Ultrasound-assisted extraction was performed using an ultrasound bath (frequency 38 kHz) (Grant Instruments™ XUB12 Digital, Cambridge, England). A sample of 0.3 ± 0.001 g of dried and milled flower heads was macerated in 10 mL of solvent. The extraction of isoflavones was performed by employing different extraction conditions: solvent (50% v/v ethanol and purified water) and extraction time (10 or 30 min), with the processing temperature of 40 ± 2 °C (the temperature is regulated automatically by the ultrasonic bath) [25 (link),26 (link)].
Thermal hydrolysis was completed by transferring the extract to a 250 mL round bottom flask. It was refluxed in a sand bath at 100 °C for 1 h. After the procedure, the mixture was left to cool down and then centrifuged with Sigma 3-18K centrifuge (Sigma, Osterode am Harz, Germany) for 10 min at 3382× g, followed by the decantation of the supernatant. The extracts were filtered through PVDF syringe filters (pore size 0.22 μm, Frisenette, Knebel, Denmark) prior to HPLC analysis. Sample preparation conditions are listed inTable 1 .
Thermal hydrolysis was completed by transferring the extract to a 250 mL round bottom flask. It was refluxed in a sand bath at 100 °C for 1 h. After the procedure, the mixture was left to cool down and then centrifuged with Sigma 3-18K centrifuge (Sigma, Osterode am Harz, Germany) for 10 min at 3382× g, followed by the decantation of the supernatant. The extracts were filtered through PVDF syringe filters (pore size 0.22 μm, Frisenette, Knebel, Denmark) prior to HPLC analysis. Sample preparation conditions are listed in
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