Study On The Antibacterial Effects Of Sanguinarine Chloride Hydrate Against Staphylococcus Aureus

Macleaya cordata（M.cordata）is a traditional medicinal herb in Macleaya（Papaveraceae）,Sanguinarine（SG）is one of the main extracts of M.cordata,which primarily accumulates in leaves,accounting for approximately 61%of the total alkaloid content,which has antibacterial,anti-inflammatory,and anti-tumour properties.Sanguinarine chloride hydrate（SGCH）is the hydrochloride form of SG,compared with SG,it has the advantages of fast absorption,rapid distribution,fast metabolism,high bioavailability,and low body residue.SG has been shown to have good antibacterial activity against Staphylococcus aureus（SA）;however,there are few reports on its antibacterial mechanism against SA.Therefore,this study investigated the in vitro antibacterial activity and antibacterial mechanism of SGCH against SA,measured the inhibitory zone,minimum inhibitory concentrations（MIC）and minimum bactericidal concentrations（MBC）,and plotted the bactericidal activity curve.In addition,the micromorphology,DNA and proteins,alkaline phosphatase（AKP）activity,Na⁺K⁺,Ca²⁺Mg²⁺-adenosine triphosphate（ATP）activity,fluorescein diacetate（FDA）,and intracellular reactive oxygen species（ROS）were observed and detected.（1）Study on the antibacterial activity of SGCH against SASA,Enterococcus faecalis（E.faecalis）,Listeria monocytogenes（L.monocytogenes）,Shigella dysenteriae（Sh.dysenteriae）,and Pseudomonas aeruginosa（P.aeruginosa）were used as test bacteria.Firstly,the inhibitory zone was measured by agar-well diffusion test.Secondly,the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of SGCH on SA were determined by broth microdilution and colony forming units（CFU）assays.Finally,the bactericidal activity curve was plotted to evaluate the antibacterial effect of SGCH on SA.The results showed that the inhibitory effect of SGCH（250,500,1000μg/m L）on gram-positive bacteria was greater than that of negative bacteria,from strong to weak were Staphylococcus aureus,Enterococcus faecalis,Listeria monocytogenes,Shigella dysenteriae,and Pseudomonas aeruginosa,and the antibacterial effect of three SGCH concentrations was lower than that of the positive control chlortetracycline hydrochloride（CTC）.The MIC and MBC values were 128 and 256,256and 512,512 and 1024,512 and>1024,>1024,and>1024μg/m L,respectively.In the time-kill curve,SGCH with 8×MIC could completely kill SA within 24 h.In vitro antibacterial activity results showed that the antibacterial effect of SGCH on SA was stronger than that of other test bacteria,and its inhibitory ability increased not only with the increase of concentration,but also with the increase of treatment time.（2）Study on the antibacterial mechanism of SGCH against SAScanning electron microscopy（SEM）was used to observe the effect of different concentrations of SGCH on the morphology and ultrastructure of SA cells.The effect of SGCH on DNA leakage in SA was studied by measuring the changes in DNA content in SA cells by OD_260nm.The BCA protein concentration assay kit was used to treat SA and the absorbance was measured at OD_562nmto explore the effect of SGCH on the changes in total protein content in SA cells.The effect of SGCH on the permeability of SA cell membrane was studied by fluorescein diacetate（FDA）staining experiment.The alkaline phosphatase（AKP）kit and Na⁺K⁺and Ca²⁺Mg²⁺-Adenosine triphosphate（ATP）enzyme test kit were used to treat SA,and the absorbance was measured at OD_520nmand OD_636nmto explore the effect of SGCH on intracellular enzyme activity.The reactive oxygen species（ROS）detection kit was used to treat SA for 10 min,20 min,and 30 min respectively,and the fluorescence intensity of different treatment times was measured at the excitation wavelength of 488 nm and the emission wavelength of 525 nm.The difference in SA intracellular green fluorescence at different treatment times was observed by laser confocal laser scanning microscope（CLEM）to explore the effect of SGCH on intracellular ROS level of SA and determine whether cells were damaged by oxidation.The SEM image results showed that SGCH could significantly destroy the microstructure of SA cells,resulting in shrinkage,collapse,leakage of cell fluid,and even adhesion and agglomeration of SA cells.In the DNA and protein assays,2×MIC,4×MIC and 8×MIC SGCH leaked3.28,3.97 and 5.19 ng/μL DNA,and 48.27,100.80,and 188.27μgprot/m L protein from SA cells after 24 h culture,respectively,which were significantly different from 0×MIC（P<0.001）and showing an obvious concentration dependence.In FDA staining experiment,SA cells with 2×MIC,4×MIC,and 8×MIC were detected significantly lower than 0×MIC（P<0.001）,indicating that the permeability of the cell membrane was severely disrupted.In the determination of enzyme activity,8×MIC SGCH significantly increased the activity of extracellular Na⁺K⁺,Ca²⁺Mg²⁺-ATP and AKP of SA after 24 h culture,and the results showed that SGCH could not only destroy the integrity of SA cell membrane,increase the permeability of Na⁺K⁺and Ca²⁺Mg²⁺ions,but also destroy the cell wall of SA and make AKP leak.In addition,2×MIC was the optimal concentration to induce ROS production in bacteria.After 30 min culture,the measured fluorescence intensity was 9.52,which was significantly higher than 0×MIC（P<0.001）,causing oxidative damage to SA cells.The in vitro antibacterial mechanism results showed that SGCH could significantly destroy the microstructure of SA cells.SGCH was also able to interfere with the integrity and permeability of the SA cell wall and membrane,as confirmed by the increase in extracellular DNA,protein,FDA,Na⁺K⁺,Ca²⁺Mg²⁺-ATP and AKP activity.Moreover,SGCH could induce SA to produce large amounts of ROS.In summary,this study revealed that SGCH has a preferable antibacterial effect on SA by destroying the morphology of SA cells,the permeability and integrity of SA cell membrane and cell wall,and oxidative damage of SA cells,which provides a theoretical basis for the use of SG as an antibiotic substitute in animal husbandry and for the clinical control and treatment of diseases caused by SA.