Hydroxychavicol (HC), found abundantly in Piper betle leaves is credited with antimicrobial property. Previously we had shown HC induces reactive oxygen species mediated DNA damage in bacterial cells. HC also resulted in membrane compromise revealing its pleiotropic effects on cellular targets. The kinetics and exact sequence of events leading to inhibition of growth and cell death in E. coli after HC treatment remains poorly understood. We show that sub-lethal concentration (125 μg/mL) of HC causes cellular filamentation within 1 h of treatment, while a higher concentration (750 μg/mL) induces cell breakage. HC-treated cells were found to experience oxidative stress as early as 10 min, while evidence of membrane damage was apparent at 30 min. DNA damage repair genes were found to be activated at 60 min. Interestingly, HC-induced cell permeabilization was inhibited and enhanced by external Mg²⁺ and EDTA, respectively, suggesting that HC damages the outer membrane. Kinetic experiments revealed that HC-treated cells underwent oxidative stress, membrane damage and DNA damage in that order. Because gram negative bacteria such as E. coli are refractory to several antibiotics due to the presence of the outer membrane, we hypothesized that HC pretreatment would sensitize E. coli to hydrophobic antibiotics. Our study reveals for the first time that HC could sensitize bacteria to clinically used antibiotics due to its outer membrane damaging property.

在Piper甲虫叶片中大量发现的羟基羟维生素（HC）被认为具有抗菌性能。以前我们已经证明HC可以诱导细菌细胞中活性氧介导的DNA损伤。HC还导致膜受损，揭示其对细胞靶标的多效作用。导致大肠杆菌生长抑制和细胞死亡的动力学和确切事件顺序HC治疗后仍知之甚少。我们显示，亚致死浓度（125μg/ mL）的HC在处理后1小时内引起细胞丝化，而更高的浓度（750μg/ mL）则导致细胞破裂。发现经过HC处理的细胞最早在10分钟时就经历了氧化应激，而在30分钟时明显出现了膜损伤的迹象。发现DNA损伤修复基因在60分钟时被激活。有趣的是，HC诱导的细胞通透性分别被外部Mg ²⁺和EDTA抑制和增强，表明HC破坏了外膜。动力学实验表明，HC处理的细胞按此顺序经历了氧化应激，膜损伤和DNA损伤。因为革兰氏阴性细菌，如大肠杆菌由于存在外膜，因此对几种抗生素难治，我们假设HC预处理会使大肠杆菌对疏水性抗生素敏感。我们的研究首次揭示了HC可以破坏细菌，因为它具有破坏外膜的特性，因此可以使细菌对临床使用的抗生素敏感。