Application of Ginsenoside C-K in preparation of gram bacterium infection resisting medicine

Abstract

The invention provides an application of Ginsenoside C-K in preparation of a medicine for resisting gram bacterial infection, and the CAS number of the Ginsenoside C-K is 39262-14-1, the CAS number of the Ginsenoside C-K is 39262-14-1, and the CAS number of the Ginsenoside C-K is 39262-14-1. The Ginsenoside C-K is used for preparing a medicine for resisting gram-positive bacterium infection, and the Ginsenoside C-K and a polypeptide antibiotic polymyxin B are combined to be used for preparing a medicine for resisting gram-negative bacterium infection. According to the technical scheme, the novel medical application of the Ginsenoside C-K. The Ginsenoside C-K has the effects of inhibiting growth of gram-positive bacteria and formation of a biofilm, and the Ginsenoside C-K and polypeptide antibiotic polymyxin B are combined to have the effect of reversing drug resistance of gram-negative bacteria to the polymyxin B. The invention further discloses a preparation method of the Ginsenoside C-K.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of Ginsenoside CK in preparing anti-Gram infection drugs. Background Art

[0002] Over the past few decades, the development of new antibiotics has slowed significantly, with only a few antibiotics with unique molecular structures or novel mechanisms of action approved for clinical use. This situation makes alternative treatments to traditional antibiotics particularly important when addressing infections caused by multidrug-resistant (MDR) bacteria. As the threat of drug-resistant bacteria continues to escalate, finding alternative therapies has become a critical issue that the global medical community urgently needs to address.

[0003] Biofilms are complex structures composed of bacterially secreted extracellular polysaccharides, proteins, and DNA, and are widely present in Gram-positive bacteria. This structure provides a protective barrier for bacteria, enabling them to resist external environmental stress and antibiotic attack, and is a key mechanism of bacterial resistance. The extracellular matrix in biofilms not only impedes antibiotic penetration but also alters the bacterial metabolic state, significantly reducing the bactericidal efficacy of antibiotics. Furthermore, biofilms protect bacteria from attack by the host immune system by preventing phagocytosis by immune cells and suppressing inflammatory responses, providing a relatively safe environment for bacteria to survive. Furthermore, biofilms are closely associated with a variety of clinical infections, such as catheter-related infections and prosthetic joint infections. In these cases, the presence of biofilms makes infection treatment particularly challenging, as traditional antibiotic therapies often fail to completely eliminate bacteria within the biofilm. Therefore, the development of novel antimicrobial strategies and treatment options is of great importance.

[0004] Gram-negative bacteria are one of the most important pathogens of bacterial infection, and their drug resistance has become a major challenge in global public health. In recent years, with the widespread prevalence of multidrug-resistant strains, especially the emergence of "super-resistant" bacteria such as carbapenem-resistant Gram-negative bacteria, clinical treatment options have become extremely limited. Polymyxin B, a cyclic peptide antibiotic, has potent antibacterial activity against most Gram-negative bacteria. Although its early clinical application was limited by toxicity issues, polymyxin B and its analogs have gradually become the "last line of defense" for treating multidrug-resistant Gram-negative infections in the current increasingly severe drug resistance situation. However, with the increasing frequency of polymyxin B use in clinical practice, the emergence and spread of drug-resistant strains has become a problem that cannot be ignored. Therefore, the development of new antimicrobial strategies is of great significance to combat the increasingly complex drug-resistant bacterial infections. Summary of the Invention

[0005] To address the above technical issues, the present invention discloses the use of Ginsenoside CK in the preparation of an anti-Gram infection drug. This represents a novel pharmaceutical application for Ginsenoside CK. Experimental findings indicate that Ginsenoside CK exhibits antibacterial activity against clinical isolates of Gram-positive bacteria, including Staphylococcus aureus and Enterococcus faecalis. It inhibits the formation of Gram-positive biofilms while simultaneously killing bacteria within mature biofilms. Furthermore, when combined with the polypeptide antibiotic polymyxin B (PB), Ginsenoside CK reverses the resistance of Gram-negative bacteria, such as Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli, to polymyxin B. This provides a reference for the development of Ginsenoside CK-based antibacterial drugs and for combination therapy against Gram-negative bacteria.

[0006] To this end, the technical solution adopted in the present invention is:

[0007] Ginsenoside CK is used to prepare a drug for treating Gram-positive infections. The Ginsenoside CK has a CAS number of 39262-14-1. The Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria are at least one of Staphylococcus aureus and Enterococcus faecalis, and the Gram-negative bacteria are at least one of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. The Ginsenoside CK is used to prepare a drug for treating Gram-positive infections. The Ginsenoside CK is combined with the polypeptide antibiotic polymyxin B to prepare a drug for treating Gram-negative infections.

[0008] The structure of Ginsenoside CK is shown in formula (1),

[0009]

[0010] The Ginsenoside CK has the effect of inhibiting the growth of Gram-positive bacteria and the formation of biofilm. The Ginsenoside CK combined with the polypeptide antibiotic polymyxin B has the effect of reversing the resistance of Gram-negative bacteria to polymyxin B.

