shown below. Figure 2 (Right). δ7.72(1H), 7.56(1H), 6.60(1H), 6.45(1H), 4.08(2H), 3.52(2H), 3.48(6H), 1.82(4H), 1.32(9H), where peaks a, b, c, and d are characteristic peaks generated by the corresponding hydrogen atoms on pyridinone, and peak h is the characteristic peak corresponding to the hydrogen atom at the ethyl terminus of triethylamine, indicating that cationic pyridinone was successfully synthesized.

[0038] Example 2: Preparation of (methyl) cationic pyridone

[0039] (1) Synthesis of 2-hydroxy-6-methyl-bromopyridone. 327.39 mg of 2-hydroxypyridine and 537.4 μL of 1,4-dibromobutane (1:1.5) were dissolved in 15 mL of tetrahydrofuran, followed by the addition of 1955 mg of cesium carbonate. The mixture was stirred at 35 °C for 24 h (the reaction volume could be increased threefold). After the reaction was complete, all the product was collected and centrifuged at 3000 rpm for 5 minutes to separate the catalyst cesium carbonate. Tetrahydrofuran was then removed by rotary evaporation to obtain the crude product. The crude product was separated by column chromatography using an eluent (methanol:dichloromethane = 1:20) to obtain 2-hydroxy-6-methyl-bromopyridone.

[0040] (3) Synthesis of (methyl) cationic pyridone.

[0041] Take 100 mg of 2-hydroxy-6-methyl-bromopyridone from step 1) and dissolve it in 4 mL of acetonitrile with 150 mg of triethylamine. Stir at 84 °C for 12 h. After the reaction is complete, remove the product and then remove the solvent by rotary evaporation. The quaternized pyridone is a red oil. Dissolve the product in a small amount of methanol, wash with isopropyl ether, remove the isopropyl ether, and wash with n-hexane. This washing process can be repeated several times. After washing, vacuum is applied to obtain the quaternary ammonium pyridone.

[0042] Characterization of (methyl)cationic pyridones. The products in this example were characterized by nuclear magnetic resonance (NMR). All products were obtained by NMR in CDCl3. 1 H NMR spectrum.

[0043] Figure 3 The NMR spectrum of the (methyl) cationic pyridinone is shown below. The 1H NMR spectrum of the 2-hydroxy-6-methyl-bromopyridinone obtained in step 1) is shown below. Figure 3 (Left). δ 7.20 (1H), 6.42 (1H), 6.02 (1H), 4.02 (2H), 3.48 (2H), 2.40 (3H), 1.94 (2H), 1.88 (2H). From the NMR peak area integration, a∶b∶c∶d∶e∶f∶g∶h=1∶1∶1∶3∶2∶2∶2∶2. This proves that 1,4-dibromobutane substituted the hydrogen atom on the nitrogen atom of pyridinone, generating 2-hydroxy-6-methyl-bromopyridinone. The 1H NMR spectrum of the final product, the (methyl)cationic pyridinone, is shown below. Figure 3 (Right). The NMR integral of (methyl) cationic pyridone yields a∶b∶c∶e∶f∶g∶i∶h=1∶1∶1∶2∶4∶6∶9, which proves the successful synthesis of (methyl) cationic pyridone.

[0044] Test Example 1: Singlet oxygen release capacity test of quaternary ammonium salt pyridinone

[0045] Singlet oxygen in quaternary ammonium pyridones (cationic pyridones and (methyl)cationic pyridones) was qualitatively detected using electron paramagnetic resonance (ESR) spectroscopy, with 2,2,6,6-tetramethylpiperidinol (HTMP) used as a probe to measure the generation of singlet oxygen.

[0046] Quaternary ammonium pyridone was dissolved in PBS (resulting in a 32 mM solution), and microbubble plasma was discharged for 10 min under atmospheric conditions. Simultaneously, an HTMP solution (400 mM) was prepared. After standing for 3 min at the beginning and end of the discharge, 100 μL of the solution was added to an equal volume of HTMP solution, and the mixture was incubated at 37°C in the dark for 30 min. A control was prepared without the addition of quaternary ammonium pyridone. Finally, electron paramagnetic resonance (ESR) spectroscopy was performed.

