Analysis of fatty acid compositions

The ground pepper samples (20.0 g) were extracted with diethyl ether in a Soxhlet apparatus for 8 h to isolate the oil in accordance with the GB/T 5009.6-2003. The fatty acid compositions of the oils were determined by converting them into fatty acid methyl esters (FAMEs), followed by gas chromatograph (GC) analysis (Yang et al. 2011; Zeng et al. 2011).

Exactly 20.0 mg of the sample was thoroughly mixed with KOH–methanol solution (2.0 mL, 0.5 M, 70 °C) for 10 min in a 50 mL round bottom flask, and BF3-methanol solution (3.0 mL) was then added. The reaction was kept at 70 °C for another 5 min. The action mixture was cooled to room temperature. The FAMEs were extracted into hexane (3 × 1.0 mL) and then dried over anhydrous sodium sulfate. 10 µL of the samples was used for GC analysis.

A Hewlett-Packard 5890 GC was used to analyze the fatty acid composition. The FAMEs were separated on a FFFAP column (PERMABOND-FFFAP DF-0.25, 25 m × 0.25 mm i.d., Macherey–Nagel, Düren, Germany) by using nitrogen as the carrier gas. The following temperature program was applied: 150 °C (2 min), raised to 230 °C at 10 °C/min and held at 230 °C for 8 min. The split ratio was 20:1. The injector and flame ionization detector temperatures were set at 250 and 300 °C, respectively. The fatty acid components of the oil were identified by comparison of the retention time with fatty acid methyl ester standards, and the relative percentage content of fatty acids was quantified by the peak area normalization method.