Phytochemical Extraction and Analysis
Desiccated leaves were homogenized under liquid nitrogen and stored at -80°C prior to extraction. To extract a broad range of polar and nonpolar metabolites, the homogenized samples were mixed with 1 mL of a 1:1 methanol-chloroform solution in conjunction with 1 µL of ethyl-decanoate (Sigma-Aldrich, Inc.) as an internal standard. Sample extracts were vortexed in an orbital shaker for 2 hours to expedite extraction at 160 rotations min-1. Post extraction, each sample was filtered through a 0.2 μm nylon filter into a 2 mL amber glass vial, where 1 µL of the sample extract was used for direct injection into a single quadrupole GCMS-QP2020 NX gas chromatograph-mass spectrometer (Shimadzu, Inc.). Each extract was injected at 230°C in splitless mode with helium carrier gas flow set to 2 mL min-1 by an AOC-6000 autosampler (Shimadzu, INC.). The oven was held isothermal at 40°C for 1 min, ramped at 15°C min-1 to 330°C where it was held isothermal for 1 min. Electron impact mass spectra were recorded in full scan mode from m/z 50 to 450 in the single quadrupole mass spectrometer. Data were initially preprocessed using GCMS solutions v1.4 (Shimadzu, Inc.). Signals were integrated using total ion count and measurements such as the area and height of chromatogram peaks were normalized to the internal standard. Compounds were putatively identified by comparing observed mass spectra with the NIST mass spectra library using a 75% significance index cutoff.