After freshly opened flowers were harvested, petals were separated, washed with deionized water and wiped dry, and the apical and basal areas were dissected. Every flower compartment was weighed and coarsely ground under liquid nitrogen. For extraction (Method 1), an amount of solvent in a 1:1 (v:v) ratio of water to methanol was added to achieve a uniform concentration of 1 mg of material per 10 µL of solvent (or, for capsules, per 5 µL). After 30 min of extraction in the ultrasonic bath, samples were centrifuged (4 °C, 13,200 rpm), and the supernatant was used for HPLC-PDA analysis. The entire procedure was carried out with three biological replicates.
The analytical HPLC-PDA system consisted of an Agilent series HP1100 (binary pump G1312A, auto sampler G1313A, and photodiode array detector G1315B, 200–700 nm) equipped with an EC250/4 Nucleodur C18 HTec column from Macherey-Nagel (5 µm; injection volume 20 µL). The method included a 21-min gradient from 20% to 67% of methanol in acidified water (0.1% trifluoroacetic acid) with a subsequent washing step (100% methanol) and equilibration to starting conditions. The flow rate was 1 mL·min−1, and the detection wavelengths were 211, 254, 281, 351, and 460 nm. The aglycone standards of kaempferol and pelargonidin chloride were purchased from Sigma-Aldrich.
The analytical HPLC-PDA system consisted of an Agilent series HP1100 (binary pump G1312A, auto sampler G1313A, and photodiode array detector G1315B, 200–700 nm) equipped with an EC250/4 Nucleodur C18 HTec column from Macherey-Nagel (5 µm; injection volume 20 µL). The method included a 21-min gradient from 20% to 67% of methanol in acidified water (0.1% trifluoroacetic acid) with a subsequent washing step (100% methanol) and equilibration to starting conditions. The flow rate was 1 mL·min−1, and the detection wavelengths were 211, 254, 281, 351, and 460 nm. The aglycone standards of kaempferol and pelargonidin chloride were purchased from Sigma-Aldrich.