Flavonoid Aglycone Profiling in Petals

The accumulation of flavonoid aglycones in the petals was analyzed as described previously [57 (link)], with some modifications. Briefly, 100 mg of ground petal sample was subjected to a 2-h acid hydrolysis in 300 μL of 50% methanol containing 2 N HCl at 90°C. After centrifugation at 15,000× g for 10 min at 4 °C, the supernatant was transferred to a new tube. The remaining pellet was rinsed with 200 μL acid hydrolysis solution, which was then combined with the first extract. A 10 μL aliquot of the extract was used in a HPLC analysis performed on an LC-20A HPLC system (Shimadzu, Kyoto, Japan) equipped with an Inertsil-ODS3 C18 column (5 μm, 250 × 4.6 mm; GL Science, Eindhoven, Netherlands). The chromatographic separation was carried out using 0.1% formic acid in water (solution A) and 0.1% formic acid in acetonitrile (solution B) with the following gradient conditions; 0–30 min, linear gradient of 5–55% (v/v) solution B; 30-45 min, linear gradient of 55–65% (v/v) solution B; and 45–50 min, linear gradient of 65–100% (v/v) solution B at a flow rate of 1 mL∙min⁻¹. The temperature of the column was maintained at 40 °C. A diode-array detector was used for compound detection. The spectra of the compounds were recorded between 210 and 800 nm, and the peak corresponding to each compound was identified by comparing the retention times and UV spectra with those of the standards.