[0011] Ginsenoside CK is the primary metabolite and final absorbed form of diol saponins (such as Rb1, Rc, and Rg3) in the human intestine. It is also the primary form that exerts its pharmacological effects and exhibits good absorption. Ginsenoside C-K exhibits multiple pharmacological actions, including anti-inflammatory effects through inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines. Furthermore, ginsenoside CK exerts its anti-inflammatory effects through mechanisms such as regulation of the NF-κB signaling pathway. Furthermore, ginsenoside CK exhibits anti-cancer properties, inducing apoptosis and inhibiting cancer cell proliferation. In human liver microsomes, ginsenoside CK inhibits the activities of cytochrome P450 enzymes CYP2C9 and CYP2A6, with IC50 values ​​of 32.0±3.6μM and 63.6±4.2μM, respectively. In addition, ginsenoside CK, a rare ginsenoside, has also been studied in areas such as neuroprotection and antiplatelet aggregation. For example, by targeting PHD2, it regulates collagen hydroxylation and inhibits platelet adhesion to collagen, thereby inhibiting thrombosis. Ginsenoside CK has attracted considerable attention due to its diverse bioactivities and potential clinical applications. Although its diverse bioactivities have been explored, its potential application in combating bacterial infections has yet to be reported.

[0012] Extensive experimental studies have revealed that ginsenoside CK exhibits excellent antibacterial activity against a variety of Gram-positive bacteria, including Staphylococcus aureus and Enterococcus faecalis. The minimum inhibitory concentration (MIC) of ginsenoside CK against 65 Gram-positive bacteria ranged from 25 to 50 μM. Notably, the MIC against 24 clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) strains was ≤50 μM. Time-kill curve analysis revealed that ginsenoside CK significantly killed S. aureus at a low concentration (1× MIC). Crystal violet staining revealed that ginsenoside CK significantly inhibited biofilm formation of S. aureus and E. faecalis at subinhibitory concentrations. Laser scanning confocal microscopy revealed that ginsenoside CK could kill S. aureus embedded in mature biofilms at a concentration of 8× MIC. In addition, adding bacterial membrane phospholipid components to the culture medium can reduce the antibacterial activity of Ginsenoside CK compounds in a dose-dependent manner, indicating that Ginsenoside CK compounds exert their antibacterial effects by targeting bacterial cell membranes, causing membrane perturbations and disruption of related physiological processes.

[0013] Ginsenoside CK combined with the peptide antibiotic polymyxin B (PB) has the potential to reverse polymyxin B resistance in Gram-negative bacteria, including Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. Broth microdilution checkerboard assays demonstrated that combined use of ginsenoside CK reduced the minimum inhibitory concentration (MIC) of polymyxin B against PB-resistant Gram-negative bacteria. Growth curves demonstrated that combined use of ginsenoside CK effectively inhibited the growth of PB-resistant Gram-negative bacteria. Kill curves demonstrated that combined use of ginsenoside CK inhibited or even killed PB-resistant Gram-negative bacteria. Furthermore, scanning electron microscopy revealed that combined use of ginsenoside CK disrupted the outer membrane of PB-resistant Gram-negative bacteria, enhancing bacterial membrane damage by PB and thus exerting its antibacterial activity. These results suggest that ginsenoside CK compounds have potential application in the treatment of bacterial infections.

[0014] As a further improvement of the present invention, the concentration of Ginsenoside CK in the treatment system is not less than 1.5625 μM. Furthermore, the concentration of Ginsenoside CK in the treatment system is 1.5625-200 μM.

[0015] As a further improvement of the present invention, the drug is a pharmaceutical composition or preparation. Further, the drug is an injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

[0016] The present invention discloses the use of Ginsenoside CK in preparing a coating for inhibiting Gram-positive bacteria. The coating is used on the surface of medical devices. The CAS number of Ginsenoside CK is 39262-14-1. The Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria are at least one of Staphylococcus aureus and Enterococcus faecalis, and the Gram-negative bacteria are at least one of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. The Ginsenoside CK is used to prepare a coating for inhibiting Gram-positive bacteria. The Ginsenoside CK is combined with the polypeptide antibiotic polymyxin B to prepare a coating for inhibiting Gram-negative bacteria.

[0017] As a further improvement of the present invention, the coating contains Ginsenoside CK and polypeptide antibiotic polymyxin B.

[0018] As a further improvement of the present invention, in the coating, the concentration of the Ginsenoside CK is not less than 1.5625 μM.

[0019] The present invention discloses the use of Ginsenoside CK for preparing a Gram-inhibiting disinfectant. The CAS number of the Ginsenoside CK is 39262-14-1. The Gram-inhibiting bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria are at least one of Staphylococcus aureus and Enterococcus faecalis, and the Gram-negative bacteria are at least one of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. The Ginsenoside CK is used for preparing a Gram-positive bacteria-inhibiting disinfectant. The Ginsenoside CK is combined with the polypeptide antibiotic polymyxin B to prepare a Gram-negative bacteria-inhibiting disinfectant.

[0020] As a further improvement of the present invention, the disinfectant contains Ginsenoside CK and polypeptide antibiotic polymyxin B.

[0021] As a further improvement of the present invention, in the disinfectant, the concentration of the Ginsenoside CK is not less than 1.5625 μM.

