*2.9. Biological Assays*

The cytotoxicity of the eight compounds was assayed by cell counting kit-8 (CCK-8) colorimetric method [71] in vitro against the human leukemia cells K562, hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line HCT-116. The cell lines were routinely in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% calf serum at 37 ◦C for 4 h in a humidified atmosphere of 5% CO2 incubator. The adherent cells at logarithmic phase were digested by pancreatic enzymes and inoculated onto 96-well culture plate at a density of 1.0 <sup>×</sup> 104 cells per/well. Test samples and control were dissolved in DMSO (dimethyl sulfoxide) and then added to the medium, incubated for 72 h. Then, the cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) reagent was added to the medium followed by further incubation for 3 h. Absorbance at 450 nm with a 600 nm reference was measured thereafter using a SpectraMax M5 microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA). The inhibitory rate of cell proliferation was expressed as IC50 values and calculated by the following formula:

$$\text{Growth inhibition (\%)} = \text{[ODcontrol-ODtreated]} \text{[ODcontrol} \times 100\%]$$

Doxorubicin was tested as a positive control, and cell solutions containing 0.5% DMSO were tested as a negative control.

The antibacterial activities of the isolated compounds were tested against Gram-positive bacteria *Staphylococcus aureus*, *Bacillus subtilis*, and *Sarcina lutea* and Gram-negative bacteria *Klebsiella pneumoniae* and *Escherichia coli* with the minimum inhibitory concentration (MIC) method recommended by the Clinical and Laboratory Standards Institute [72].
