Graviola extracts preparation and GPE chemical characterization

Samples of Annona muricata plant parts (i.e. seeds, pulps, exocarp, leaves, and twigs) were acquired from the Philippines via Trish Flaster of Botanical Liaisons (Boulder, CO), and a voucher specimen (NCU633520) was deposited in the herbarium of the University of North Carolina at Chapel Hill. The samples were processed using procedures described in detail previously⁴⁰. Briefly, to each 1000 mL flask containing a sample of ~25 g of powdered plant material (where all parts were treated separately) 240 mL of 1:1 MeOH/CHCl₃ were added. The samples were stirred overnight at room temperature, filtered using vacuum filtration, and the remaining residues were washed with MeOH. To the filtrate, 360 mL of CHCl₃ and 600 mL of water were added. The mixtures were stirred for ½ h, and then transferred into separatory funnels. The bottom layers were drawn off into round-bottom flasks, which were evaporated to dryness. The dried organic extracts were re-constituted in 200 mL of 1:1 MeOH/CH₃CN and 200 mL of hexanes and transferred to separatory funnels. The biphasic solutions were shaken vigorously. The MeOH/CH₃CN layers were evaporated to dryness under vacuum.

HRESIMS was performed in positive ionization mode on a Thermo Q Exactive Plus mass spectrometer (ThermoFisher, San Jose, CA) equipped with an electrospray ionization source. UPLC was carried out on a Waters Acquity system [using a BEH C18 (2.1 × 100 mm, 1.7 μm) column (Waters Corp., Massachusetts, USA) equilibrated at 40 °C]. A mobile phase consisting of CH₃CN:H₂O (both acidified with 0.1% formic acid) at a flow rate of 300 μL/min was used for analysis with lithium fluoride (2 mM in MeOH) infused post-column at a flow rate of 5 μL/min. The gradient started with 70:30 (CH₃CN:H₂O) for 1 min then increased linearly to 100% CH₃CN over 7 min, holding for 1.5 min and then returning to the starting conditions within 0.5 min. An Acquity UPLC photodiode array detector was used to acquire PDA spectra, which were collected from 190–500 nm with 4 nm resolution. Higher-energy collisional dissociation (HCD) was performed using normalized collision energy (NCE) at 60%.