3.5. Sample and Standard Preparation

Whole fruit powder of 3 mg was taken while preparing samples for extraction and quantification of flavonoid content. Dimethyl sulfoxide (DMSO) 5 mL was added and the whole solution was sonicated for about 20 min in sonicator 2K1008008 series (Life-Care Equipments Pvt. Ltd., Mumbai, India). Then this was filtered through 0.45 micron nylon filter and injected into HPLC Agilent Model No. 1260 Infinity System (M/s. Agilent Technologies Pvt. Ltd., Bangaluru, India) for flavonoids analysis [10] and their relative percentage was reported.

Standard stock solution of 600 ppm was prepared by dissolving each flavonoid (hesperidin, narirutin/isonaringin, diosmin and didymin/neoponcirin) in dimethyl sulfoxide. For calibration purposes, stock solution was diluted to further lower concentrations using mobile phase.