[0047] Figure 4 The figures show the singlet oxygen ESR of two quaternary ammonium pyridinone salts. From the figures, we can see that:

[0048] Figure 4 The left side shows the addition of cationic pyridone, denoted as + cationic pyridone in the figure, which shows a typical 1:1:1 triplet peak. The control group has no obvious signal peak.

[0049] Figure 4 The right side shows the addition of (methyl) cationic pyridone, denoted as +(methyl) cationic pyridone in the figure. It exhibits a more significant 1:1:1 triplet with a longer signal duration (the baseline did not drop rapidly after the peak). The control group showed no obvious signal peak.

[0050] This demonstrates the successful loading and release of singlet oxygen by the two quaternary ammonium pyridinone salts of the present invention.

[0051] Quantitative detection of singlet oxygen in quaternary ammonium pyridones (cationic pyridones and (methyl)cationic pyridones) using the Singlet Oxygen Sensor Green Fluorescent Probe (SOSG). 1 The release of O2. SOSG is a probe that is highly selective in binding to singlet oxygen, and is effective against hydroxyl radicals (·OH) and superoxide anion radicals (·O). 2- There was no obvious reaction between [acid] and nitric oxide (NO). [With] 1 After the O2 reaction, the generated SOSG endoperoxide (SOSG-EP) emits a green fluorescence similar to fluorescein, with a maximum excitation wavelength of 504 nm and a maximum emission wavelength of 525 nm.

[0052] Set up three groups, namely:

[0053] Group 1: Quaternary ammonium pyridone is dissolved in physiological saline (the concentration of the quaternary ammonium pyridone solution is 32mM), and then discharged by a plasma microbubble generator, denoted as: +(methyl) cationic pyridone or + cationic pyridone.

[0054] Group 2: First, discharge the physiological salt for 3 minutes. After the discharge is complete, add quaternary ammonium salt pyridone (the concentration of the quaternary ammonium salt pyridone solution is 32mM), which is recorded as: -(methyl) cationic pyridone or -cationic pyridone.

[0055] Group 3: Direct discharge group with physiological saline, denoted as: control group.

[0056] Prepare SOSG solution (5 μM concentration), and load singlet states using microbubble plasma discharge for 10 min. 1 After O2 discharge, 100 μL of the discharged liquid was mixed with an equal volume of SOSG solution in the dark, and added parallel to a 96-well plate. The absorbance difference of singlet oxygen over time was measured using a microplate reader at a fluorescence wavelength (λex / em = 488 / 525 nm) to determine the result. 1 O2 release. Half-life is defined as the time required for the instantaneous rate of singlet oxygen production to decrease from its initial maximum to half; or indirectly reflected by analyzing the time it takes for the cumulative release to reach half of its maximum value.

[0057] Figure 5 This is a diagram showing the singlet oxygen release of two quaternary ammonium salt pyridinones.

[0058] from Figure 5 As can be seen from a, the release of singlet oxygen by adding +(methyl)cationic pyridone to the discharger is significantly higher than that by -(methyl)cationic pyridone, proving that (methyl)cationic pyridone effectively loads singlet oxygen and prolongs its release. Analysis shows that the half-life of (methyl)cationic pyridone is approximately 90 minutes.

[0059] from Figure 5 As can be seen from b, the release of singlet oxygen by adding +cation pyridone to the discharger is significantly higher than that by -cation pyridone, proving that cationic pyridone effectively loads singlet oxygen and prolongs its release. Analysis shows that the half-life of cationic pyridone is approximately 20 minutes.

[0060] from Figure 5 As can be seen from c, the release rate per minute of (methyl) cationic pyridone is four times that of cationic pyridone. Fitting the fluorescence intensity data to time yields an exponential growth curve, eventually reaching a plateau. The growth rate constant of this curve reflects the release rate of singlet oxygen. Therefore, (methyl) cationic pyridone results in a greater release of singlet oxygen and a longer survival time.

