2.5. Quantitative analysis of flavonol O-glycosides by HPLC

The seeds, sprouts, and aerial parts of indigo leaves were harvested as described in the section of 2.2 and used for the preparation of dried materials by lyophilization. The dry materials ground in a mortar using a pestle from the seeds (900 mg dry weight (DW)), sprouts (100 mg DW), or aerial parts (100 mg DW) were individually mixed with 10 ml of 80% methanol and 200 μl of dimethyl sulfoxide containing 200 μg of hesperetin as an internal standard. The mixture was vortexed vigorously for 30 sec and then sonicated for 20 min at 20 °C in a Bransonic sonicator (Emerson Japan, Tokyo, Japan) for the extraction of flavonoids. The resulting supernatant was collected after centrifugation at 1500 × g for 5 min. The same extraction procedure was repeated again by mixing the pellets with 10 ml of 80% methanol. The combined supernatants were filtrated through a Kiriyama funnel filter paper No. 4, after which the extracts were filled up to 20 ml with 80% methanol. The 1-ml aliquot of the filled extracts was evaporated into dryness and redissolved in 1 ml of a mixture of 0.1% formic acid/acetonitrile (95:5, v/v). A portion of the extracts was applied to the analysis by reverse-phase HPLC following filtration through a membrane filter with a pore size of 0.22 μm. The HPLC analysis was performed using a YMC-Pack ODS-AM column (150 mm × 3 mm i.d., 5-μm particle size) on a Shimadzu LC-2010A system equipped with a Chromatopac C-R8A recorder. The column was eluted with a flow rate of 0.4 ml/min at 40 °C for a total of 40 min with a mobile phase of 0.1% formic acid/acetonitrile in a linear gradient system from 95:5 (v/v) to 60:40 (v/v). The quantified value was expressed as hesperetin equivalent (HesE) (μg HesE/g DW).