[0022] The present invention discloses a pharmaceutical composition comprising ginsenoside CK and the polypeptide antibiotic polymyxin B. Ginsenoside CK has a CAS number of 39262-14-1. This technical solution inhibits the growth of gram-negative bacteria and can combat infections caused by both gram-positive and gram-negative bacteria. The gram-positive bacteria are at least one of Staphylococcus aureus and Enterococcus faecalis, and the gram-negative bacteria are at least one of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The technical solution of the present invention discloses a new medical use of Ginsenoside CK. Ginsenoside CK exhibits antibacterial activity against clinical isolates of Gram-positive bacteria including Staphylococcus aureus and Enterococcus faecalis, can inhibit the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis, and kill bacteria embedded in the biofilms. In addition, Ginsenoside CK combined with the polypeptide antibiotic polymyxin B exhibits antibacterial activity against polymyxin B-resistant Gram-negative bacteria such as Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. These results suggest that Ginsenoside CK has the potential to treat clinical bacterial infections. Finally, at low concentrations, Ginsenoside CK has low hemolysis of mammalian cells and is suitable for clinical use. It can be seen that Ginsenoside CK has potential application value in the clinical treatment of antibacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The figures show the growth and sterilization curves of Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis after adding Ginsenoside CK to the samples of the present invention. (a) shows the growth curve of methicillin-sensitive Staphylococcus aureus (MSSA) SA113 after adding Ginsenoside CK, (b) to (c) show the growth curves of methicillin-resistant Staphylococcus aureus (MRSA) USA300 and YUSA145 after adding Ginsenoside CK, (d) shows the growth curve of Enterococcus faecalis OG1RF after adding Ginsenoside CK; (e) shows the sterilization curve of methicillin-sensitive Staphylococcus aureus (MSSA) SA113 after adding Ginsenoside CK, and (f) shows the sterilization curve of methicillin-resistant Staphylococcus aureus (MRSA) USA300 after adding Ginsenoside CK. MIC stands for minimum inhibitory concentration, and Van stands for vancomycin.

[0026] Figure 2 This is an analysis of the inhibition of biofilm formation by Ginsenoside CK on Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis by Ginsenoside CK according to an embodiment of the present invention. (a) is a crystal violet staining quantitative analysis of the effect of Etalocib on biofilm formation by methicillin-sensitive Staphylococcus aureus (MSSA); (b) is a crystal violet staining quantitative analysis of the effect of Etalocib on biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA); and (c) is a crystal violet staining quantitative analysis of the effect of Etalocib on biofilm formation by Enterococcus faecalis. MIC stands for minimum inhibitory concentration in the figures, and Van stands for vancomycin.

[0027] Figure 3 The effects of Ginsenoside CK on the bacterial membrane of Staphylococcus aureus according to an embodiment of the present invention are shown in Figures 1 and 2. (a) shows a fluorescence intensity quantitative analysis of the effect of Ginsenoside CK on the cell membrane permeability of methicillin-resistant Grapefruit Aureus (MRSA) YUSA145; (b) shows a fluorescence intensity quantitative analysis of the effect of Ginsenoside CK on the cell membrane potential of methicillin-resistant Grapefruit Aureus (MRSA) YUSA145; and (c) shows the effect of adding different bacterial membrane phospholipid components on the antibacterial activity of Ginsenoside CK. In the figure, MIC stands for minimum inhibitory concentration, Van stands for vancomycin, CL stands for cardiolipin, PG stands for phosphatidylglycerol, and PE stands for phosphatidylethanolamine.

[0028] Figure 4This is a checkerboard broth microdilution test of Ginsenoside CK combined with polymyxin B against polymyxin B-resistant Gram-negative bacteria according to an embodiment of the present invention. (a) is polymyxin B-resistant Escherichia coli MG1655-PB; (b) is polymyxin B-resistant Klebsiella pneumoniae K2044-PB; (c) is polymyxin B-resistant Acinetobacter baumannii AB2260-PB; (d) is MCR-1-mediated polymyxin B-resistant Escherichia coli MG1655-MCR-1; (e) is MCR-1-mediated polymyxin B-resistant Klebsiella pneumoniae K2044-MCR-1; and (f) is MCR-1-mediated polymyxin B-resistant Escherichia coli ATCC25922-MCR-1. In the figure, PB represents polymyxin B, and CK represents Ginsenoside CK. The color depth represents the bacterial growth level (i.e., OD600) in that well of the checkerboard, and the lighter the color, the more inhibited the bacterial growth.

[0029] Figure 5 The figures are the growth curves and sterilization curves of Ginsenoside CK combined with polymyxin B against polymyxin B-resistant Gram-negative bacteria in an embodiment of the present invention. (a) shows the growth curve analysis of Ginsenoside CK combined with polymyxin B against MCR-1-mediated polymyxin B-resistant Escherichia coli MG1655-MCR-1, (b) shows the growth curve analysis of Ginsenoside CK combined with polymyxin B against polymyxin B-resistant Escherichia coli MG1655-PB, (c) shows the sterilization curve analysis of Ginsenoside CK combined with polymyxin B against MCR-1-mediated polymyxin B-resistant Escherichia coli MG1655-MCR-1, and (d) shows the sterilization curve analysis of Ginsenoside CK combined with polymyxin B against polymyxin B-resistant Escherichia coli MG1655-PB. In the figure, Tic represents tigecycline.

[0030] Figure 6 This is an analysis of the bacterial membranes of polymyxin B-resistant Gram-negative bacteria following the use of Ginsenoside CK in combination with polymyxin B in an example of the present invention. (a) shows the morphological changes of untreated Escherichia coli MG1655-MCR-1 observed using a laser scanning confocal microscope; (b) shows the morphological changes of polymyxin B-treated Escherichia coli MG1655-MCR-1 observed using a laser scanning confocal microscope; (c) shows the morphological changes of Ginsenoside CK in combination with polymyxin B observed using a laser scanning confocal microscope; (d) to (f) are enlarged views of the red boxes in (a) to (c), respectively; the damaged outer membrane is marked with a red arrow.