[0061] Test Example 2: Pathogen Film Integrity Test

[0062] The cell membrane is the "barrier and foundation" for pathogens to maintain their physiological functions. Maintaining membrane integrity is a prerequisite for pathogen material transport, energy metabolism, and osmotic pressure balance. Therefore, detecting the integrity of the pathogen membrane can determine whether singlet oxygen is effectively bactericidal.

[0063] Test method: Overnight cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were washed three times with PBS buffer and then resuspended. Three groups of materials were prepared:

[0064] Group 1: Quaternary ammonium pyridone is dissolved in physiological saline (the concentration of the quaternary ammonium pyridone solution is 32mM), and then discharged by a plasma microbubble generator, denoted as: +(methyl) cationic pyridone or + cationic pyridone.

[0065] Group 2: First, discharge the saline solution for 3 minutes. After the discharge is complete, add quaternary ammonium salt pyridone (the concentration of the quaternary ammonium salt pyridone solution is 32mM), which is recorded as: -(methyl) cationic pyridone or -cationic pyridone.

[0066] Group 3: Direct discharge group with physiological saline, denoted as: control group.

[0067] Each discharge lasted 10 minutes. After discharge, 50 μL of bacterial suspension and 50 μL of the three material solutions were added to a 96-well plate. The plates were then incubated in a shaker at 37°C for 6 hours under dark conditions. Finally, 25 μL of a 1×10⁻⁶ solution was added. -8 A mol / L solution of propidium iodide (PI) was prepared. The fluorescence intensity after co-culturing with different groups was then measured using a microplate reader (excitation wavelength: 535 nm, emission wavelength: 620 nm). PI can penetrate the cell membrane disrupted by bacterial death, embedding itself into the DNA double strand to stain the cell nucleus, thus producing a fluorescence intensity value. Figure 6 The image shows the results of endothelial damage in Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans after different treatment groups.

[0068] Figure 6 As can be seen from a, the fluorescence intensity of +(methyl)cationic pyridone was significantly higher than that of -(methyl)cationic pyridone and the control group, indicating that (methyl)cationic pyridone can significantly disrupt the membrane integrity of Staphylococcus aureus after being subjected to singlet oxygen generated by a loaded plasma microbubble discharge device.

[0069] Figure 6 As can be seen from b, the fluorescence intensity of the + cationic pyridone is higher than that of the - cationic pyridone and the control group, indicating that the cationic pyridone can effectively release singlet oxygen.

[0070] Figure 6c shows that the fluorescence intensity of +(methyl)cation pyridone is much higher than that of -(methyl)cation pyridone and the control group, indicating that (methyl)cation pyridone can significantly disrupt the membrane integrity of Pseudomonas aeruginosa after being subjected to singlet oxygen generated by a loaded plasma microbubble discharge device.

[0071] Figure 6 As can be seen from d, the fluorescence intensity of the + cationic pyridone is higher than that of the - cationic pyridone and the control group, indicating that the cationic pyridone can effectively release singlet oxygen.

[0072] Figure 6 As can be seen from the results, the fluorescence intensity of the +(methyl) cationic pyridone was significantly higher than that of the other groups, indicating that the +(methyl) cationic pyridone can load more singlet oxygen and effectively release singlet oxygen during the co-action process, thereby damaging the Candida albicans membrane.

[0073] Test Example 3: Pathogen Infection Reactive Oxygen Species (ROS) Level Experiment

[0074] Test objective: Two quaternary ammonium salts, pyridinones, can enter pathogens and release singlet oxygen. Singlet oxygen, as an important ROS, can damage internal bacterial biomolecules, thereby affecting the normal metabolism of pathogens and causing bacterial death.

[0075] Test method: Wash the overnight cultured Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans three times with PBS buffer, and then resuspend.

[0076] The materials are set into three groups, namely:

[0077] Group 1: Quaternary ammonium pyridone is dissolved in physiological saline (the concentration of the quaternary ammonium pyridone solution is 32mM), and then discharged by a plasma microbubble generator, denoted as: +(methyl)cationic pyridone.

[0078] Group 2: First, discharge the saline solution for 3 minutes. After the discharge is complete, add quaternary ammonium salt pyridone (the concentration of the quaternary ammonium salt pyridone solution is 32mM), which is denoted as: -(methyl)cationic pyridone.

