Actus triart c18

Acetonitrile (MeCN), formic acid (HCOOH), and water (H₂O) were LC/MS-grade and obtained from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The ¹H and ¹³C NMR spectra were measured on a JEOL ECA-500 spectrometer (Tokyo, Japan) with the measuring deuterated solvent as the internal reference, and the chemical shifts were expressed in ppm units. Column chromatography was performed on Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan) and ODS (100–200 mesh, Chromatorex DM1020T ODS, Fuji Silysia Chemical Co., Ltd., Aichi, Japan). Semi-preparative HPLC was performed on a Waters 600E HPLC pump (Waters Corp., Milford, MA, USA) with a Shimadzu SPD-10A (Kyoto, Japan) intelligent ultraviolet/visible (UV/vis) detector, a Shodex RI-72 differential refractometer detector (Shoko Science Co., Ltd., Tokyo, Japan), and an RP-C18 silica gel column (YMC Actus Triart C18, 150 × 20 mm I.D., Kyoto, Japan).

The ethanolic CS crude extract was prepared by dissolving it in 10% methanol to a final concentration of 10 mg/mL before passing through 0.45 and 0.22 µm filters. The filtrated CS crude extract was fractionated using a YMC LC-Forte/R recycling preparative HPLC (YMC, Kyoto, Japan) with a photodiode array detector (YMC-YUV-3400). It was operated at 18 mL/min using 1% acetic acid in distilled water (solvent A) and 1% acetic acid in methanol (solvent B). The linear gradient used was as follows: 0 min, 5% B; 5 min, 20% B; 10 min, 30% B; 15 min, 35% B; 20 min, 45% B; 25 min, 75% B; 30 min, 95% B; 35 min, 95% B; and 45 min, 5% B. The preparative HPLC column was a YMC-Actus Triart C18 (250 mm × 20 mm, 5 μm, pore size 12 nm) (YMC, Kyoto, Japan). The detection wavelength was set to 280 and 330 nm. Each injection volume was equivalent to 100 mg/load. Each semi-purified CS extract fraction was collected and evaporated using a rotary evaporator before drying the sample with nitrogen blown through needles onto the samples in tubes. Next, the samples were weighed and kept at −20 °C until they were used for the antiviral bioassay and phytocomposition determination by LC-QTOF-MS.