Warangalone (Scandenolone)

Kinase experiment:

The enzyme activities of caspase-3 and caspase-9 are measured using a caspase fluorometric assay kit. Cells are seeded in 24-well plates at a density of 3×106 cells per well. After exposure of the cells to Warangalone for the allotted time periods, the cells are washed three times with PBS, and then lysed in a lysis buffer for 10 min on ice. The protein content of the cell lysates is assayed with a Micro BCA reagent. Cell lysates containing 50 μg of protein are incubated with a caspase-3 fluorogenic substrate (DEVD-AFC) or a caspase-9 fluorogenic substrate (LEHD-AFC) for 1 h at 37°C. Caspase activity is measured by fluorometric detection[3].

Cell experiment:

Cell viability is determined using the Cell Titer 96 Aqueous assay kit. Cells are seeded in 96-well plates at a density of 1×105 cells per well. The cells are maintained for 24 h at 37°C and then Warangalone (30 μM) is added to the culture medium. MTS solution is added to the 96-well plates at the indicated time points, and the cells are incubated for 1 h at 37°C. The absorbance is measured at a wavelength of 490 nm with a microplate counter[3].

References:

[1]. Tati Herlina, et al. ANTI-MALARIAL COMPOUND FROM THE STEM BARK OF Erythrina variegate. Indo. J. Chem., 2009, 9 (2), 308-311.
[2]. Wang BH, et al. Specific inhibition of cyclic AMP-dependent protein kinase by warangalone and robustic acid. Phytochemistry. 1997 Mar;44(5):787-96.
[3]. Induction of apoptosis by isoflavonoids from the leaves of Millettia taiwaniana in human leukemia HL-60 cells. Planta Med. 2006 Apr;72(5):424-9.