[0031] Figure 7This is an analysis of the hemolytic activity of Ginsenoside CK combined with polymyxin B on mammalian erythrocytes according to an embodiment of the present invention. (a) shows the hemolytic activity of Ginsenoside CK or polymyxin B at different concentrations on mammalian erythrocytes; (b) shows the hemolytic activity of Ginsenoside CK combined with polymyxin B on mammalian erythrocytes.

[0032] Figure 8 The results of a chessboard broth microdilution test of the Ginsenoside CK analogs combined with polymyxin B against polymyxin B-resistant Gram-negative Escherichia coli MG1655-PB in Comparative Example 2 of the present invention are shown. The color depth represents the bacterial growth level in that well on the chessboard (i.e., absorbance OD600 at a wavelength of 600 nm), with lighter colors indicating that bacterial growth is inhibited. The Ginsenoside CK analogs (a) to (o) are Ginsenoside F2, Ginsenoside Rb1, Ginsenoside Rh1, Ginsenoside F1, Notoginsenoside R1, Ginsenoside Rb3, Ginsenoside Rg2, Ginsenoside Re, Ginsenoside Rd, Ginsenoside Rh2, Ginsenoside Rc, Ginsenoside Rf, Ginsenoside Rg1, Ginsenoside F3, and Ginsenoside Rb2, respectively. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention are described in further detail below.

[0034] The 65 Gram-positive bacteria (including 20 MSSA, 24 MRSA, and 21 Enterococcus faecalis) and 6 Gram-negative bacteria (including 1 Acinetobacter baumannii, 2 Klebsiella pneumoniae, and 3 Escherichia coli) used in the following examples were clinical strains collected from different inpatients in the hospital or experimental strains stored in our laboratory. All clinical strains were identified using the Phoenix 100 automated microbial identification system and re-identified using matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI-TOF-MS) after subculture.

[0035] Example 1

[0036] In this example, the minimum inhibitory concentration (MIC) of Ginsenoside CK against 65 strains of Gram-positive bacteria (including 20 strains of MSSA, 24 strains of MRSA, and 21 strains of Enterococcus faecalis) was detected by the broth microdilution method. The specific steps are as follows:

[0037] Adjust the turbidity of the overnight culture to 0.5 McFarland (approximately 1.0-1.5 × 108 CFU / mL) using a turbidimetric cup. Dilute the culture 1:100 with CAMHB medium and add to a 96-well plate. Set up a 7-well gradient (100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM) per row. Add 200 μL of the above culture to the 8th well as a positive control, and add 200 μL of CAMHB medium to the 9th well as a negative control. Incubate at 37°C for 18 hours and observe the results. Calculate the MIC value based on the drug concentration at which no bacterial precipitation is visible to the naked eye.

[0038] In this example, the statistical results of the MIC values ​​of Ginsenoside CK against Gram-positive bacteria such as Staphylococcus aureus and Enterococcus faecalis are shown in Table 1. It can be seen that Ginsenoside CK has a relatively broad spectrum of antibacterial activity against Gram-positive bacteria, and the MIC values ​​are mainly distributed between 25 μM and 50 μM.

[0039] Table 1 Distribution of MIC values ​​of common Gram-positive bacteria to Ginsenoside CK

[0040]

[0041] Note: MIC: Minimum Inhibitory Concentration; MSSA: Methicillin-Susceptible Staphylococcus aureus; MRSA: Methicillin-Resistant Staphylococcus aureus; E. faecalis: Enterococcus faecalis; No: Number of strains tested. MIC50: Drug concentration required to inhibit 50% growth of the test bacteria; MIC90: Drug concentration required to inhibit 90% growth of the test bacteria.

[0042] Example 2

[0043] Experiment on the effect of Ginsenoside CK on the growth curve of Gram-positive bacteria.

[0044] To verify whether Ginsenoside CK can inhibit the growth of Gram-positive bacteria, in this example, three strains of Staphylococcus aureus (SA113, USA300, and YUSA145) and one strain of Enterococcus faecalis (OG1RF) were treated with Ginsenoside CK at different subinhibitory concentrations, and their absorbance at 600 nm (OD600) was measured at different time points. The specific steps were as follows:

[0045] The overnight culture was diluted 200-fold with tryptone soy broth (TSB) medium and added to a 96-well plate. After adding different concentrations (1 / 8×, 1 / 4×, 1 / 2× and 1×MIC) of Ginsenoside CK, the plate was placed in an automatic growth curve analyzer. The incubation temperature was set to 37°C and the shaking condition was 200 rpm. The OD600 absorbance value was monitored every 1 hour to detect the planktonic bacterial content in the culture supernatant and draw a growth curve.

[0046] Growth curve analysis Figure 1 (a)~ Figure 1 As shown in (d), ginsenoside CK can delay the growth of Staphylococcus aureus and Enterococcus faecalis even at a concentration of 1 / 2×MIC. At a concentration of 1×MIC, the growth of Staphylococcus aureus and Enterococcus faecalis is completely inhibited within 20 to 24 hours. These results preliminarily suggest that ginsenoside CK has potential as an antibacterial drug.

[0047] Example 3

[0048] Experiment on the effect of time and dosage of Ginsenoside CK on the bactericidal activity of Gram-positive bacteria.

[0049] In this example, the time- and dose-dependent effects of the antibacterial activity of Ginsenoside CK against Gram-positive bacteria were studied and compared with the activity of the clinical antibiotic vancomycin. The specific steps included:

[0050] Logarithmic phase (OD600 = 0.5) MSSA strain SA113 and MRSA strain USA300 were diluted 100-fold and incubated with 1×, 2×, and 4× the MIC of ginsenoside CK and 4× the MIC of vancomycin, respectively, on a shaker at 200 rpm. Samples were collected at 0, 2, 6, and 24 hours, serially diluted with sterile saline, and plated on TSB plates, incubated at 37°C, and colony counts were obtained after 24 hours. Colony counts are expressed as CFU / mL.

