枸杞槲皮素对金黄色葡萄球菌产β-内酰胺酶的抑制作用

Inhibitory Effect of Lycium barbarum Quercetin on β-lactamase from Staphylococcus aureus

【目的】探讨枸杞槲皮素对金黄色葡萄球菌产生的β-内酰胺酶的抑制作用机制,为临床研发β-内酰胺酶抑制剂提供基础依据。【方法】通过青霉素抑菌圈边缘试验筛选产β-内酰胺酶阳性菌株,通过药敏纸片法检测阳性菌株在32、64、128μg/mL枸杞槲皮素作用下对青霉素的敏感性变化;借助多功能酶标仪检测在32、64、128、256μg/mL枸杞槲皮素处理下青霉素含量的变化,并以此来评估枸杞槲皮素对β-内酰胺酶合成及在64、128、256μg/mL枸杞槲皮素作用下对β-内酰胺酶活性的影响。通过AutoDockTools 1.5.6软件对枸杞槲皮素与BlaZ蛋白进行分子对接,采用Discovery Studio 2019 Client软件对分子对接结果进行可视化分析。【结果】从36株金黄色葡萄球菌中共筛选出23株产β-内酰胺酶阳性菌株。药敏纸片结果表明,随着枸杞槲皮素浓度的升高,阳性菌株对青霉素的敏感性增加。多功能酶标仪检测结果表明,枸杞槲皮素可以抑制β-内酰胺酶的合成,并显著抑制β-内酰胺酶的活性(P<0.05)。分子对接结果表明,枸杞槲皮素通过与BlaZ蛋白Ω-loop区域内的保守残基Asn161以氢键结合,影响BlaZ蛋白与β-内酰胺类抗生素的结合。【结论】枸杞槲皮素可以减少β-内酰胺酶的合成,还可通过降低β-内酰胺酶的活性增强青霉素的抑菌作用,具有作为β-内酰胺酶抑制剂的潜在活性,为后续开发新型β-内酰胺酶抑制剂提供了参考依据。

【Objective】This study was aimed to investigate the inhibitory mechanism of Lycium barbarum quercetin onβ-lactamase produced by Staphylococcus aureus,so as to provide a basic basis for the clinical development ofβ-lactamase inhibitors.【Method】β-lactamase positive strains were screened by penicillin inhibition zone edge test,and the sensitivity of positive strains to penicillin was detected by drug sensitivity paper method under the action of Lycium barbarum quercetin at concentrations of 32,64 and 128μg/mL.The changes of penicillin content under the treatment of Lycium barbarum quercetin at concentrations of 32,64,128 and 256μg/mL were detected by multifunctional microplate reader,so as to evaluate the effect of Lycium barbarum quercetin onβ-lactamase synthesis andβ-lactamase activity under the treatment of Lycium barbarum quercetin at concentrations of 64,128 and 256μg/mL.The molecular docking of Lycium barbarum quercetin and BlaZ protein was performed by AutoDockTools 1.5.6 software,and the results of molecular docking were visualized by Discovery Studio 2019 Client software.【Result】A total of 23 strains producingβ-lactamase positive strains were screened from 36 strains of Staphylococcus aureus.The results of drug sensitivity paper showed that the sensitivity of positive strains to penicillin increased with the increase of Lycium barbarum quercetin concentration.The results of multifunctional microplate reader showed that Lycium barbarum quercetin could inhibit the synthesis ofβ-lactamase and significantly inhibit the activity ofβ-lactamase(P<0.05).Molecular docking results showed that Lycium barbarum quercetin affected the binding of BlaZ protein toβ-lactam antibiotics by hydrogen bonding with the conserved residue Asn161 in theΩ-loop region of BlaZ protein.【Conclusion】Lycium barbarum quercetin could reduce the synthesis ofβ-lactamase,and also enhance the antibacterial effect of penicillin by reducing the activity ofβ-lactamase,which had potential activity as aβ-lactamase inhibitor.The results provided a reference for the subsequent development of newβ-lactamase inhibitors.