Cytotoxic and antibacterial properties
The cytotoxicity of extracts was tested in triplicate in MCF7 breast cancer cell line using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium chloride (MTT) assay. Briefly, MCF7 cells were cultured up to 50% confluence in a 96-well microplate containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS). The cells were washed copiously with phosphate-buffered saline (PBS), and the medium was exchanged with DMEM (10 ml) containing test compounds (20 ppm). Cells exposed to DMEM or DMSO alone were maintained in separate wells as control. The cells were incubated for 24 h at 37 °C and subjected for MTT assay following standard protocol. Briefly, cells treated with test compounds were supplemented with 50 µl of MTT solution (5 mg ml−1) prepared in PBS and kept for incubation under dark at 37 °C for 3 h. Subsequently, the viability of the cells was measured as a function of reduction of MTT to insoluble formazan by mitochondrial dehydrogenase enzyme of healthy cells. Formazan crystals were dissolved in Dimethyl Sulphoxide and the absorbance was recorded at 570 nm using a microplate reader (Biotech, USA).
The antibacterial property of the organic extracts against four isolates of multiple drug resistant bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio cholera and Escherichia coli, was tested using standard disc diffusion assay. Briefly, the 6 mm diameter paper discs impregnated with 100 µl test compound (20 ppm) were placed over the surface of a Muller Hinton agar plate swabbed with test organisms. Paper discs impregnated with 100 µl DMSO were also used as control. The plates were incubated for 24 h at 28 ± 2 °C and the formation of clear zones around the discs was considered as the positive indication of inhibitory activity. The zone of inhibition around the discs was recorded after 24 h using Hi Antibiotic Zone Scale (Himedia, India).