[0051] The sterilization curve analysis obtained is as follows Figure 1 (e) and Figure 1 As shown in (f), it can be seen that Ginsenoside CK has a significant bactericidal effect against Staphylococcus aureus at 1×MIC. Within 2 hours, the bactericidal effect of 2×MIC and 4×MIC Ginsenoside CK on MSSA strains is reduced to the detection limit; within 6 hours, the bacterial count of 4×MIC Ginsenoside C-K on MRSA strains is also reduced to the detection limit, and the bactericidal effect is stronger than that of 4×MIC vancomycin.

[0052] Example 4

[0053] This example uses the crystal violet method to study the biofilm-inhibiting activity of Ginsenoside CK against Staphylococcus aureus and Enterococcus faecalis. The specific steps are as follows:

[0054] To monitor biofilm formation, changes in biofilm volume were assessed using crystal violet staining. The procedure is briefly described as follows: overnight bacterial culture was diluted 1000-fold in TSBG medium containing 1% glucose and plated in 96-well plates. Ginsenoside CK was added at varying subinhibitory concentrations in triplicate. A blank culture without bacterial culture served as a negative control, and DMSO as a positive control. The plates were incubated in a 37°C incubator for 24 hours. The culture medium was gently aspirated, and the plates were rinsed two to three times with sterile PBS and air-dried at room temperature. The plates were fixed with methanol for 15 minutes, stained with 0.1% crystal violet for 15 minutes, and rinsed three times with sterile water until the control wells were colorless. The plates were then air-dried at room temperature. The absorbance at 570 nm (OD570) was measured using a microplate reader.

[0055] Crystal violet staining results Figure 2 As shown in the results, it can be seen that different sub-inhibitory concentrations of Ginsenoside CK have a significant inhibitory effect on the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis; when Ginsenoside CK is 1 / 4×MIC, the biofilm biomass formed by almost all Enterococcus faecalis and Staphylococcus aureus strains is significantly reduced; when the concentration of Ginsenoside CK is 1 / 2×MIC, the biofilm formation of 11 of the 12 strains tested is inhibited by more than 50%, indicating that Ginsenoside CK can inhibit the formation of biofilms of Enterococcus faecalis and Staphylococcus aureus.

[0056] Example 5

[0057] This example is a study on the cell membrane permeability of Staphylococcus aureus YUSA145 by Ginsenoside CK. The specific steps are as follows:

[0058] The logarithmic phase Gram-positive bacteria YUSA145 cells were adjusted to OD600 = 0.05 and incubated with propidium iodide (PI) fluorescent dye in the dark for 20 minutes. After the incubation, the suspended bacteria were treated with different concentrations of Ginsenoside CK (final concentrations of 1× and 2× MIC), with 0.1% DMSO as a control. After 1 hour, the propidium iodide (PI) staining fluorescence value was monitored using a microplate reader.

[0059] The fluorescence intensity monitoring results are as follows Figure 3As shown in (a), the fluorescence intensity of Staphylococcus aureus YUSA145 was significantly increased after treatment with Ginsenoside CK, and the fluorescence value also increased in a gradient, indicating that the cell membrane permeability changed, indicating that Ginsenoside CK had a damaging effect on the cell membrane of Staphylococcus aureus.

[0060] Example 6

[0061] This example is a study on the cell membrane permeability of Staphylococcus aureus YUSA145 by Ginsenoside CK. The specific steps are as follows:

[0062] The logarithmic phase Gram-positive bacteria YUSA145 cells were adjusted to OD600 = 0.05 and incubated with DiBAC4 (3) fluorescent dye in the dark for 20 minutes. After the incubation, the suspended bacteria were treated with different concentrations of Ginsenoside CK (final concentrations of 1× and 2× MIC), with 0.1% DMSO as a control. After 1 hour, the fluorescence value of DiBAC4 (3) fluorescent staining was monitored using a microplate reader.

[0063] The fluorescence intensity monitoring results are as follows Figure 3 As shown in (b), the fluorescence intensity of Staphylococcus aureus YUSA145 was significantly increased after treatment with Ginsenoside CK, indicating that the cell membrane potential changed, indicating that Ginsenoside CK had a damaging effect on the cell membrane of Staphylococcus aureus.

[0064] Example 7

[0065] In this example, the antibacterial activity of Ginsenoside CK on the phospholipid components of bacterial cell membranes was studied. The specific steps included:

[0066] In this example, to analyze whether the antibacterial activity of Ginsenoside CK is related to the cell membrane, the checkerboard broth microdilution method was used to detect the effects of different concentrations of bacterial cell membrane phospholipid components phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL) on the minimum inhibitory concentration (MIC) of Ginsenoside CK. The specific steps are as follows:

[0067] An overnight culture of Staphylococcus aureus (MRSA) clinical strain YUSA145 was adjusted to a turbidity of 0.5 McFarland (approximately 1.0-1.5 × 108 CFU / mL) using a turbidimetric cup. The culture was diluted 1:100 with CAMHB medium and plated in a 96-well plate. Each row contained 12 wells. An 8-well gradient of Etalocib was set up (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μM). 200 μL of the above culture was added to the 9th well as a positive control, and 200 μL of CAMHB medium was added to the 10th well as a negative control. A 7-well gradient of cell membrane phospholipids was set up (128, 64, 32, 16, 8, 4, and 2 μg / mL). The culture conditions and time for the determination of the MIC values ​​of each antimicrobial drug were carried out in accordance with the CLSI guidelines. The results were observed after incubation at 37°C for 18 hours, and the drug concentration well at which no bacterial precipitation could be seen with the naked eye was calculated as the MIC value.

