ABSTRACT
Objective To study the effects of four cytotoxicity evaluation methods on the inhibition rate of shikonin (SK) in vitro, and to compare the pseudo-negative phenomenon often found in the evaluation of cytotoxic activity of natural pigments represented by naphthoquinones in Lithospermum erythrorhizon. Methods SK was co-cultured with its high-sensitive strain HL-60 cells and low-sensitivity strain A549 cells, trypan blue method, sulforhodamine B (SRB) method, Cell Counting Kit-8 (CCK-8) and MTT cytotoxicity test were used for parallel experiments to determine the dose-effect relationship curve of SK (0.4-128 μmol/L) inhibiting the growth of cells. Results The half-inhibitory concentration (IC50) of shikonin on HL-60 cells was determined by trypan blue method, SRB method, CCK-8 method, and MTT method, which was 0.57, 0.77, 1.36, and 1.01 μmol/L, respectively. For A549 cells, the IC50 was 6.30, 10.38, 13.48, and 15.24 μmol/L, respectively. When the concentration of shikonin was below 3.2 μmol/L and 32 μmol/L, the inhibition rate of the two kinds of cells increased linearly by the four methods, followed by differences. Among them, the results of the trypan blue method and the SRB method are in good agreement, while the MTT method and the CCK-8 method have lower inhibition rates. At 12.8 μmol/L, the inhibitory rate of SK on HL-60 cell measured by CCK-8 was 81%, while the inhibitory rate measured by trypan blue method was 96%, and at 128 μmol/L the inhibitory rate of SK on A549 cells measured by MTT method was 89%, however, the inhibitory rate measured by trypan blue method was 99%. The absorption spectrum of SK overlapped with formazan at the wavelength from 400 to 600 nm, with the maximum overlap peak from 550 to 570 nm, and CCK-8 reagent had a synergistic inhibitory effect on HL-60 with SK. The results of trypan blue method showed that SK at the highest dose almost completely killed cells in the plate wells, which was significantly different from the control group, but both MTT method and CCK-8 method resulted in a pseudo-negative phenomenon. Conclusion Therefore, cytotoxicity test of natural pigments represented by naphthoquinones in L. erythrorhizon, MTT method, and CCK-8 method are not recommended, while SRB method and trypan blue method are suggested.