Application of piceatannol in the preparation of Staphylococcus aureus infection inhibitors

Abstract

The present invention discloses the application of piceatannol in the preparation of a Staphylococcus aureus infection inhibitor, specifically including: (1) piceatannol inhibits β-lactamase activity, restores the sensitivity of bacteria to β-lactam antibiotics, and enhances the bactericidal ability of ampicillin; (2) piceatannol inhibits Staphylococcus aureus biofilm, controls the formation of persisters, and reduces the persistence of infection; (3) piceatannol combined with ampicillin shows a systemic protective effect on bacterial infection of greater wax moth. The present invention expands the application scope of piceatannol, proposes a new idea for the inhibition of Staphylococcus aureus infection, provides a lead compound with good application prospects, helps to alleviate or solve the infection problem of Staphylococcus aureus, and has important practical significance.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of piceatannol in preparing a Staphylococcus aureus infection inhibitor. Background Art

[0002] Staphylococcus aureus is an important foodborne zoonotic pathogen that can cause pneumonia, meningitis, bacteremia and other infectious diseases in humans and livestock and poultry, seriously affecting the healthy development of livestock and poultry farming and public health. Antibiotics play an important role in ensuring the healthy development of livestock and poultry farming and public health. However, antibiotics promote the generation and development of bacterial resistance, especially the emergence of super-resistant Staphylococcus aureus (MRSA), which poses a huge challenge to its infection suppression. After my country implemented the policy of "limiting, reducing and banning antibiotics" on the feed side, livestock and poultry lost the "protection" of antibiotics, bacterial infectious diseases increased, the economic benefits of the breeding industry declined, and the demand for anti-infective drugs on the treatment side was huge. However, the development of new drugs was slow, and the development of new anti-Staphylococcus aureus infection inhibitors became a "bottleneck" problem in the prevention and control of Staphylococcus aureus infection. Therefore, exploring new anti-infection alternative strategies and developing new auxiliary drugs to inhibit Staphylococcus aureus infection are of great significance to the healthy development of livestock and poultry farming and public health.

[0003] The emergence of β-lactamase has made β-lactam antibiotics challenging in the treatment of bacterial infections. In addition, the biofilm formed by Staphylococcus aureus significantly increases antibiotic resistance and the severity of infection. The formation of biofilms can cause bacteria to form persisters, leading to persistent infections, causing the disease to evolve into a chronic disease, and causing serious complications, increasing the cycle and difficulty of treatment. The ability to form biofilms makes Staphylococcus aureus the most common pathogen in health care and community settings. Studies have shown that more than 80% of bacterial infections are accompanied by the formation of biofilms, and there are approximately 17 million new biofilm-related infections in the United States each year. Therefore, it is of great significance to explore and develop auxiliary preparations that can restore the sensitivity of bacteria to antibiotics, especially β-lactam antibiotics, and inhibit the formation of bacterial biofilms for the inhibition of Staphylococcus aureus infections.

[0004] Piceatannol (Pit) is a natural compound widely found in fruits and vegetables such as grapes, blueberries, and passion fruit. It has multiple biological activities, including anti-inflammatory, antioxidant, anti-aging, anti-allergic, anti-diabetic, and neuroprotective. However, there are currently no reports on piceatannol targeting β-lactamase and inhibiting Staphylococcus aureus biofilm. Summary of the invention

[0005] Technical problems to be solved: In view of the huge challenges currently faced by the inhibition of Staphylococcus aureus infection, the present invention aims to provide a lead compound with good application prospects for the development of Staphylococcus aureus infection inhibitors. The present invention describes the application of piceatannol in the development of Staphylococcus aureus infection inhibitors, specifically including the following aspects: (1) piceatannol inhibits β-lactamase activity, restores the sensitivity of bacteria to β-lactam antibiotics, and enhances the bactericidal ability of ampicillin; (2) piceatannol inhibits Staphylococcus aureus biofilm, controls the formation of persisters, and reduces the persistence of infection; (3) piceatannol combined with ampicillin shows a systemic protective effect against bacterial infection of greater wax moth. The present invention expands the scope of application of piceatannol, provides a new idea for the development of Staphylococcus aureus infection inhibitors, and helps to alleviate or solve the infection problem of Staphylococcus aureus.

[0006] Technical solution: Application of piceatannol in the preparation of Staphylococcus aureus infection inhibitors.

