Application of alpha-hederin in preparation of medicine for preventing or treating staphylococcus aureus infectious diseases

Abstract

The invention discloses application of alpha-hederin in preparation of a medicine for preventing or treating staphylococcus aureus infectious diseases, and belongs to the technical field of medicines. The staphylococcus aureus infectious disease is one of skin or soft tissue infection, pneumonia, osteoarthritis, mastitis, enteritis, endocarditis, urinary tract infection and enteritis. The medicine does not have an in-vitro direct sterilization function on sensitive and methicillin-resistant staphylococcus aureus, but can remarkably promote infected cells to remove thalli under a cell infection condition, so that the medicine plays a role in resisting sensitive and methicillin-resistant staphylococcus aureus infection by improving the anti-infection immune level of the cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of an α-hederone in the preparation of a medicament for the prevention or treatment of Staphylococcus aureus infections. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Antimicrobial cysts (SA) are a leading cause of hospital-acquired and community-acquired infections globally, and have become one of the most dangerous and deadly pathogens worldwide. SA has long been considered a typical extracellular pathogen, causing acute infections such as skin abscesses and sepsis by producing various toxins and enzymes. However, recent studies have shown that this pathogen can invade and survive in various host cells, including epithelial cells, endothelial cells, and macrophages, thus forming an intracellular infection phase and ultimately leading to chronic infectious diseases such as pneumonia. This dual extracellular and intracellular approach explains why some infections are persistent, increasing the complexity of clinical treatment.

[0003] Currently, clinical treatment of SA infection mainly relies on antibiotics, but these easily induce the development of drug-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus MRSA (metastatic antibiotic resistance) is a typical example of antibiotic-resistant SA, causing bacteremia with a mortality rate twice that of susceptible strains, posing a significant threat. Furthermore, antibiotics have limited effectiveness against intracellular bacteria, and long-term use can disrupt the host's normal flora, increasing the risk of reinfection. With in-depth research into pathogen-host interactions, it has become increasingly clear that antibiotic-only treatment models are insufficient to address complex infection scenarios and the continuous emergence of drug-resistant bacteria. Targeting host immune function and enhancing the host's anti-infective immune response are becoming important strategies for anti-infective therapy. This approach holds promise for activating the host immune response, thereby achieving broad-spectrum defense against extracellular, intracellular, and drug-resistant pathogens, while minimizing the likelihood of inducing resistance and maintaining host microecological balance. It provides a novel and sustainable approach to addressing the increasingly severe problem of drug-resistant bacterial infections. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides the application of α-hederin in the preparation of drugs for the prevention or treatment of Staphylococcus aureus infections. α-Hederin can effectively enhance the host's immune response against Staphylococcus aureus, thereby effectively resisting infections caused by susceptible and drug-resistant Staphylococcus aureus.

[0005] The technical solution of the present invention is as follows: The first objective of this invention is to provide the use of α-hederone in the preparation of medicaments for the prevention or treatment of Staphylococcus aureus infections.

[0006] In one embodiment of the present invention, Staphylococcus aureus infectious diseases include skin or soft tissue infections (such as folliculitis, abscess, wound infection, cellulitis), pneumonia, osteoarthritis, mastitis, enteritis, endocarditis, urinary tract infection, and endophthalmitis.

[0007] The second objective of this invention is to provide a Staphylococcus aureus inhibitor with α-hederone as the active ingredient.

[0008] In one embodiment of the present invention, the Staphylococcus aureus inhibitor further includes a pharmaceutically acceptable carrier or excipient.

[0009] In one embodiment of the present invention, the carrier includes, but is not limited to, starch and cellulose.

[0010] In one embodiment of the present invention, the excipients include, but are not limited to, magnesium stearate, talc, sodium carboxymethyl starch, and crospovidone.

[0011] In one embodiment of the present invention, the dosage form is an oral preparation, a topical preparation, or an injectable preparation.

[0012] In one embodiment of the present invention, the oral preparation is a tablet, granule, capsule, powder, or oral liquid.

[0013] In one embodiment of the present invention, the topical skin preparation is a lotion, ointment, or gel.