[0079] Group 3: Direct discharge group with physiological saline, denoted as: control group.

[0080] The discharge time for each group was 10 minutes. After discharge, 50 μL of bacterial suspension and 50 μL of material solution were added to a 96-well plate, along with 25 μL of a 1×10⁻⁶ solution. -5 A mol / L solution of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was prepared. After incubation at 37°C for 0, 1, 2, 4, and 6 hours in the dark, the fluorescence intensity was measured using a microplate reader (excitation wavelength: 488 nm, emission wavelength: 525 nm).

[0081] DCFH-DA is a fluorescent probe. After being taken up by bacteria, DCFH-DA can be deacetylated into a non-fluorescent compound by cellular esterases. Subsequently, as the ROS content inside the bacteria increases, it can be oxidized by ROS to 2′-7′ dichlorofluorescein (DCF). Therefore, we chose DCFH-DA as a fluorescent probe to detect the ROS content in bacteria.

[0082] Figure 7 The results of ROS levels in Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans after different treatment groups were statistically analyzed.

[0083] Figure 7 As can be seen from a, the fluorescence intensity increase of +(methyl) cationic pyridone is much greater than that of other groups, indicating that the internal peroxide formed by the loading of singlet oxygen by +(methyl) cationic pyridone effectively releases singlet oxygen, increases the internal ROS level of Staphylococcus aureus, and thus causes damage.

[0084] Figure 7 b shows that the fluorescence intensity increase of +(methyl) cationic pyridone is much greater than that of other groups, indicating that the internal peroxide formed by the loading of singlet oxygen by +(methyl) cationic pyridone effectively releases singlet oxygen, increases the internal ROS level of Pseudomonas aeruginosa, and thus causes damage.

[0085] Figure 7 c shows that the fluorescence intensity increase of +(methyl) cationic pyridone is much greater than that of other groups, indicating that the internal peroxide formed by the loading of singlet oxygen by +(methyl) cationic pyridone effectively releases singlet oxygen, increases the internal ROS level of Candida albicans, and thus causes damage.

[0086] Test Example 4: Test on the bactericidal performance of quaternary ammonium salt pyridinone-enhanced plasma.

[0087] The study tested quaternary ammonium pyridone as a cationic singlet oxygen carrier to enhance the sterilization performance of plasma against Staphylococcus aureus and Pseudomonas aeruginosa.

[0088] The bacterial strain was incubated in lysozyme (LB) medium at 37°C in a shaker for 12 hours, and then the OD was read using a micro spectrophotometer. 600 To the logarithmic growth phase, i.e. 0.6 × 10 8 -0.8×10 8 The optimal concentration is CFU / mL. Then dilute with physiological saline to an initial bacterial concentration of 2 × 10⁻⁶. 5 CFU / mL.

[0089] The materials are set into three groups, namely:

[0090] Group 1: Quaternary ammonium pyridone is dissolved in physiological saline (the concentration of the quaternary ammonium pyridone solution is 32mM), and then discharged by a plasma microbubble generator, denoted as: +(methyl) cationic pyridone or + cationic pyridone.

[0091] Group 2: First, discharge the saline solution for 3 minutes. After the discharge is complete, add quaternary ammonium salt pyridone (the concentration of the quaternary ammonium salt pyridone solution is 32mM), which is recorded as: -(methyl) cationic pyridone or -cationic pyridone.

[0092] Group 3: Direct discharge group with physiological saline, denoted as: control group.

[0093] The discharge time for each group was set to 10 minutes. After discharge, the three groups of material solutions were serially diluted in 96-well plates to concentrations of 32, 16, 8, 4, 2, 1, 0.5, and 0.25 mM, respectively. An equal volume of bacterial culture was then added, and the plates were co-cultured for 6 hours, followed by 12 hours in an incubator at 37°C. Three parallel dilutions were performed for each concentration (10-fold dilution for Staphylococcus aureus and 20-fold dilution for Pseudomonas aeruginosa), and 50 μL of each was plated. The antibacterial properties of the agar plates were recorded by photographing them with a digital camera.

