Ultrasonic-assisted acidified aqueous two-phase extraction for one-step production and recovery of flavonoid aglycones from Malvaviscus arboreus Cav. flower: Process exploration, composition analysis and activity validation

Using a polyethylene glycol (PEG)/ammonium sulfate system, a novel approach for one-step extraction and separation of the flavonoid aglycones from Malvaviscus arboreus Cav. flower (MACF) was developed by combining ultrasonic-assisted extraction (UAE) with acidified aqueous two-phase extraction (AATPE). Under the optimized UAE-AATPE conditions of PEG 600 of 22.8 % (w/w), ammonium sulfate of 22.5 % (w/w), 1.5 mol/L HCl, hydrolysis temperature of 85 ℃, and reaction time of 60 min, MACF flavonoid glycosides were not only fully extracted but also completely hydrolyzed, and the produced aglycones were purified by biphasic separation with a yield of 35.90 ± 0.96 mg/g. The mechanism in the UAE-AATPE process including UAE and ultrasonic-assisted acid hydrolysis (UAAH), was explored by investigating the different procedures and chemical composition under an ultrasonic field. The deglycosylated/glycosylated flavonoids were identified and quantified by high-resolution ultrahigh-performance liquid chromatography with quadrupole orbitrap mass spectrometry (UHPLC-Q/Orbitrap-MS) and UHPLC-DAD, revealing that MACF flavonoids were mainly composed of pelargonidin, kaempferol, catechin, cyanidin and quercetin in descending order of their content. The inhibitory assay on pancreatic lipase (PL) confirmed that the activity of the extracted aglycones with IC₅₀ of 563.01 ± 280.63 μg/mL was stronger than their glycosides. Kinetic and molecular docking analyses further explored and validated the PL inhibitory mechanism. The above results proved that UAE-AATPE integrating the multiple procedures, including extraction, hydrolysis and separation, greatly simplified the complex procedures and significantly improved extraction efficiency, providing a simple, rapid and efficient alternative for direct obtainment of the flavonoid aglycones from MACF.