[0068] The results were plotted with the concentration of different phospholipids as the horizontal axis and the MIC concentration fold change of Ginsenoside CK as the vertical axis. Figure 3 As shown in (c), it can be seen that the exogenous addition of bacterial cell membrane phospholipids, phosphatidylethanolamine (PE) to the culture medium has no effect on the antibacterial activity of Ginsenoside CK, while phosphatidylglycerol (PG) and cardiolipin (CL) reduce the antibacterial activity of Ginsenoside CK in a dose-dependent manner. It can be seen that Ginsenoside CK exerts its antibacterial activity by acting on the bacterial cell membrane.

[0069] Example 8

[0070] Experiment on the combined effect of Ginsenoside CK and polypeptide antibiotic polymyxin B.

[0071] This example is an experiment to analyze the combined antibacterial effect of Ginsenoside CK and the polypeptide antibiotic polymyxin B against 6 polymyxin B-resistant Gram-negative bacteria (including 1 Acinetobacter baumannii, 2 Klebsiella pneumoniae, and 3 Escherichia coli) using the checkerboard microbroth dilution method. The specific steps are as follows:

[0072] Adjust the turbidity of the overnight culture to 0.5 McFarland (approximately 1.0-1.5 × 108 CFU / mL) using a turbidimetric cup. Dilute the culture 1:200 with CAMHB medium and add 200 μL to a 96-well plate, with 8 wells per row. Add ginsenoside CK at different concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 μM) to each row. Add polymyxin B at different concentrations (32, 16, 8, 4, 2, 1, 0.5, 0 μg / mL) to each column. Incubate at 37°C for 18 hours and observe the results. The MIC value is calculated as the well at which no precipitation is visible to the naked eye.

[0073] The results were plotted with different concentrations of Ginsenoside CK as the horizontal axis and the concentration of Polymyxin B as the vertical axis, and a chessboard graph was drawn to show the effect of combined use on the growth level of drug-resistant strains (represented by OD600). Figure 4 As shown in the results, the MICs of Ginsenoside CK against the six polymyxin B-resistant Gram-negative bacteria were all greater than 100 μM, indicating that it had essentially no antibacterial activity. The MICs of polymyxin B against the six polymyxin B-resistant Gram-negative bacteria ranged from 4 to 256 μg / mL. However, the addition of 25 to 50 μM Ginsenoside CK reversed the resistance of Gram-negative bacteria to polymyxin B, with the MICs dropping to between 2 μg / mL and 4 μg / mL. This indicates that Ginsenoside CK combined with polymyxin B has the effect of reversing the resistance of Gram-negative bacteria to polymyxin B. In the future, Ginsenoside CK can be used as an antibacterial adjuvant for polymyxin B.

[0074] Example 9

[0075] Experiment on the effect of Ginsenoside CK combined with polymyxin B on the growth curve of polymyxin B-resistant Gram-negative bacteria.

[0076] To verify whether Ginsenoside CK combined with polymyxin B can inhibit the growth of polymyxin B-resistant Gram-negative bacteria, we used polymyxin B combined with different concentrations of Ginsenoside CK to treat two polymyxin B-resistant Escherichia coli strains and measured their absorbance at 600 nm (OD600) at different time points. The specific steps were as follows:

[0077] The overnight culture was diluted 200-fold with Luria-Bertani (LB) medium and added to a 96-well plate. The plates were then treated with different concentrations of polymyxin B alone, different concentrations of ginsenoside CK alone, and different concentrations of polymyxin B combined with ginsenoside CK. The plates were then placed in an automatic growth curve analyzer with the incubation temperature set at 37°C and the shaking condition set at 200 rpm. The OD600 absorbance was monitored every 1 hour to detect the planktonic bacterial content in the culture supernatant and to draw a growth curve.

[0078] Growth curve analysis Figure 5 As shown in Figures 5(a) and 5(b), it can be seen that polymyxin B and Ginsenoside CK alone had no inhibitory activity on the growth of polymyxin B-resistant Escherichia coli MG1655-PB and MCR-1-mediated polymyxin B-resistant Escherichia coli MG1655-MCR-1. After combining with polymyxin B (1μg / mL and 2μg / mL), Ginsenoside CK at 12.5μM could delay the growth of Escherichia coli MG1655-PB and MG1655-MCR-1, and at 50μM, Ginsenoside CK could significantly inhibit the growth of Escherichia coli MG1655-PB and MG1655-MCR-1. These results preliminarily indicate that Ginsenoside C-K combined with polymyxin B has the potential to be used as an antibacterial infection drug.

[0079] Example 10

[0080] Experiment on the effects of time and dosage of Ginsenoside CK combined with polymyxin B on the bactericidal activity of polymyxin B-resistant Gram-negative bacteria.