[0094] Figure 8 The image shows the killing effect of quaternary ammonium salt pyridinone on Staphylococcus aureus and Pseudomonas aeruginosa.

[0095] from Figure 8 As can be seen from a, the minimum bactericidal concentration for the control group and the -cationic pyridone group was 16 mM, while that for the +cationic pyridone group was 4 mM, representing a 4-fold reduction in the bactericidal concentration against Staphylococcus aureus. This indicates that cationic pyridone successfully prolonged the survival lifetime of singlet oxygen generated by the plasma microbubble discharger, significantly improving its bactericidal effect against Staphylococcus aureus.

[0096] from Figure 8 As shown in b, the minimum bactericidal concentration for the control group was 16 mM, for -(methyl)cationic pyridone it was 8 mM, and for +cationic pyridone it was 2 mM, representing a 4-fold reduction in the bactericidal concentration against Staphylococcus aureus. This indicates that (methyl)cationic pyridone successfully prolonged the survival lifetime of singlet oxygen generated by the plasma microbubble discharger, significantly improving its bactericidal effect against Staphylococcus aureus.

[0097] from Figure 8 As shown in c, the minimum bactericidal concentration for the control group was 32 mM, for -cationic pyridone it was 16 mM, and for +cationic pyridone it was 2 mM, representing an 8-fold reduction in the bactericidal concentration for Pseudomonas aeruginosa. This indicates that cationic pyridone successfully prolonged the survival lifetime of singlet oxygen generated by the plasma microbubble discharger, significantly improving its bactericidal effect against Pseudomonas aeruginosa.

[0098] fromFigure 8 As can be seen from d, the minimum bactericidal concentration for the control group was 32 mM; for -(methyl)cationic pyridone it was 4 mM; and for +(methyl)cationic pyridone it was 0.5 mM, representing an 8-fold reduction in the bactericidal concentration for Pseudomonas aeruginosa. This indicates that (methyl)cationic pyridone successfully prolonged the survival lifetime of singlet oxygen generated by the plasma microbubble discharger, significantly improving its bactericidal effect against Pseudomonas aeruginosa.

[0099] Test Example 5: Test of the fungicidal performance of quaternary ammonium salt pyridinone-enhanced plasma.

[0100] The study tested the use of quaternary ammonium pyridone as a cationic singlet oxygen carrier to enhance the antifungal properties of plasma, using Candida albicans as an example.

[0101] The *Candida albicans* strain was cultured in lysate broth (LB) at 37°C in a shaker for 24 hours, then plated and counted to confirm that it had reached the logarithmic growth phase. The culture was then diluted with physiological saline to an initial fungal concentration of 2 × 10⁵ CFU / mL.

[0102] The materials are set into three groups, namely:

[0103] Group 1: Quaternary ammonium pyridone is dissolved in physiological saline (the concentration of the quaternary ammonium pyridone solution is 32mM), and then discharged by a plasma microbubble generator, denoted as: +(methyl) cationic pyridone or + cationic pyridone.

[0104] Group 2: First, discharge the saline solution for 3 minutes. After the discharge is complete, add quaternary ammonium salt pyridone (the concentration of the quaternary ammonium salt pyridone solution is 32mM), which is recorded as: -(methyl) cationic pyridone or -cationic pyridone.

[0105] Group 3: Direct discharge group with physiological saline, denoted as: control group.

[0106] The discharge time for each group was 10 minutes. After discharge, the three groups of material solutions were serially diluted in 96-well plates to concentrations of 16, 8, 4, 2, 1, 0.5, and 0.25 mM, respectively. An equal volume of bacterial culture was then added, and the plates were co-cultured for 6 hours, followed by 12 hours in an incubator at 37°C. Three parallel dilutions of each concentration (20-fold dilution) were performed, and 50 μL of each was plated. The antibacterial properties of the agar plates were recorded using a digital camera.

[0107] Figure 9 The results show the killing effect of quaternary ammonium salt pyridinone on Candida albicans.