[0007] Furthermore, the structural formula of the piceatannol is:

[0008]

[0009] Furthermore, the application includes inhibiting the biological activity of β-lactamase, synergistic effect with β-lactam antibiotics and inhibiting biofilm formation of Staphylococcus aureus.

[0010] Furthermore, when the piceatannol is used to inhibit the biological activity of β-lactamase, the concentration of the piceatannol is ≥32 μg / mL.

[0011] Furthermore, the β-lactam antibiotics in the synergistic effect with β-lactam antibiotics include ampicillin, penicillin, penicillin G sodium, nitrocefin, and ceftriaxone sodium.

[0012] Furthermore, the synergistic effect condition with ampicillin is: the minimum inhibitory concentration of piceatannol is 64 μg / mL, and the minimum inhibitory concentration of ampicillin is 128 μg / mL.

[0013] Furthermore, when the piceatannol is used to inhibit biofilm formation of Staphylococcus aureus, the concentration of the piceatannol is ≥4 μg / mL.

[0014] Beneficial effects:

[0015] 1. The present invention found that when the concentration of piceatannol is ≥32 μg / mL, it can significantly inhibit β-lactamase activity, restore the sensitivity of bacteria to β-lactam antibiotics (reducing the minimum inhibitory concentration of bacteria to ampicillin from 128 μg / mL to 8 μg / mL), and enhance the bactericidal ability of ampicillin; when the concentration of piceatannol is ≥4 μg / mL, it can inhibit Staphylococcus aureus biofilm (when the concentration of piceatannol is 32 μg / mL, the formation of Staphylococcus aureus biofilm is reduced by more than 90%), control the formation of persisters, and reduce the persistence of infection; in addition, piceatannol combined with ampicillin shows a systemic protective effect on bacterial infection of greater wax moth (the survival rate is significantly improved);

[0016] 2. The present invention expands the application scope of piceatannol, proposes a new idea for inhibiting Staphylococcus aureus infection, and provides a compound with good application prospects, which helps to alleviate or solve the infection problem of Staphylococcus aureus and has important practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The biological activity of piceatannol in inhibiting β-lactamase of Staphylococcus aureus;

[0018] Figure 2 is the binding site of piceatannol and β-lactamase of Staphylococcus aureus, wherein a is the binding site of piceatannol and β-lactamase; b is the binding site of piceatannol and β-lactamase;

[0019] Figure 3 Piceatannol enhances the bactericidal ability of ampicillin;

[0020] Figure 4 Restoration of bacterial sensitivity to ampicillin for piceatannol;

[0021] Figure 5 Piceatannol inhibits the formation of Staphylococcus aureus persisters;

[0022] Figure 6 Piceatannol inhibits biofilm formation of Staphylococcus aureus;

[0023] Figure 7 Piceatannol synergizes with ampicillin to protect Staphylococcus aureus from infecting G. mellonella;

[0024] Figure 8 This is the minimum inhibitory concentration experiment of resveratrol synergistically with ampicillin. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments:

[0026] Example 1: Biological activity of piceatannol in inhibiting β-lactamase of Staphylococcus aureus

[0027] Experimental steps: Different concentrations of piceatannol (0, 32, 64, 128 μg / mL) were incubated with β-lactamase at 37°C for 20 min, and then the substrate nitrocefin was added and incubated for another 10 min. OD492 was detected to analyze the inhibitory effect of piceatannol on the hydrolysis of nitrocefin by β-lactamase. The binding mode and binding site of β-lactamase and piceatannol were analyzed based on molecular docking.

[0028] Figure 1 Studies have shown that piceatannol can significantly inhibit the biological activity of β-lactamase from Staphylococcus aureus. When the concentration of piceatannol is 128 μg / mL, the biological activity of β-lactamase is reduced by nearly 80%. Figure 2 The binding mode and binding site of piceatannol and Staphylococcus aureus β-lactamase. Piceatannol binds to the β-lactamase binding pocket, and LYS-66, ASN-207, SER-226, ARG-235, ASN-161, SER-63 and ASN-123 residues are involved in the interaction with piceatannol.