[0014] Beneficial effects: The traditional Chinese medicine compound α-Hederin provided by this invention can effectively enhance the host's immune response against Staphylococcus aureus, thereby effectively resisting infections caused by susceptible and drug-resistant Staphylococcus aureus. Attached Figure Description

[0015] Figure 1 To evaluate the anti-SA infection efficacy of traditional Chinese medicine monomer compounds through cellular-level CFU experiments.

[0016] Figure 2 To compare the anti-SA infection effects of the traditional Chinese medicine monomer compound α-Hederin and the antibiotic methicillin using a cellular-level CFU assay; ns: no significant difference.

[0017] Figure 3 To detect the anti-MRSA infection effect of the traditional Chinese medicine monomer compound α-Hederin using a cellular-level CFU assay; MRSA: methicillin-resistant Staphylococcus aureus.

[0018] Figure 4To determine the minimum inhibitory concentration (MIC) of the traditional Chinese medicine monomer compound α-Hederin against SA and MRSA in vitro. Detailed Implementation

[0019] Example 1 Bacterial and cell resuscitation, culture, cryopreservation and counting 1.1 Resuscitation, culture, cryopreservation, and counting of SA and MRSA (1) Preparation of LB liquid and solid culture media: LB liquid medium: Weigh 10 g Tryptone, 5 g Yeast extract powder, and 10 g NaCl into 1 L of single-distilled water, autoclave at 121℃ for 30 min, and store at 4℃.

[0020] LB solid medium: Weigh 10 g Tryptone, 5 g Yeast extract powder, 10 g NaCl and 15 g Agar into 1 L of single-distilled water, autoclave at 121℃ for 30 min, cool and pour into bacterial culture dishes, and then store at 4℃.

[0021] (2) Resuscitation and culture of SA and MRSA: Remove the cryovials from the -80°C freezer, disinfect them with alcohol, open them in a biosafety cabinet, pick up glycerol bacteria with a sterile inoculation loop, streak them in four zones on LB solid medium, and incubate overnight at 37°C. Subsequently, pick up single colonies of bacteria with a sterile inoculation loop and culture them in LB liquid medium for scale-up in a 37°C incubator.

[0022] (3) Cryopreservation and counting of SA and MRSA: After the bacteria reached the logarithmic growth phase, glycerol was added to the bacterial culture to a final concentration of 15%, and the glycerol-containing bacteria were transferred to cryovials and stored at -80°C. In addition to bacterial cryopreservation, the bacterial culture that had reached the logarithmic growth phase was aliquoted into 1.5 mL centrifuge tubes, flash-frozen in liquid nitrogen, and then stored at -80°C. After overnight storage, the frozen bacteria were thawed rapidly in a 37°C water bath, centrifuged, and the supernatant was discarded. The culture was then resuspended in LB liquid medium and serially diluted 10-fold. The diluted bacterial culture was then spread onto LB solid medium, incubated overnight at 37°C, and colony counts were performed to calculate the concentration of the frozen bacteria for subsequent cell infection and in vitro bacterial culture experiments.

[0023] 1.2 Resuscitation, culture, and cryopreservation of immortalized bone marrow-derived macrophage cell line (iBMDM) (1) Resuscitation and culture of iBMDM cell line: Remove the cryopreserved cells from liquid nitrogen, thaw them rapidly in a 37°C water bath, and then add them to DMEM cell culture medium containing 10% fetal bovine serum (FBS) in a biosafety cabinet. Culture overnight in a 37°C, 5% CO2 cell incubator, then replace the medium with fresh medium. When the cells reach approximately 80% confluence, passage them into new cell culture dishes.

[0024] (2) Cryopreservation of iBMDM cell line: When iBMDM cells reach a concentration of 90%-95%, discard the old culture medium, wash with 1×PBS, and resuspend the cells in cryopreservation solution. Then, aliquot the cell suspension into labeled cell cryovials, place the cryovials in a cryopreservation box containing isopropanol, incubate overnight at -80°C, and then transfer to liquid nitrogen for long-term storage.

[0025] Example 2. Evaluation of the anti-SA infection efficacy of traditional Chinese medicine monomer compounds at the cellular level 2.1 Resuscitation and Cultivation of SA and iBMDM The method is the same as in Example 1.