[0108] As shown in the figure, the control group and -(methyl)cationic pyridone had high minimum inhibitory concentrations (MICs) and poor killing effects; while +(methyl)cationic pyridone had a MIC of 16 mM against Candida albicans, exhibiting the best killing effect. This indicates that adding the (methyl)cationic pyridone of this invention simultaneously with plasma microbubble generator discharge can successfully prolong the survival lifetime of singlet oxygen in PAW and significantly improve the killing effect on Candida albicans.

[0109] Test Example 6: Virus disinfection performance test of cationic singlet oxygen carrier-enhanced plasma.

[0110] CFRK cells, the host cells for the experiment, were removed from liquid nitrogen and rapidly thawed in 37°C water. They were then transferred using a capillary pipette into cell culture tubes containing cell maintenance medium, mixed by pipetting several times, and immediately centrifuged to remove the supernatant. Appropriate cell maintenance medium was added again, mixed by pipetting several times, centrifuged again, and then transferred to culture flasks containing 10 mL of complete culture medium. Cell growth was observed daily, and when a monolayer of cells was formed, it was used for sterilization experiments. Feline calicivirus seed, the cryopreserved strain, was thawed in a 37°C water bath, diluted 10-fold with cell maintenance medium, and then inoculated into cell culture flasks containing a monolayer of cells. The flasks were placed in a 37°C incubator to allow for cell adsorption and growth. Pathogenesis was observed daily, and the virus was harvested when 3 / 4 of the cells showed pathogenesis. The culture medium containing the virus and host cells was used to disrupt the host cells under ice bath conditions using ultrasound (or repeated freeze-thaw cycles) to release the virus. The virus suspension was then quickly centrifuged to remove the precipitate (mainly cell debris), and the supernatant was the desired virus suspension. Aliquot 1.0 mL into sterile centrifuge tubes (1.5 mL each). Take one vial of feline calicivirus suspension and determine its viral titer according to the viral titer assay method. Store the remainder frozen at -80°C for later use. After passing the identification test using the residual disinfectant chemical neutralization method, the feline calicivirus experiment will officially begin.

[0111] The materials are set into three groups, namely:

[0112] Group 1: Quaternary ammonium pyridone is dissolved in physiological saline (the concentration of the quaternary ammonium pyridone solution is 32mM), and plasma microbubble generator discharge is performed simultaneously. It is denoted as: +(methyl) cationic pyridone or + cationic pyridone.

[0113] Group 2: First, discharge the saline solution for 3 minutes. After the discharge is complete, add quaternary ammonium salt pyridone (the concentration of the quaternary ammonium salt pyridone solution is 32mM), which is recorded as: -(methyl) cationic pyridone or -cationic pyridone.

[0114] Group 3: Direct discharge group with physiological saline, denoted as: control group.

[0115] Each discharge lasted 10 minutes. After discharge, 0.9 mL of material from each of the three groups was placed in a test tube and incubated in a 20℃±1℃ water bath for 5 minutes. Then, 0.1 mL of feline calicivirus suspension was added and mixed thoroughly. Positive and negative controls were also set up. Cell maintenance medium was added to the virus suspension, without any disinfectant or drug removal treatment, and complete cell culture medium was added to uninoculated cell tubes for culture. After 6 hours of inactivation, the solution was treated with a neutralizing agent. According to the experimental specifications, appropriate virus-free diluents were used to create serially diluted samples for subsequent virus titer determination. The average inactivation logarithmic value was calculated using the following formula: Let the average viral infection titer of the positive (virus) control group (TCID) be... 50 The value of N0 (or PFU) in the test (disinfection) group was the average viral infection titer (TCID). 50 (or pfu) is Nx. Average inactivation log value = log No - log Nx. Under normal circumstances, an average inactivation log value of ≥4.00 from 3 tests is considered a qualified killing test for feline calicivirus.

[0116] Figure 10 Statistical graph showing the killing effect of (methyl) cationic pyridone on feline calicivirus.

[0117] As can be seen from the figure:

[0118] TCID of (methyl) cationic pyridone 50 The highest value, 5.4, indicates that adding the (methyl)cationic pyridone of this invention simultaneously with plasma microbubble generator discharge has the best virus-killing effect. The TCID values ​​of (methyl)cationic pyridone and the control group are... 50 A value below 1 indicates that it has no antiviral effect.

