All reagents and solvents for extraction, as well as UPLC and HPLC-MS analyses (methanol, acetonitrile, formic acid, dimethylsulfoxide, polyamide and chloroform), were from Sigma-Aldrich (Poznań, Poland), and ultrapure water was obtained from a Millipore Direct Q3 device. Standards of flavonoids (apigenin, apigenin-7-O-glucoside, apigenin-6-C-glucoside, apigenin-8-C-glucoside, isorhamnetin, luteolin, luteolin 7-O-glucoside, luteolin 4′-O-glucoside, luteolin-8-C-glucoside, luteolin-6-C-glucoside, quercetin 3-O-galactoside, quercetin, rutin, isorhamnetin, isorhamnetin 7-O-glucoside) were purchased from Extrasynthese (Genay, France).
Extracts were prepared according to [21 (link)]. Weighted samples of frozen leaves (about 100 mg) were placed in Eppendorf tubes with 1.4 mL of 80% methanol and the internal standard, homogenized using a ball mill (MM 400, Retsch, Haan, Germany) and subsequently placed in an ultrasonic bath for 30 min and centrifuged (11,000× g) for 10 min. apigenin (5 µL of 1 mg/mL solution in dimethylsulfoxide) was used as the internal standard in extracts prepared for the UPLC analyses. Samples of the secondary metabolite extract were directly subjected to HPLC-MS analysis and, prior to the UPLC analysis, 200 µL were diluted with 80% methanol (800 µL).
Extracts were prepared according to [21 (link)]. Weighted samples of frozen leaves (about 100 mg) were placed in Eppendorf tubes with 1.4 mL of 80% methanol and the internal standard, homogenized using a ball mill (MM 400, Retsch, Haan, Germany) and subsequently placed in an ultrasonic bath for 30 min and centrifuged (11,000× g) for 10 min. apigenin (5 µL of 1 mg/mL solution in dimethylsulfoxide) was used as the internal standard in extracts prepared for the UPLC analyses. Samples of the secondary metabolite extract were directly subjected to HPLC-MS analysis and, prior to the UPLC analysis, 200 µL were diluted with 80% methanol (800 µL).