[0026] 2.2 Establishment of a cell infection model and the anti-SA infection effect of traditional Chinese medicine monomer compounds iBMDM cells were divided into 5×10 5 The cells were seeded into 12-well plates. After overnight culture, counted SA were used to infect iBMDM cells with an MOI of 10, and 10 μM α-Hederin, Triptolide, and Quercetin were added for treatment (DMSO treatment group served as negative control). Two hours after infection, the old culture medium was discarded, and the cells were washed three times with 1×PBS. Fresh culture medium containing 10% FBS, 10 μg / mL gentamicin, and 10 μM of different Chinese herbal monomers was added, and the cells were cultured for another 10 hours. Subsequently, the supernatant was discarded, and the cells were washed three times with 1×PBS. 0.05% sodium dodecyl sulfate (SDS) was added to lyse the cells and release the bacteria. 100 μL of cell lysate was serially diluted 10-fold in 900 μL LB liquid medium, and the diluted solution was spread on LB solid medium. After overnight incubation at 37 °C, colony counts were performed, and the number of intracellular viable cells (CFU) was calculated.

[0027] 2.3 Experimental Results Experimental results showed that quercetin, triptolide, and α-hederin promoted intracellular clearance of SA, with α-hederin showing the most significant effect in clearing intracellular SA. Figure 1 ).

[0028] Example 3. Comparison of the anti-SA infection effects of the traditional Chinese medicine monomer compound α-Hederin and the antibiotic Methicillin at the cellular level. 3.1 Resuscitation and Cultivation of SA and iBMDM The method is the same as in Example 1.

[0029] 3.2 Establishment of cell infection model and detection of bacterial intracellular survival iBMDM cells were divided into 5×10 5 Seeded into 12-well plates. After overnight culture, counted SA were used to infect iBMDM cells with an MOI of 10, along with treatment with 2.5 μM or 5 μM α-Hederin. Additionally, treatment with 2.5 μM or 5 μM Methicillin served as a positive control (Methicillin is a widely used clinical antibiotic against penicillinase-producing Staphylococcus infections), and DMSO treatment served as a negative control. Two hours after infection, the old culture medium was discarded, and the cells were washed three times with 1×PBS. Fresh culture medium containing 10% FBS, 10 μg / mL gentamicin, and 2.5 μM or 5 μM α-Hederin or Methicillin was added, and the cells were cultured for another 10 hours. Subsequently, the supernatant was discarded, and the cells were washed three times with 1×PBS. Cell lysis was performed with 0.05% SDS to release intracellular bacteria. Take 100 μL of cell lysate and serially dilute it 10-fold in 900 μL of LB liquid medium. Spread the diluted solution onto LB solid medium. After incubation overnight at 37 °C, count the colonies and calculate the number of intracellular viable cells (CFU).

[0030] 3.3 Experimental Results The experimental results showed that compared with the control group treated with DMSO, the bacterial load (CFU) in cells treated with 5 μM α-Hederin decreased by approximately 50%, while the CFU in cells treated with 2.5 μM α-Hederin decreased by approximately 70%. In the positive control group, both 2.5 μM and 5 μM Methicillin showed significant anti-SA infection effects (CFU in cells decreased by approximately 90%), suggesting that α-Hederin and the clinically used antibiotic Methicillin both have significant anti-Staphylococcus aureus infection effects. Figure 2 ).

[0031] Example 4. Detection of the anti-infective effect of the traditional Chinese medicine monomer compound α-Hederin on MRSA at the cellular level. 4.1 Resuscitation and Cultivation of MRSA and iBMDM The method for reviving and culturing MRSA is the same as that for reviving and culturing SA in Example 1.

[0032] The resuscitation and culture methods for iBMDM are the same as in Example 1.

[0033] 4.2 Establishment of cell infection model and detection of bacterial intracellular survival iBMDM cells were divided into 5×10 5 The cells were seeded into 12-well plates. After overnight culture, counted MRSA were used to infect iBMDM cells with an MOI of 10, and 2.5 μM, 5 μM, or 10 μM α-Hederin or Methicillin were added for treatment (DMSO treatment group served as negative control). Two hours after infection, the old culture medium was discarded, and the cells were washed three times with 1×PBS. Fresh culture medium containing 10% FBS, 10 μg / mL gentamicin, and 2.5 μM, 5 μM, or 10 μM α-Hederin or Methicillin was added, and the cells were cultured for another 10 hours. Subsequently, the supernatant was discarded, and the cells were washed three times with 1×PBS. Cells were lysed with 0.05% SDS to release intracellular bacteria. 100 μL of cell lysate was serially diluted 10-fold in 900 μL LB liquid medium, and the diluted solution was spread onto LB solid medium. After overnight incubation at 37 °C, colony counts were performed, and the number of intracellular viable cells (CFU) was calculated.

