2.3.2.2 Prepare the standard working stock solution

2.3.2.2.1 Preparation of standard working stock solution

0.1g of the mixed standard solution of 10 kinds of mixed elements and 0.1g of the nitrogen standard of Se element were respectively accurately weighed, and diluted to 10 mL with 2% nitric acid to form the mixed standard stock solution (1000 ng/mL) containing 11 metal elements.

2.3.2.2.2 Preparation of series of standard stocking solution

Appropriate amount of the above standard working stock solution was weighed accurately, and which was diluted with 2% nitric acid to a standard series of mixed solutions containing 0.2ng, 1ng, 5ng, 10ng, 50ng, 100ng and 500ng of each element per 1mL.

2.3.2.2.3 Preparation of internal standard working solution

Appropriate amount of the mixed internal standard solution was accurately weighed and diluted with 2% nitric acid to 50 ng/mL.

2.3 In vitro biological evaluation

2.3.1 In vitro antioxidant effect of the P. nagi kernel oil

The scavenging ability of P. nagi kernel oil to DPPH⸱ was carried out in this work. Briefly, 5mg DPPH was added into a 25mL volumetric flask, and diluted with DMSO to 25mL. The volumetric flask was covered by a black plastic bag and shaken fully to obtain the DPPH stock solution. Then, taking out 1.8mL DPPH stock solution and diluting with 8.2mL DMSO to 10mL as the working solution, which was measured the absorbance as A₀ at 519nm. Subsequently, 40µL P. nagi kernel oil was added into 5mL fresh DPPH stock solution and shaken to mix well, which was also measured the absorbance as A at 519nm. The DPPH scavenging ability was calculated at the ratio [(A₀-A) / A₀ × 100], each experiment was repeated for 5 times.

2.3.2 Preliminary in vitro anticancer evaluation

The anticancer activity of this oil was evaluated against gastric cancer, breast cancer (MCF-7), lung cancer(A549) and Hela cell lines using the counting kit-8 (CCK-8) method ⁷. The evaluation process was described elsewhere with some modifications. Briefly, the oil was dissolved in DMSO at a concentration of 100mg/mL, then diluted successively with DMSO for eight different concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, 3.125 mg/mL, 1.5625 mg/mL and 0.78125 mg/mL, respectively) as stock solution for below experiment.