[0119] In summary, the addition of the (methyl) cationic pyridone of this invention during plasma microbubble generator discharge can successfully prolong the lifespan of internal peroxides and significantly improve the killing effect on feline calicivirus.

[0120] Test Example 7: Biofilm Damage Test

[0121] Bacterial biofilms are highly organized, systematic, three-dimensional multicellular communities formed by bacteria attaching to surfaces and secreting extracellular polymeric substances (EPS) during their growth. These EPS include extracellular DNA (eDNA), proteins, polysaccharides, and other biomolecules, encapsulating the bacteria. The formation of bacterial biofilms mainly involves five stages: bacterial adhesion; microcolony formation; EPS secretion; formation of the mature biofilm; and bacterial detachment and dispersal. Due to the unique structure and formation mechanism of bacterial biofilms, the bacteria within them exhibit significantly enhanced tolerance to heterogeneous external environments. Compared to planktonic bacteria, bacteria within biofilms can show 10 to 1000 times greater drug tolerance. Therefore, understanding the specific drug tolerance mechanisms of bacterial biofilms is crucial for preventing and controlling biofilm formation, inhibiting bacteria within biofilms, and treating bacterial biofilm-related infections.

[0122] Test method: Staphylococcus aureus and Pseudomonas aeruginosa were inoculated into lysate broth (LB) and cultured in a shaker at 37°C for 12 h. The OD600 was then measured using a micro spectrophotometer until it reached the logarithmic growth phase, i.e., 0.6 × 10⁸–0.8 × 10⁸ CFU / mL. The cultured bacterial suspension was then resuspended in LB medium containing 2% glucose to a concentration of 1 × 10⁸ CFU / mL. 800 μL of the resuspended bacterial suspension was then added to each well of a 48-well plate and incubated at 37°C for 3 days to allow for natural film formation and adhesion to the bottom of the wells. Three sets of materials were prepared:

[0123] Group 1: Quaternary ammonium pyridone is dissolved in physiological saline (the concentration of the quaternary ammonium pyridone solution is 32mM), and then discharged by a plasma microbubble generator, denoted as: +(methyl) cationic pyridone or + cationic pyridone.

[0124] Group 2: First, discharge the saline solution for 3 minutes. After the discharge is complete, add quaternary ammonium salt pyridone (the concentration of the quaternary ammonium salt pyridone solution is 32mM), which is recorded as: -(methyl) cationic pyridone or -cationic pyridone.

[0125] Group 3: Direct discharge group with physiological saline, denoted as: control group.

[0126] The discharge time for each group was 10 minutes. After discharge, the supernatant of the cultured biofilm was slowly aspirated, retaining the bottom membrane structure. Equal volumes of the three material solutions were added to each group, and the mixture was cultured for 6 hours. The biofilm was then dispersed using a pipette and diluted to 1×10⁴ CFU / mL. 50 μL of the diluted solution was then taken from each well and plated onto the agar plate. The biofilm damage performance was tested by photographing the agar plates with a digital camera.

[0127] Crystal violet staining was used to assess the destructive effect on biofilms. The procedure was as follows: The supernatant of the cultured biofilm was slowly aspirated, preserving the bottom membrane structure. The membrane was gently washed three times with PBS, and 200 μL of methanol was added to each well. The mixture was incubated for half an hour to fix the biofilm. The methanol was then aspirated, and the plate was allowed to dry at room temperature. 200 μL of 0.1% crystal violet solution was added, and the plate was incubated in the dark for 10 minutes. Unbound free dye was then slowly and repeatedly washed away with deionized water. After the plate dried, 200 μL of 33% glacial acetic acid solution was added to each well, and the plate was incubated at 37°C for 20-30 minutes to fully dissolve the crystal violet. Finally, the absorbance at 590 nm was read using a microplate reader; lower absorbance indicated better biofilm destruction. This method was used to evaluate the destructive effect of +(methyl)cationic pyridinone on biofilms.

[0128] Figure 11 This is a statistical graph showing the damage to biofilms caused by (methyl)cationic pyridones. Staphylococcus aureus is on the left, and Pseudomonas aeruginosa is on the right.

