Quantification of Resveratrol and Piceid Content

Harvested samples were dried in an oven at 60°C for 24 h or until the dry weight was stable. The dried sample (0.1 g) was macerated with 1 mL of 80% methanol and then placed in an ultrasonic bath (Jinwoo-Alex Company, Korea) at 40°C for 30 min. The fluid extract was filtered through a 0.45-μm nylon filter (Hyundai Micro Co., Ltd.). The resveratrol and piceid content during germination and in the presence of elicitors was determined using an HPLC system (Waters separation module e2695) with a reverse-phase column (C18, 4.6 × 150 mm, Waters). The mobile phase consisted of 100% acetonitrile as solvent A and water as solvent B, and the solvents were transported at a flow rate of 1 mL/min.

A gradient elution program was carried out by changing the ratio of solvent A and solvent B, from 10: 90, solvent A: B for 37 min, then 30: 70 for 1 min, 100: 0 for 7 min. then return to the initial condition 10:90 for 5 min. The UV-visible detector was operated at 308 nm. Trans-piceid and trans-resveratrol standards were obtained from Sigma-Aldrich. We analyzed piceid and resveratrol levels simultaneously using a standard mixture of piceid and resveratrol. The peaks of piceid and resveratrol were observed at retention times of approximately 16.2–16.8 and 26.7–27.3 min, respectively. The P:R ratio was determined to represent glycosyltransferase activity. In this ratio, P represents the piceid content, and R denotes the resveratrol content. If the P:R ratio exceeds 1, then it is assumed that glycosyltransferase can actively convert resveratrol to piceid, whereas a P:R ratio of less than 1 indicates that glycosyltransferase activity is suppressed because the resveratrol concentration is lower than the piceid concentration.