2.5 Flavonoids measurement
Flavonoids contents in tobacco tissues were determined as described in
our previous studies (Z. Wang et al.,
2018; Z. Wang et al., 2020). In brief,
fresh tobacco tissues were frozen in liquid nitrogen, and then
freeze-dried till constant weight. The tissues were grinded to powder,
and suspended with 1.5 ml 80% ethanol (with 0.012 g/L vitexin as
internal standard). The mix was ultrasonicated for 30 min, and then
centrifuged (14,000 rpm) for 10 min. A 0.22 μm membrane was adapted to
filter the supernatant, which was used for flavonoids content
determination by HPLC-UV. The experimental parameters are as follows:
chromatographic column-ACQUITY UPLC®BEH Phenyl (1.7 μm 2.1mm×150 mm,
Waters); injection volume-1μl; gas temperature-350°C; flow-0.3 mL/min;
wave length-230 nm, 260nm, 360 nm, and 570 nm; mobile phase-A 100%
water, B 100% acetonitrile; gradient elution-8 min 85% A+15% B, 5 min
58% A+42% B, 0.01 min 100% B, 3 min 95% A+5% B, 18 min stop.