Fresh C. oleifera seeds collected at different treatment groups were dried in an oven at 60°C to a constant weight. All dried seeds were powdered by a mill (FOSS Scino (Suzhou) Co Ltd., Suzhou, China), and 5 g of three independent samples of dry seeds were weighed. Total tea oil was extracted according to the manufacturer’s protocol (Gong et al., 2020 (link)). Approximately 50 mL of petroleum ether was added to the aluminum cup in Soxtec device for extraction, and the machine temperature was set to 75°C. The program was as follows: 30 minutes of digestion, 150 minutes of extraction, and 60 minutes of solvent evaporation and recovery. The extracted oil is stored in the centrifuge tube for further use. Oil content was calculated according to the following formula: Oil content = [(weight of filter paper bag before extraction − weight of filter paper bag after extraction)/weight of powder] × 100%. Each sample had three biological replicates, and all oil contents in this study are performed with three biological replicates.
Fatty acid composition was determined using gas chromatography (Shimadzu GC-2014, Shimadzu, Kyoto, Japan) following the manufacturer’s instructions (Gong et al., 2020 (link)). Each sample had three biological replicates. Fatty acid methyl esters were prepared using NaOH/methanol method. A total of 60 mg of oil was transferred into a ground glass stoppered test tube, dissolved by 4 mL isooctane. Internal standard solution (triglyceride undecanoate, 100 μL) and 200 μL of potassium hydroxide methanol solution were added to the sample. The samples were mixed for 30 s on vortex mixer, and then allowed to clarify. One gram of sodium bisulfate was added, and the test tube was shaken vigorously to neutralize the potassium hydroxide. After the salt settled, the upper layer solution was used for chromatographic analysis. The gas chromatograph program was as follows: flame ionization detector temperature, 250°C; sample inlet temperature, 250°C; chromatographic column, 60 cm × 0.25 mm × 0.2 μm; carrier gas, nitrogen; split ratio, 1:50; sample injection volume, 1 μL; heating process, 50°C (2 min), 170°C (10°C/min, stored for 10 min), 180°C (2°C/min, stored for 10 min), and 220°C (4°C/min, stored for 22 min).
Fatty acid composition was determined using gas chromatography (Shimadzu GC-2014, Shimadzu, Kyoto, Japan) following the manufacturer’s instructions (Gong et al., 2020 (link)). Each sample had three biological replicates. Fatty acid methyl esters were prepared using NaOH/methanol method. A total of 60 mg of oil was transferred into a ground glass stoppered test tube, dissolved by 4 mL isooctane. Internal standard solution (triglyceride undecanoate, 100 μL) and 200 μL of potassium hydroxide methanol solution were added to the sample. The samples were mixed for 30 s on vortex mixer, and then allowed to clarify. One gram of sodium bisulfate was added, and the test tube was shaken vigorously to neutralize the potassium hydroxide. After the salt settled, the upper layer solution was used for chromatographic analysis. The gas chromatograph program was as follows: flame ionization detector temperature, 250°C; sample inlet temperature, 250°C; chromatographic column, 60 cm × 0.25 mm × 0.2 μm; carrier gas, nitrogen; split ratio, 1:50; sample injection volume, 1 μL; heating process, 50°C (2 min), 170°C (10°C/min, stored for 10 min), 180°C (2°C/min, stored for 10 min), and 220°C (4°C/min, stored for 22 min).
Free full text: Click here