[0029] Example 2: Piceatannol enhances the bactericidal ability of ampicillin

[0030] Experimental procedures: Bacteria in the logarithmic growth phase were treated with 64 μg / mL ampicillin, 32 μg / mL piceatannol, 64 μg / mL ampicillin and 32 μg / mL piceatannol, respectively, and cultured at 37°C with shaking. Samples were taken every 2 hours, diluted and plated on agar medium, and cultured statically at 37°C. The number of colonies was counted the next day to analyze the effect of piceatannol on the bactericidal ability of ampicillin.

[0031] Figure 3 The effect of piceatannol on enhancing the bactericidal ability of ampicillin against Staphylococcus aureus was studied, and the effect of piceatannol on the bactericidal effect of the β-lactam antibiotic ampicillin was evaluated by a bactericidal curve test. The results showed that there was no significant difference in the growth trend between the 32μg / mL piceatannol treatment group and the control group (culture medium and bacteria), indicating that piceatannol did not affect the normal growth of bacteria; for the ampicillin (64μg / mL) treatment group, the bacteria grew slower than the control group in the first 10 hours, but almost reached the same growth point after 24 hours; after co-treatment with 32μg / mL piceatannol and ampicillin (64μg / mL), the growth of bacteria was close to that of the ampicillin treatment group in the first 8 hours, and no bacteria were detected in the culture environment when the culture time reached 10 hours, indicating that 32μg / mL piceatannol can significantly enhance the bactericidal ability of ampicillin and is expected to synergize with ampicillin to treat Staphylococcus aureus infection.

[0032] Example 3: Piceatannol restores bacterial sensitivity to ampicillin

[0033] Experimental steps: LB medium containing a series of different concentrations (0-128 μg / mL) of ampicillin was obtained by serial dilution method, and bacteria were added to make the final concentration of 5×10 5 CFUs / mL, then piceatannol was added to make the final concentration of 32 μg / mL, and cultured overnight at 37°C for 24 hours. The synergistic effect of piceatannol and ampicillin was analyzed by detecting OD600.

[0034] Figure 4 The synergistic effect of piceatannol and ampicillin was further analyzed. Based on the minimum inhibitory concentration experiment, it was found that with the gradual increase of ampicillin concentration, the absorbance value of bacteria at OD600 gradually decreased, indicating that ampicillin had an inhibitory effect on bacterial growth, and the minimum inhibitory concentration was 128μg / mL; only the bacterial growth of the piceatannol-treated group was slower than that of the ampicillin-treated group, indicating that piceatannol can also slow down bacterial proliferation; when 32μg / mL piceatannol was added to the ampicillin treatment groups with different concentrations, the bacterial growth rate was significantly slowed. After treatment with 32μg / mL piceatannol, the minimum inhibitory concentration of ampicillin for bacteria decreased from 128μg / mL to 8μg / mL, a 16-fold decrease, indicating that piceatannol and ampicillin have a good synergistic effect.

[0035] Example 4: Piceatannol inhibits the formation of Staphylococcus aureus persisters

[0036] Experimental procedures: Bacteria in the logarithmic growth phase were treated with 10 μg / mL gentamicin, 10 μg / mL gentamicin and 32 μg / mL piceatannol, respectively, and cultured at 37°C with shaking. Samples were taken every hour, diluted, spread on agar medium and cultured overnight at 37°C. The effect of piceatannol on the formation of persister bacteria of Staphylococcus aureus was analyzed by counting the number of colonies.

[0037] During Staphylococcus aureus infection, biofilms are formed to resist the killing effect of antibiotics and increase the persistence of infection. This study further evaluated the effect of piceatannol on the formation of persisters of Staphylococcus aureus. Figure 5 As shown in the results, it was found that after treatment with 10 μg / mL gentamicin, the number of bacteria in the culture system gradually decreased, but a part of them still survived and formed persisters. After treatment with 32 μg / mL piceatannol and 10 μg / mL gentamicin, all the bacteria were killed at 2 hours, indicating that piceatannol can inhibit the formation of persisters of Staphylococcus aureus and reduce the persistence of infection.

[0038] Example 5: Piceatannol inhibits Staphylococcus aureus biofilm formation

[0039] Experimental steps: Bacteria in logarithmic growth phase were added to 96-well culture plates (1×10 7 CFUs / well), and different concentrations of piceatannol (0, 4, 8, 16, 32 μg / mL) were added and cultured at 37°C for 24 hours; the culture medium was discarded, the cells were washed three times with sterile phosphate buffer, fixed with 4% formaldehyde, stained with 0.1% crystal violet for 15 minutes, the crystal violet was discarded and washed, and then dissolved with 33% glacial acetic acid, and OD570 was detected to analyze the effect of piceatannol on bacterial biofilm formation.

