Waters 600 pump system

All solvents and reagents were of analytical grade and purchased from commercial sources. The UV spectra and optical rotations were recorded using a UV-1900i UV-Vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan) and a HORIBA SEPA-300 high-sensitive polarimeter (HORIBA, Kyoto, Japan). The IR spectra were recorded using an FT-720 spectrometer equipped with a DuraSampl IR II ATR instrument (HORIBA). MPLC was performed using the Teledyne ISCO CombiFlash Companion (Teledyne ISCO, Lincoln, NE, USA). Preparative HPLC was conducted using a Waters 600 pump system (Waters, Milford, MA, USA) equipped with a Cosmosil MS-II C18 column (5 μm, 10 mm i.d. × 250 mm; Nacalai, Kyoto, Japan). DAD-LC-MS analyses were conducted using a Waters UPLC H-class system with a Waters UPLC BEH C18-column (1.7 μm, 2.1 mm i.d. × 50 mm; Waters) connected to an AB Sciex API3200 MS/MS system (AB Sciex, Framingham, MA, USA) equipped with an electrospray ionization (ESI) probe. HR–ESI–TOFMS was measured using a Vion IMS QTOF Mass Spectrometer (Waters). NMR spectra were recorded using a JEOL ECA500 FT-NMR spectrometer (JEOL, Tokyo, Japan) at 500 MHz for ¹H NMR and 125 MHz for ¹³C NMR. The chemical shifts were referenced to the corresponding solvent signals (δ_H 7.24 and δ_C 77.23 in CDCl₃).

Quantification of the possible active constituents in ARE, aloe-emodin, chrysophanol, emodin, physcion, rhein, and psoralen, was provided by Herbiotek Co., Ltd., New Taipei City, Taiwan. Twenty milliliter of ARE was filtered through 0.22-μm membranes before analyzed. The Waters HPLC system (Milford, Massachusetts, USA) included Waters 600 pump system, Waters 2996 photodiode array detector, Waters 717 plus autosampler, and Sugai U-620 column oven (Wakayama City, Japan). Cosmosil 5C18-MS-II reversed phase column (5 μm, 4.6 mm × 250 mm, Nacalai Tesque, Japan) equipped with LiChrospher RP-18 end-capped guard column (5 μm, 4.0 mm × 10 mm, Merck, Germany) was used as the stationary phase. The gradient elution was consisted of 10 mM phosphate buffer, acetonitrile, and water. The flow rate was 1 mL/min, and the column temperature was maintained at 35 °C. UV 246 nm was used for detection of psoralen (a furanocoumarin isomer), with a retention time of 14.5 min. UV 270 nm was used for detection of aloe-emodin, rhein, emodin, chrysophanol, and physcion, with a retention time of 89.1, 94.1, 107.4, 114.9, and 118.1 min, respectively. Data are presented as the concentration (μg/mL) of each constituent in ARE.