Si gel

The air-dried and finely powdered rhizomes of B. rotunda (2.5 kg) were percolated with 95% EtOH (6 L, 4 times × 7 days) at room temperature to give a crude EtOH extract (190.5 g) after solvent removal. The obtained EtOH extract was divided into two portions. Each portion was separated by VLC over Si-gel (250 g each, Merck Art. No. 7731), packing on a sintered glass funnel (i.d. 12.5 cm × packing height 4.5 cm), using EtOAc-hexanes and MeOH-EtOAc gradients as eluents, respectively. Fractions (500 mL each) were collected and combined based on their TLC behaviors to give frs. A₁–A₅. Fr. A₄ (60.1 g, eluted with 25–100% EtOAc-hexanes), after three further consecutive Si-gel CC (Si-gel: Merck, Art. No 7734, 1st CC: 20% EtOAc-hexanes; 2nd CC: 60% CH₂Cl₂-hexanes; 3rd CC: 10% CH₃COCH₃-hexanes) afforded three separated frs. B₁–B₃. Fr. B₃ (5.37 g) was further purified by Sephadex LH-20 CC (Sephadex LH-20: GE Healthcare Bio-Sciences AB, 10% MeOH-CH₂Cl₂), followed by recrystallization from EtOH-CH₂Cl₂ to provide pure panduratin A (3.18 g).

For column chromatography (CC), Si-gel (107734, 107741, and 107749, Merck) and Sephadex LH-20 (Sigma–Aldrich) were used. For TLC chromatography, Si-gel (105554 and 105715; Merck) plates were used and visualized with óleum solution (sesquiterpenes) and Dragendorff’s reagent (alkaloids). The prep. HPLC chromatography was carried out on a Beckman 125P system equipped with an Ultrasphere semiprep column (10 × 250 mm) and a UV/visible diode array detector 168. Optical rotations were determined at 20 °C on a Perkin-Elmer 343 Plus polarimeter. IR Spectra were recorded in CHCl₃ on a Perkin Elmer 1600 spectrophotometer. NMR spectra were recorded on a pulsed-field gradient Bruker Advance II-500 MHz spectrometer (solvent as internal standard CDCl₃, at δ_H 7.26 and δ_C 77.0) and the Bruker software was used for DEPT, ¹H, ¹H-COSY (Homonuclear correlation spectroscopy), NOESY (Nuclear Overhauser Effect Spectroscopy), HSQC (Heteronuclear single quantum coherence spectroscopy) and HMBC (Heteronuclear Multiple Bond Correlation). EI and HR-EI-MS spectra were recorded in m/z on a Micromass Autospec spectrometer.

Optical rotations and IR spectra were measured on a JASCO P-1020 polarimeter and a JASCO FT/IR-4100 (JASCO Corporation, Tokyo, Japan) spectrophotometer, respectively. HRESIMS spectra were measured on a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). ¹H and ¹³C NMR spectra were obtained on a Varian Unity INOVA 600 FT-NMR (or Varian Unity INOVA500 FT-NMR) instruments (Varian Inc., Palo Alto, CA, USA) at 600 MHz (or 500 MHz) for ¹H, and 150 MHz (or 125 MHz) for ¹³C in CDCl₃. Thin-layer chromatography (TLC) analyses were performed on precoated silica (Si) gel plates (Kieselgel 60 F-254, 0.2 mm), and Si gel (230–400 mesh) (Merck, Darmstadt, Germany) and C18-reversed phase Si gel (RP-18; 40–63 µM) (Parc-Technologique BLVD, Quebec, Canada) were used for column chromatography. Further purification and the isolation of compounds were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Hitachi L-2455 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 μm).