[0040] Biofilm formation is key to enhancing persistent infection, and this study analyzed the effects of piceatannol on biofilm formation of Staphylococcus aureus. Figure 6 As shown, the results showed that piceatannol could inhibit the formation of biofilm in a dose-dependent manner. When the concentration of piceatannol was 32 μg / mL, the bacterial biofilm was reduced by more than 90% compared with the control group (the concentration of piceatannol was 0 μg / mL).

[0041] Example 6: Piceatannol cooperates with ampicillin to protect Staphylococcus aureus from infecting Glechoma gmelinii

[0042] Experimental steps: Inject bacteria into the body of G. mellonella (5×10 5 The infection model was constructed by adding 100 μg / kg BSA into the culture medium and 80 μg / kg CFUs / each. The experiment was randomly divided into 4 groups: infection group, 50 mg / kg piceatannol treatment group, 12.5 mg / kg ampicillin treatment group, and piceatannol and ampicillin combined treatment group. The constructed wax moth model was placed at 37°C, and the survival of wax moth was counted at designated time points to analyze the protective effect of piceatannol combined with ampicillin on wax moth infected with Staphylococcus aureus.

[0043] like Figure 7 As shown in the figure, the study constructed an experimental model of Staphylococcus aureus infection of wax moth. The experiment was divided into four groups, namely, piceatannol treatment group (50 mg / kg), ampicillin treatment group (12.5 mg / kg), piceatannol and ampicillin combined treatment group, and the control group injected with equal volume of solvent. The survival of wax moth was monitored at the specified time points, and it was found that the infection group began to die at 12 hours, and the survival rate was 11.11% at 84 hours. The ampicillin and piceatannol treatment groups both began to die at 24 hours, and the final survival rates were 11.11% and 44.44%, respectively, while the combined treatment group began to die at 28 hours, and the final survival rate was 55.56%. The above results show that both the single treatment group and the combined treatment group delayed the death time of wax moth, and the piceatannol treatment group and the combined treatment group significantly improved the survival rate.

[0044] Comparative Example 1

[0045] The difference between this comparative example and Example 2 is that piceatannol and ampicillin are not added, and the details are as follows:

[0046] Staphylococcus aureus was inoculated into agar medium and cultured at 37°C. The colony count was counted the next day to form the control group.

[0047] Comparative Example 2

[0048] The difference between this comparative example and Example 3 is that piceatannol is replaced by resveratrol, as follows:

[0049] Experimental steps: LB medium containing a series of different concentrations (0-128 μg / mL) of ampicillin was obtained by serial dilution method, and bacteria were added to make the final concentration of 5×10 5 CFUs / mL, and then resveratrol was added to make the final concentration of 32 μg / mL, and cultured overnight at 37°C for 24 hours. The synergistic effect of resveratrol and ampicillin was analyzed by detecting OD600.

[0050] Figure 8 The synergistic effect of resveratrol and ampicillin was further analyzed. Based on the minimum inhibitory concentration experiment, it was found that as the concentration of ampicillin gradually increased, the absorbance value of bacteria at OD600 gradually decreased, indicating that ampicillin had an inhibitory effect on bacterial growth, and the minimum inhibitory concentration was 128 μg / mL; only the bacterial growth of the resveratrol-treated group was slower than that of the ampicillin-treated group, indicating that resveratrol can also slow down bacterial proliferation; when 32 μg / mL resveratrol was added to the ampicillin-treated groups with different concentrations, the bacterial growth rate did not change much. After treatment with 32 μg / mL resveratrol, the minimum inhibitory concentration of ampicillin for bacteria decreased from 128 μg / mL to 64 μg / mL, which was reduced by 2 times, which was poorer than the synergistic effect of piceatannol and ampicillin in the present invention.

[0051] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. Use of piceatannol in combination with β-lactam antibiotics in the preparation of Staphylococcus aureus infection inhibitors, characterized in that: The β-lactam antibiotic is ampicillin, and the mass ratio of piceatannol to ampicillin is 1:

2.

2. The use according to claim 1, characterized in that: The structural formula of the piceatannol is:

Images