[0129] from Figure 11 As shown in a and b, the +(methyl) cationic pyridone exhibits a biofilm removal rate of up to 99.9% against Staphylococcus aureus and Pseudomonas aeruginosa. In contrast, the -(methyl) cationic pyridone and the control group showed a biofilm removal rate of less than 50%.

[0130] from Figure 11 As shown in c and d, the absorbance of +(methyl)cationic pyridone decreased significantly, indicating that the addition of (methyl)cationic pyridone during plasma microbubble generator discharge, and the resulting (methyl)cationic pyridone loaded with singlet oxygen, had a significant destructive effect on biofilms. In contrast, -(methyl)cationic pyridone and the control group showed higher absorbance, indicating almost no removal effect on biofilms.

[0131] In summary, the addition of the (methyl) cationic pyridone of this invention during the discharge of the plasma microbubble generator can successfully prolong the lifespan of internal peroxides and significantly improve the removal effect on biofilms.

[0132] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The application of quaternary ammonium salt pyridinone as a broad-spectrum bactericidal enhancer in plasma microbubble discharge devices, characterized in that, Quaternary ammonium pyridinone was dissolved in physiological saline and discharged in a plasma microbubble discharger for 5–15 minutes to obtain plasma microbubble-activated water. This plasma microbubble-activated water was then mixed with an equal volume of pathogenic bacterial solution for sterilization. The quaternary ammonium pyridinone was either a cationic pyridinone or a (methyl)cationic pyridinone, and the cationic pyridinone had the structural formula C1. 15 H 27 The structural formula of N2O, (methyl)cationic pyridone is C 16 H 29 N2O.

2. The application of the quaternary ammonium salt pyridinone according to claim 1 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, The concentration of quaternary ammonium salt pyridinone in physiological saline is 32-64 mM.

3. The application of the quaternary ammonium salt pyridinone according to claim 1 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, The pH of physiological saline is 8.0-8.

5.

4. The application of the quaternary ammonium salt pyridinone according to claim 1 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, The method for preparing the cationic pyridone is as follows: 1) 2-hydroxypyridine is reacted with 1,4-dibromobutane and cesium carbonate to obtain bromopyridone; 2) the bromopyridone obtained in step 1) is grafted with triethylamine to obtain cationic pyridone.

5. The application of the quaternary ammonium salt pyridinone according to claim 2 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, In step 1), the molar ratio of the three reactants, 2-hydroxypyridine, 1,4-dibromobutane, and cesium carbonate, is 1:1.5:1, the reaction temperature is 25-35℃, and the reaction time is 12-24 hours.

6. The application of the quaternary ammonium salt pyridinone according to claim 2 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, In step 2), the molar ratio of bromopyridone to triethylamine is 1:1.5; the reaction temperature in step 1) is 25-35℃, and the reaction time is 12-24 hours.

7. The application of the quaternary ammonium salt pyridinone according to claim 1 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, The preparation method of the (methyl) cationic pyridone is as follows: 1) 2-hydroxy-6-methylpyridine is reacted with 1,4-dibromobutane to obtain bromopyridone; 2) the bromopyridone obtained in step 1) is grafted with triethylamine to obtain (methyl) cationic pyridone.

8. The application of the quaternary ammonium salt pyridinone according to claim 5 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, In step 1), the molar ratio of the three reactants, 2-hydroxy-6-methylpyridine, 1,4-dibromobutane, and cesium carbonate, is 1:1.5:1, the reaction temperature is 25-35℃, and the reaction time is 12-24 hours.

9. The application of the quaternary ammonium salt pyridinone according to claim 5 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, In step 2), the molar ratio of bromopyridone to triethylamine is 1:1.5; the reaction temperature in step 1) is 25-35℃, and the reaction time is 12-24 hours.

10. The application of the quaternary ammonium salt pyridinone according to claim 1 as a broad-spectrum bactericidal enhancer for plasma microbubble discharge devices, characterized in that, The pathogenic microorganisms mentioned are Staphylococcus aureus, Pseudomonas aeruginosa, or Candida albicans.

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