Context and objective: Zerumbone has been reported to exert anti-microbial effects, but the mechanism by which the compound exerts its action is not known. Thus, this study aimed to investigate the mechanism of action of zerumbone against methicillin-resistance Staphylococcus aureus (MRSA), using the atomic force microscopy (AFM), scanning electron microscopy (SEM), and flow cytometry techniques. Methods: MRSA (NCTC 13277) cell viability was determined using the microplate AlamarBlue assay. AFM and SEM were used to determine the morphology of zerumbone-treated MRSA cells. Flow cytometric analysis was used to determine the effect of zerumbone on bacterial membrane permeability and membrane potential, using the propidium iodide (PI) staining method, membrane potential-sensitive fluorescence probe, and DiBAC4(3) dye. DCFDA dye was used to determine the generation of reactive oxygen species (ROS) by MRSA. Results: Zerumbone significantly inhibited MRSA growth with a minimum inhibitory concentration (MIC) of 125 μg/ml. The AFM analysis showed that zerumbone caused leakage of cytoplasmic content from the bacterial cells. Ultrastructure analysis showed small colonies of the bacteria with pores on the membrane surface. There were increases in zerumbone-treated MRSA PI and DiBAC4(3) fluorescence, indicating an increase in cell membrane permeability and a decrease in membrane potential that culminated in the loss of membrane structural integrity and bacterial death. Based on DCFDA dye analysis, zerumbone also reduced ROS production by MRSA. Conclusions: Zerumbone exerts anti-MRSA effects by causing membrane depolarization, increasing membrane permeability, and finally disrupting cell membrane and bacterial killing.
Context: Clausena excavata Burm. f is a plant used in folklore medicine for the treatment of various ailments in South East Asia. The plant parts contain chemical components that are cytotoxic to many cancer cells. Objective: The study investigated the cytotoxic effects of ethyl acetate, methanol and chloroform C. excavata leaf extracts on the non-small-lung cancer, NCI-H460, cell line. Methods: Based on the 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay, among extracts, ethyl acetate C. excavata leaf extract (EACE) was the most potent anti-NCI-H460 cells, with IC50 value of 47.1 ± 6.1 μg/ml. The effects of EACE on NCI-H460 cells were also determined by clonogenic, 4', 6-diamidino-2-phenylindole (DAPI), and annexin-V-fluorescein isothiocyanate/propidium iodide-PI flow cytometric assays. Reactive oxygen species (ROS) production and apoptotic gene expressions was determined via flow cytometry and real-time quantitative PCR, respectively. Results: EACE-treated NCI-H460 cells after 48 h underwent apoptosis as evident by loss of cell viability, cell shrinkage, and chromatin condensation. The results also showed EACE mediated increase in ROS production by the NCI-H460 cells. After 48 h treatment, EACE increased the pro-apoptotic BAX and decreased the anti-apoptotic Bcl-2, Survivin and c-Myc gene expressions. Conclusions: EACE is a potential anti-lung cancer by increasing cancer cell ROS production and apoptosis.
