The Procedure for Anticancer Evaluation
The target cancer cell lines were seeded in 96-well plates (5000cells/well) with 100μL DMEM supplemented with 10% fetal bovine serum, and cultured at 37 oC in a humidified CO2 incubator (95% air, 5% CO2) for 24h. While the cell lines grew to 90% in logarithmic growth, the culture medium was removed from each well, and 100 μL fresh DEME was added to each well. Then, 10μL different concentrations of P. nagi kernel oil solutions were added into each well (every concentration was repeated for 5 times) and the plates were incubated for another 48h at 37 oC. Subsequently, 10μL CCK8 was added to each well, and the plates were cultured at 37 oC for another 4 hours. The optical density was measured at a wave-length of 450 nm on an ELISA microplate reader. DMEM and DMSO solution (V/V: 10/1) was used as a negative control. The results were expressed as the inhibition calculated at the ratio [(1-(OD450 treated/ OD450negative control)) ×100].
3. Results and discussion
3.1 The influence of steaming time on the yield and quality of P. nagi kernel oil
The P. nagi kernel oil was obtained through two steps: (1) steaming the powder of P. nagi kernel; (2) squeezing the steamed powder of P. nagi kernel. During the experiment, we found that the time of steaming of the powder of P. nagi kernel is extremely important for the yield and the quality of the oil, the short time steaming does not lead to the higher yield, while long time steaming will lead to the higher yield, but the oil also contains higher water content, which affects the quality of the oil, making the oil very turbid. Thus, we optimized the experimental conditions about the steaming time, the yield and the quality of oil. The results (see totable 3 ) exhibited that the yield of oil increased with the increasing of the steaming time, and the steaming time is 20min with the 90% yield of oil, and the oil quality is normal; even if the steaming time is 25min with the 93% yield, the oil quality is turbid and the yield increased only 3% compared with the 20min steaming time. Thus, we selected steaming time 20min as the optimized condition.
Table 3 . Steaming time of P. nagi kernel, yield and oil quality