[0081] In this example, the time- and dose-dependent effects of the antibacterial activity of Ginsenoside CK combined with polymyxin B against polymyxin B-resistant Gram-negative bacteria were studied, and the activity was compared with that of the clinical antibiotic tigecycline. The specific steps included:

[0082] Polymyxin B-resistant E. coli MG1655-PB and MCR-1-mediated polymyxin B-resistant E. coli MG1655-MCR-1 in the logarithmic phase (OD600 = 0.5) were diluted 100-fold and treated with polymyxin B, tigecycline, ginsenoside CK, or ginsenoside CK combined with polymyxin B, respectively. The cells were then incubated on a shaker at 200 rpm. Subsequently, samples were collected at 0, 2, 4, 6, 8, 10, and 12 hours, serially diluted with sterile saline, and plated on LB plates. The plates were incubated at 37°C and colonies were counted after 24 hours. Colony counts are expressed as CFU / mL.

[0083] The sterilization curve analysis obtained is as follows Figure 5 As shown in Figures 5(c) and 5(d), it can be seen that ginsenoside CK and polymyxin B alone cannot kill polymyxin B-resistant E. coli. Ginsenoside CK combined with polymyxin B can significantly kill polymyxin B-resistant E. coli. The bactericidal effect of ginsenoside CK combined with polymyxin B against polymyxin B-resistant E. coli MG1655-PB is the same as that of tigecycline. At 4 hours, ginsenoside CK combined with polymyxin B has a better bactericidal effect than tigecycline against MCR-1-mediated polymyxin B-resistant E. coli MG1655-MCR-1.

[0084] Example 11

[0085] Experiment on the effects of time and dosage of Ginsenoside CK combined with polymyxin B on the bactericidal activity of polymyxin B-resistant Gram-negative bacteria.

[0086] In this example, scanning electron microscopy (SEM) was used to observe the morphological changes of Ginsenoside CK combined with polymyxin B on polymyxin B-resistant Gram-negative bacteria. The specific steps were as follows:

[0087] Escherichia coli MG1655-MCR-1, which is MCR-1-mediated and polymyxin B-resistant in the logarithmic phase (OD600 = 0.5), was inoculated into LB medium and treated with either polymyxin B or ginsenoside CK combined with polymyxin B. The cells were then incubated at 200 rpm on a shaker for 2 hours. The cells were then collected, the pellet washed twice with pre-chilled PBS, and fixed overnight at room temperature with 4% glutaraldehyde. Finally, SEM samples were prepared and the morphological changes of the cells were observed under SEM.

[0088] Scanning electron microscopy analysis Figure 6 As shown in the results, it can be seen that Ginsenoside CK and polymyxin B alone cannot cause changes in the cell morphology of polymyxin B-resistant Escherichia coli, while Ginsenoside CK combined with polymyxin B can be observed to have destroyed the outer membrane of polymyxin B-resistant Escherichia coli, thereby causing damage to the bacterial cell membrane.

[0089] Example 12

[0090] This example is an experiment on the effects of Ginsenoside CK and Ginsenoside CK combined with polymyxin B on mammalian cells. The specific steps are as follows:

[0091] To investigate the potential cytotoxicity of ginsenoside CK and ginsenoside CK combined with polymyxin B against mammalian cells, hemolytic activity was measured to evaluate their effects on mammalian cells. Briefly, fresh sheep erythrocytes were washed three times with sterile PBS and suspended at a 4% concentration. Then, 200 μL of erythrocytes were added to a 96-well plate and treated with a series of different concentrations of polymyxin B, ginsenoside CK, and ginsenoside CK combined with polymyxin B. The plates were then incubated at 37°C for 30 minutes. The plates were then centrifuged at 1000 × g for 5 minutes, and 100 μL of the supernatant was transferred to a new 96-well microplate. Finally, the absorbance at 540 nm (OD540) was measured using a microplate reader.

[0092] Hemolytic activity assay Figure 7 As shown, it can be seen that hemolysis does not occur when polymyxin B is used alone, and Ginsenoside CK does not cause hemolytic activity of mammalian erythrocytes at antibacterial concentrations (<50 μM), which indicates that Ginsenoside CK combined with polymyxin B has good biosafety.

[0093] Comparative Example 1

[0094] On the basis of the above examples, this comparative example selected 15 Ginsenoside CK structural analogs as research objects, Ginsenoside CK structural analogs including Ginsenoside F2 (CAS No.: 62025-49-4), Ginsenoside Rb1 (CAS No.: 41753-43-9), Ginsenoside Rh1 (CAS No.: 63223-86-9), Ginsenoside F1 (CAS No.: 53963-43-2), Notoginsenoside R1 (CAS No.: 80418-24-2), Ginsenoside Rb3 (CAS No.: 68406-26-8), Ginsenoside Rg2 (CAS No.: 52286-74-5), Ginsenoside Re (CAS No.: 52286-59-6), Ginsenoside Rd (CAS No.: 53963-43-2), Notoginsenoside R1 (CAS No.: 80418-24-2), Ginsenoside Rb3 (CAS No.: 68406-26-8), Ginsenoside Rg2 (CAS No.: 52286-74-5), Ginsenoside Re (CAS No.: 52286-59-6), Ginsenoside Rd (CAS No.: 53963-43-2), Notoginsenoside R No.:52705-93-8), Ginsenoside Rh2 (CAS No.:78214-33-2), Ginsenoside Rc (CAS No.:11021-14-0), Ginsenoside Rf (CAS No.:52286-58-5), Ginsenoside Rg1 (CAS No.:22427-39-0), Ginsenoside F3 (CAS No.:62025-50-7), Ginsenoside Rb2 (CAS No.:11021-13-9). The structural formulas of the 15 Ginsenoside CK analogs are as follows:

[0095]

[0096] The experiment was carried out according to the steps of Example 1, and the results were as follows:

[0097] In this comparative example, the MICs of 15 ginsenoside CK structural analogs against various Gram-positive bacteria, including the MSSA standard strain ATCC29213 of Staphylococcus aureus, the MRSA standard strain USA300, and the Enterococcus faecalis standard strain ATCC29212, were determined using the broth microdilution method. The statistical results of the MIC values ​​of the 15 ginsenoside CK structural analogs against these strains are shown in Table 2. It can be seen that, with the exception of ginsenoside CK and ginsenoside F2, the ginsenoside CK structural analogs had no antibacterial activity against S. aureus and E. faecalis, with MIC values ​​greater than 200 μM. The MIC of ginsenoside F2 was higher than that of ginsenoside CK, indicating that its antibacterial effect was significantly inferior to that of ginsenoside CK.

