Cytotoxicity determination by the MTT assay

The MTT assay was used to detect the cytotoxic activity and MDR-reversal activity of uncaria alkaloids and VRP. Cells (5×10³/well) were distributed into 96-well plates and were maintained overnight. For the cytotoxicity experiments, cells were cultured with different concentrations of uncaria alkaloids (Rhy, Irhy, Cory, Icory, HS and HST) or with VRP for 72 h. To evaluate the reversal activity of these alkaloids, various concentrations of the chemotherapeutic drugs with or without alkaloids at sub-toxic concentrations were added into the designated wells. After 72 h of incubation, 30 μl of MTT (5 mg/ml) was added to each well, and the plates were further incubated at 37°C for 4 h. The medium was then removed, and 100 μl of DMSO was added into each well to dissolve the formazan crystals. The absorbance was read at 570 nm using a DTX 880 Multimode Detector (Beckman Coulter, Brea, CA, USA). The IC₅₀ value was calculated from the survival curves using GraphPad Prism 5.0 software. The reversal fold was calculated by dividing the IC₅₀ of antineoplastic agents alone by the IC₅₀ of antineoplastic agents in the presence of alkaloids. VRP was used as a positive control.