[0034] 4.3 Experimental Results The experimental results showed that, compared with the DMSO control group, 5 μM or 10 μM α-Hederin and Methicillin were significantly effective in clearing MRSA from cells. Furthermore, there was no significant difference in bacterial load (CFU) between cells treated with 10 μM α-Hederin and 10 μM Methicillin, suggesting that both treatments had comparable efficacy against MRSA infection. Figure 3 ).

[0035] Example 5. Determination of the minimum inhibitory concentration (MIC) of the herbal monomer compound α-Hederin against SA and MRSA in vitro. Add 200 µL of LB liquid medium to the wells around the perimeter of a 96-well plate to prevent evaporation of the liquid from the central experimental group. Then, add 100 µL of LB liquid medium to each well in the remaining columns 1-9. Add 100 µL of the prepared drug working solution (initial concentration 60 μg / mL) to column 1, mix well, and then aspirate 100 µL from the previous well to the next well, serially diluting until column 9. Discard any excess 100 µL. Column 10 is the drug-free group. Afterward, add 100 μL of bacterial culture (5 × 10⁻⁶) to each well in columns 1-10. 4 Mix CFU thoroughly. After incubating in a bacterial incubator at 37°C for 24 hours, observe the turbidity of the bacterial solution. The lowest drug concentration that inhibits bacterial growth is its MIC value.

[0036] Experimental results showed that the minimum inhibitory concentration (MIC) of α-Hederin against both SA and MRSA was greater than 15 μg / mL (equivalent to a molar concentration of 25.86 μM). Meanwhile, the antibiotic Methicillin, used as a positive control, exhibited significant in vitro bactericidal activity: a MIC of 1 μM against SA and 10 μM against MRSA. Combined with the previous CFU experiments, the conclusion is that since 10 μM α-Hederin had no direct in vitro bactericidal effect against SA and MRSA, this indicates that in the cellular-level CFU experiments, the herbal monomer compound α-Hederin promoted the clearance of SA and MRSA through the regulation of host immunity, rather than exerting a direct bactericidal function. Figure 4 ).

[0037] Therefore, this invention suggests that in the future, it is possible to consider combining α-Hederin, which is based on host immune resistance to infection, and Methicillin, an antibiotic that exerts a direct bactericidal function, to achieve antibacterial effects from the two perspectives of enhancing host anti-infection immunity and direct bactericidal action, respectively.

[0038] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Application of α-hederone in the preparation of drugs for the prevention or treatment of Staphylococcus aureus infections.

2. The application according to claim 1, characterized in that, Staphylococcal infections can be one of the following: skin or soft tissue infections, pneumonia, osteoarthritis, mastitis, enteritis, endocarditis, urinary tract infections, or endophthalmitis.

3. The application according to claim 2, characterized in that, Skin or soft tissue infections include folliculitis, abscesses, wound infections, or cellulitis.

4. A Staphylococcus aureus inhibitor, characterized in that, The active ingredient is α-hederone.

5. The Staphylococcus aureus inhibitor according to claim 4, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

6. The Staphylococcus aureus inhibitor according to claim 5, characterized in that, Carriers include, but are not limited to, starch and cellulose.

7. The Staphylococcus aureus inhibitor according to claim 5, characterized in that, Excipients include, but are not limited to, magnesium stearate, talc, sodium carboxymethyl starch, and crospovidone.

8. The Staphylococcus aureus inhibitor according to claim 4, characterized in that, Dosage forms include oral preparations, topical preparations, or injectable preparations.

9. The Staphylococcus aureus inhibitor according to claim 4, characterized in that, Oral preparations include tablets, granules, capsules, powders, or oral liquids.

10. The Staphylococcus aureus inhibitor according to claim 4, characterized in that, Topical skin preparations include lotions, ointments, or gels.

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