METHOD: Bacterial cell viability was performed by using microplate AlamarBlue assay. Atomic force microscopy was used to determine morphological changes in the surface of bacterial cells. Cytotoxicity and phytotoxicity were determined by brine shrimp lethality and Lemna minor bioassay. Caco-2 (colorectal adenocarcinoma) cell line was used for the evaluation of the anticancer effects.
RESULT: Among the fractions tested, ethyl acetate (EA) fraction was found to be active with minimum inhibitory concentration (MIC) of 750 μg/mL against E. faecalis, but other fractions were found to be insensitive to bacterial growth. Microscopically, the EA fraction-treated bacteria showed highly damaged cells with their cytoplasmic content scattered all over. The LC50 value of the EA fraction against brine shrimp was more than 1000 μg/mL showing the nontoxic nature of this fraction. Chloroform (CH), EA, and methanol (MOH) fractions of C. excavata were highly herbicidal at the concentration of 1000 μg/mL. EA inhibited Caco-2 cell line with an IC50 of 20 μg/mL.
CONCLUSIONS: This study is the first to reveal anti-E. faecalis property of EA fraction of C. excavata leaves, natural herbicidal, and anticancer agents thus highlight the potential compound present in its leaf which needs to be isolated and tested against multidrug-resistant E. faecalis.
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.