Dd2 500 nmr spectrometer

Flowers, leaves, and twigs collected during spring and young fruits collected in early summer 2020 were firstly air-dried in the dark at room temperature and then comminuted and extracted separately with aqueous methanol as follows: 1 g of dried and comminuted plant tissue was extracted with 30 mL of MeOH_(aq) 70% v/v for 10 min in a sonicator bath at room temperature. The extracts were filtered through a paper filter, and the sediment was re-extracted a second time with an additional 30 mL of the extraction solution. The filtrates were combined and dried in vacuo (40 °C, Rotavapor^® R-210, Büchi, Labortechnik, Flawil. Switzerland). ¹H-NMR (Nuclear Magnetic Resonance) spectra of all extracts were recorded in an AGILENT DD2 500 NMR spectrometer (20 mg of extract dissolved in 500 μL of CD₃OD) operating at 500 MHz. Chemical shifts are reported in ppm (δ). Before the in vitro analyses, the extracts were reconstituted with DMSO at a concentration of 5 mg mL⁻¹.

UV and IR spectra were obtained on a HP 8452A UV-Vis spectrometer (Hewlett-Packard, Palo Alto, CA, USA) and an Cary 630 FTIR spectrometer (Agilent Technologies, Santa Clara, CA, USA), respectively. High-resolution mass spectra were obtained on an Agilent TOF LC/MS spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source and operated with the Analyst QS 1.1 software for data acquisition and processing. NMR spectra were acquired on an Agilent DD2-500 NMR spectrometer with a OneNMR probe at 500 MHz for ¹H and 125 MHz for ¹³C using the pulse programs provided by the Agilent Vnmrj 4.0 software. Silica gel (J. T. Baker, 40 μm for flash chromatography) and Sephadex LH-20 were purchased from Fisher Scientific Co. (Waltham, MA, USA), and used for column chromatographic separations. Biotage Isolera^TM Prime flash chromatography system (Biotage Co, Charlotte, NC, USA) was used for further separation and purification. TLC was performed on silica gel 60 GF 254 plates (Millipore Sigma, Burlington, MA, USA) with the TLC spots being observed at 254 nm, followed by spraying with 1% vanillin-H₂SO₄ derivatization reagent.