Silica gel

Reagents and chemicals, including silica gel (60 Å, 230–400 mesh), were purchased from VWR (Radnor, PA, USA) and used without further purification unless otherwise noted. Nuclear magnetic resonance (NMR) spectra were recorded on an Avance III (400 MHz, Bruker, Billerica, MA, USA) spectrophotometer. ¹⁹F NMR spectra were recorded using trifluoroacetic acid as a standard (δ = −76.55 ppm). Mass spectrometry was performed on an LC/MS 6545 Q-TOF (Agilent, Santa Clara, CA, USA) in APCI mode. Purity analysis by HPLC was performed on an Agilent 1100 system with a diode array detector and C8 ZORBAX Eclipse Plus column (Agilent). Absorption data was collected on a Cary-100 UV-vis spectrophotometer (Agilent) in double-beam mode using 1-cm path quartz cuvettes. Corrected fluorescence spectra were collected on a Fluorolog 3 fluorometer (Horiba Jobin-Yvon, Kyoto, Japan) equipped with an R928 PMT (Hamamatsu, Bridgewater Township, NJ, USA). Solutions were prepared such that absorption remained below 0.1 AU to prevent reabsorption and self-quenching.

Chemicals were purchased from following suppliers: Manchester Organics, abcr, VWR, Fluka, Acros, Roth, ChemPure, Merck, Sigma Aldrich, Apollo scientific, and Fluorochem. Solvents were dried before use, if required. Air- and moisture-sensitive reactions were carried out under argon atmosphere. Room temperature (rt) refers to 20–25 °C. The progress of a reaction was monitored by thin layer chromatography using pre-coated TLC sheets POLYGRAM^® SIL G/UV254 purchased from Macherey-Nagel. Detected spots were observed under UV light at λ 254 nm and 365 nm. Flash chromatography was performed with silica gel 40–63 μm from VWR Chemicals.
NMR spectra (¹H, ¹³C, ¹⁹F) were recorded on Mercury 300/Mercury 400 (Varian, Palo Alto, CA, USA) or Fourier 300/Avance DRX 400 Bruker (Billerica, MA, USA) instruments. Signals of solvents were used as internal standards for ¹H-NMR (CDCl₃, δ_H = 7.26; DMSO-d₆, δ_H = 2.50) and ¹³C-NMR (CDCl₃, δ_C = 77.16; DMSO-d₆, δ_C = 39.52). The chemical shifts (δ) are reported in ppm as follows: s, singlet; d, doublet; t, triplet; m, multiplet; and the coupling constants (J) are reported in Hz. Mass spectra were recorded on an ESQUIRE 3000 Plus (ESI, low resolution) and a 7 T APEX II (ESI, high resolution) from Bruker Daltonics.

Manual column
chromatography
was performed with silica gel, 60 Å, 230–400 mesh particle
size from VWR International GmbH (Darmstadt, Germany) or silica gel
(w/0.1% Ca), 60 Å, 230–400 mesh particle size from Sigma-Aldrich
GmbH (Steinheim, Germany). Automated column chromatography was performed
on a Büchi Pure C-815 flash system (Büchi Labortechnik
GmbH, Essen, Germany) using Reveleris C₁₈ reversed phase
cartridges (Büchi Labortechnik GmbH, Essen, Germany).

Dried hooks and stems of US were purchased from Hwalim Natural Drug (Busan, Korea) in September 2010 and authenticated by one of the study authors (Jin Woo Hong). A voucher specimen (accession number PDRLCW-1) has been deposited in the Plant Drug Research Laboratory of Pusan National University (Mirang, Korea). The dried hooks and stems of US (2.0 kg) were ground to a fine powder and then successively extracted at room temperature with n-hexane, ethyl acetate and methanol. Briefly, hexane extracts of US were filtered and evaporated under reduced pressure at 45 °C and then lyophilized, which yielded a white powder of hexane extract (14.54 g). The hexane extract (11.31 g) was subjected to chromatography on a silica gel (40 μm; J.T. Baker, Phillipsburg, NJ, USA) column (70 x 8.0 cm) with a step gradient of 50 % CHCl₂ in hexane, 100 % CHCl₂, 5 and 20 % acetone in CHCl₂ and 5, 25 and 50 % MeOH in CHCl₃ to obtain 62 fractions. Of these extracts, the solid form of fraction 43 (23.1 mg) was dissolved with dimethyl sulfoxide (DMSO) for further experiments.