Lc 6a system

Rv-PEM01-99 extract was analyzed by HPLC using Agilent 1100 series HPLC system (Agilent Technologies Japan Ltd., Tokyo, Japan) equipped with a DAD G1315B photodiode array detector and TSKgel ODS-80Ts (4.6 i.d.× 250 mm) HPLC column (Tosoh, Tokyo, Japan). The mobile phase consisted of a linear gradient of 10 mM phosphoric acid–acetonitrile (95:5→5:95, 60 min), and flow rate was set at 0.8 mL/min (21 (link)). Measurement of urushiols was performed using the method of Du et al. (22 (link)) with some modification. The urushiols were measured using Develosil ODS-5 (10 i.d. × 250 mm) column (Nomura Chemical, Aichi, Japan) and Shimadzu LC6A system (Kyoto, Japan) equipped with an SPD-6AV UV-visible detector. The mobile phase consisted of 90% acetonitrile, and the flow rate was 1 mL/min with UV detection at 272 nm.

A rotary evaporator, R-200 (Büchi, Flawil, Switzerland), a vacuum pump V-700 (Büchi), a vacuum controller V-850 (Büchi), and a heating bath B-491 (Büchi) were used for evaporating solvents from crude extract and subsequent fractions. Various chromatographic techniques were used for separating fractions and isolating compounds, for example, a Sephadex LH-20 (Sigma, St. Louis, MO, USA), 270 × 45 mm glass chromatographic column, silica gel (60 Å pore size, Merck, Germany), and rotating disc chromatography (RDC) (1 mm). Further separations and isolation of compounds were performed by HPLC, consisting of a two-pumps LC-8A unit (Shimadzu, Kyoto, Japan), by using Activon Gold pack normal phase (NP)-semi-preparative (25 × 1 cm, silica) HPLC columns on a Shimadzu LC-6A system and reverse phase (RP) analytical HPLC column (250 × 4.60 mm, 3 µ, C18) on Shimadzu UFLC system with a UV/VIS detector SPD-20A (Shimadzu), a communication bus module CBM-20A (Shimadzu), fraction collector FRC-10A (Shimadzu), software LC Solution (Shimadzu). For identifying the structures of compounds, NMR data (both one- and two- dimensional spectra) were obtained on a Brüker Avance III 500 MHz spectrometer.

Optical rotations were measured on a JASCO P-1020 automatic digital polarimeter at room temperature. IR spectra were recorded on a Bruker Tensor 27 spectrometer using KBr pellets. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan). High-resolution electrospray ionization mass spectrometry (HRESIMS) was obtained on an Agilent 6529B Q-TOF mass instrument using electrospray ionization. The 1D and 2D nuclear magnetic resonance (NMR) spectra were obtained on Bruker AVANCE III 500 MHz or Bruker AVIIIHD 600 MHz spectrometers in CDCl₃ with TMS as an internal standard. Analytical HPLC was conducted on an Agilent 1260 infinity system equipped with a DAD-UV detector. Preparative HPLC was carried out using a Shimadzu LC-6A system (Shimadzu, Tokyo, Japan) equipped with a Shim-pack RP-C₁₈ column (20 × 200 mm, i.d. 10 μm, Shimadzu, Tokyo, Japan) with a flow rate of 10.0 mL/min, detected by a binary channel UV detector. Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., China), MCI (Mitsubishi, Japan), and RP-C₁₈ silica (40–63 μm, FuJi, Japan) were used for column chromatography.