Diterpene resin acid extraction and analysis
Diterpenes were extracted from lyophilized ground tissue in 1 ml of methanol, as adapted from Mullin et al . (2021). Samples were vortexed for 30 sec, then sonicated for 10 min, and left for 24 h in the dark at room temperature, and then vortexed for 30 sec and centrifuged (18,213 rcf) for 15 min. Extracts were transferred into 2 mL glass vials and stored at −40 °C. Diterpenes were analyzed in an UHPLC, fitted with a reverse-phase InfinityLab Poroshell 120 EC-C18 column (2.1 x 150 mm 1.9µm, Agilent Tech.) and a diode array detector (UV/Vis, 1290 DAD, Agilent Tech.). We performed a gradient analysis with a binary solvent system, a 1.7% v/v glacial acetic acid in distilled deionized water (channel A) and 100% pure methanol (channel B) flowing at 0.3 mL min-1 for 17 min with solvent gradients. A 5 µl injection volume was used. The system began at 75% B for 1 min, then increasing to 85% B over 9 min, held for 2 min, then decreased to 75% B over 14-17 min, and held for 3 min.
Diterpenes were quantified using analyte absorbance at wavelengths of 240, 268, and 282 nm (Kersten et al. 2006) and applying standard curves. The curves were generated from dilutions prepared from analytical standards of dehydroabietic acid (CP: >99%), sandaracopiramic acid (CP: >90%), levopiramic acid (CP: >95%), neoabietic acid (CP: >99%), palustric acid (CP: >92%), and abietic acid (CP: >75%). All chemicals were obtained from CanSynth Chem. Corporation (Toronto, ON, CAN), except for abietic acid from Sigma Aldrich. Diterpene concentrations were reported as µg mg-1 of dried weight.