*4.5. E*ff*ect of Compounds on Anti-Microbial and Antiproliferative Bioactivities*

Antiproliferative activities against HeLa and MCF-7 cell lines were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described [9]. Briefly, 6000 cells were plated into 96 well plate cultured with 90 μL DMEM (Dulbecco's Modified Eagle Medium) medium supplemented with 10% fetal bovine serum (FBS). After overnight culture, 10 μL compounds in 5% DMSO culture solutions with gradient final concentrations of 0.39, 0.78, 1.56, 3.13, 6.26, 12.5, 25 and 50 μg/mL were added into each well in triplicates, using positive control of paclitaxel. After 48 h incubation, 12 μL MTT solutions (final concentration of 0.5 mg/mL in PBS - phosphate buffered saline) were added and plates were further incubated for 4 h. Then, the medium was replaced gently by 100 μL DMSO. Plates were shaken and read by a Tecan Infinite M200 Pro reader at 570 nm, and the reference wavelength was 690 nm. The values of IC50 were calculated by GraphPad Prism 7.0 by applying nonlinear regression with (inhibitor) versus normalized response.

Anti-microbial activities against *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 51650 and *Candida albicans* ATCC 10231 were evaluated by microtiter broth dilution method as described [16,17] with some modifications. Briefly, 75 μL Lysogeny broth (LB) or yeast extract-peptone-dextrose (YPD) medium and 20 <sup>μ</sup>L inoculums (5 <sup>×</sup> 105 CFU/mL, colony-forming unit/mL) was plated into each well in 96 well plate. Then 5 μL test compounds with gradient final concentrations of 0.78, 1.56, 3.13, 6.26, 12.5, 25, 50 and 100 μg/mL was added into each well with three copies, using the positive control of kanamycin (for bacteria) and nystatin (for fungus). Plates were shaken at 200 rpm and cultured at 30 ◦C for 20 h. At last, plates were examined for bacteria growth by turbidity in daylight. The MICs were defined as the lowest concentration at which no microbial growth could be detected.