[0098] Table 2 MIC values ​​of Ginsenoside CK and its structural analogs against standard strains of Staphylococcus aureus and Enterococcus faecalis

[0099]

[0100] Comparative Example 2

[0101] The method of Example 8 was used to analyze the synergistic antibacterial effect of the 15 Ginsenoside CK structural analogs in Comparative Example 1 combined with the polypeptide antibiotic polymyxin B against the polymyxin B-resistant strain Escherichia coli MG1655-PB. The results were plotted with the concentration of the different Ginsenoside CK structural analogs as the abscissa and the concentration of polymyxin B as the ordinate. A chessboard diagram was drawn based on the bacterial growth level (expressed as OD600) in each well after the combination treatment. The results are shown in Figure 1. Figure 8 As shown in the figure, with the exception of Ginsenoside F2, the other Ginsenoside CK structural analogs failed to enhance the antibacterial activity of polymyxin B against drug-resistant strains. Furthermore, Ginsenoside F2's enhancement of polymyxin B activity was weaker than that of Ginsenoside CK. This suggests that Ginsenoside CK, when combined with polymyxin B, has the best effect in reversing polymyxin B resistance in Gram-negative bacteria and has potential application as an antibacterial adjuvant for polymyxin B.

[0102] All experiments were performed using GraphPad Prism 8.0 software for data processing and plotting. P < 0.05 was considered statistically significant.

[0103] The above experimental results show that ginsenoside CK exhibits excellent antibacterial activity against Gram-positive bacteria, with significant bactericidal activity against Staphylococcus aureus, surpassing vancomycin in cell counts after 2 hours. Ginsenoside CK also exhibits strong antibiofilm activity, significantly inhibiting biofilm formation of clinical strains of Staphylococcus aureus and Enterococcus faecalis. Furthermore, ginsenoside CK combined with polymyxin B reverses polymyxin resistance in Gram-negative bacteria. This combination effectively inhibits the growth of polymyxin-resistant Gram-negative bacteria and induces bacterial membrane damage in these bacteria. Finally, ginsenoside CK exhibits low hemolytic activity against mammalian erythrocytes at the MIC dose. These results suggest that ginsenoside CK has the potential to treat Gram-bacterial infections.

[0104] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. Use of Ginsenoside CK in the preparation of an anti-Gram infection drug, characterized in that: The Ginsenoside CK has a CAS number of 39262-14-1; the Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria, the Gram-positive bacteria are at least one of Staphylococcus aureus and Enterococcus faecalis, and the Gram-negative bacteria are at least one of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii; the Ginsenoside CK is used to prepare a drug for treating Gram-positive bacterial infections, and the Ginsenoside CK is combined with the polypeptide antibiotic polymyxin B to prepare a drug for treating Gram-negative bacterial infections.

2. The use of Ginsenoside CK according to claim 1 for preparing an anti-Gram infection drug, characterized in that: The concentration of Ginsenoside CK in the treatment system is not less than 1.5625 μM.

3. The use of Ginsenoside CK according to claim 1 for preparing an anti-Gram infection drug, characterized in that: The medicine is in the form of an injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

4. Use of Ginsenoside CK in the preparation of a coating for inhibiting Gram-bacteria, characterized in that: The coating is used for the surface of medical devices. The CAS number of the Ginsenoside CK is 39262-14-1. The Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria are at least one of Staphylococcus aureus and Enterococcus faecalis, and the Gram-negative bacteria are at least one of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. The Ginsenoside CK is used to prepare a coating that inhibits Gram-positive bacteria. The Ginsenoside CK is combined with the polypeptide antibiotic polymyxin B to prepare a coating that inhibits Gram-negative bacteria.

5. Use of Ginsenoside CK according to claim 4 for preparing a coating for inhibiting Gram bacteria, characterized in that: In the coating, the concentration of the Ginsenoside CK is not less than 1.5625 μM.

6. Use of Ginsenoside CK for the preparation of a Gram-inhibiting disinfectant, characterized in that: The CAS number of the Ginsenoside CK is 39262-14-1, and the Gram-positive bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria are at least one of Staphylococcus aureus and Enterococcus faecalis, and the Gram-negative bacteria are at least one of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. The Ginsenoside CK is used to prepare a disinfectant that inhibits Gram-positive bacteria, and the Ginsenoside CK is combined with the polypeptide antibiotic polymyxin B to prepare a disinfectant that inhibits Gram-negative bacteria.

7. Use of Ginsenoside CK according to claim 6 for preparing a Gram-inhibiting disinfectant, characterized in that: In the disinfectant, the concentration of Ginsenoside CK is not less than 1.5625 μM.

8. A pharmaceutical composition, characterized in that: The invention comprises Ginsenoside CK and polypeptide antibiotic polymyxin B, wherein the CAS number of the Ginsenoside CK is 39262-14-1.

Images