Cosmosil 75c18 prep

The lyophilized blueberry powders were subjected to extraction with a mixed solvent (MeOH:H₂O:acetic acid = 85:15:0.5); then, the extracts were purified using solid-phase extraction (SPE) cartridges with a 60-mL tube (Supelco, Milwaukee, WI, USA) filled with C-18 (Cosmosil 75 C18-PREP; Nacalai Tesque, Kyoto, Japan). The cartridge was equilibrated with 100 mL MeOH and washed with 100 mL 0.2% formic acid in H₂O. The blueberry extract was loaded onto the column and washed with 100 mL 0.2% formic acid in H₂O. Then, the polyphenol-enriched fraction was collected by washing the column with 100 mL 0.2% formic acid in MeOH. Finally, the sample was dried under nitrogen flow.
The dried sample was ready for DPPH assay, enzyme-linked immunosorbent assay, and the expression of miR-21, miR-125b, and miR-146a.
For the HPLC/MS² analyses, the dried sample was reconstituted with 0.2 mg extract/mL in MeOH.

Cosmosil 75C18-PREP (Nacalai Tesque Inc., 75 µm) was used for ODS flash chromatography. NMR spectra were obtained by a Bruker AVANCE 500 spectrometer at 500 MHz for ¹H. Residual solvent peaks at δ_H/δ_C 7.27/77.0 ppm in CDCl₃ were used as chemical shift reference signals. HRMS–ESI–TOF and LC–MS analyses were carried out by an Agilent 1200 HPLC-DAD system coupled with a Bruker micrOTOF mass spectrometer. Optical rotation and UV spectra were recorded on a JASCO DIP-3000 polarimeter and a Hitachi U-3210 spectrophotometer, respectively.

The roots (1.5 kg) of barley ‘Shunrei’ seedlings were inoculated with F. culmorum and incubated at 23 °C with a 14-h photoperiod. After 72 h, the root metabolites were extracted in 80% methanol for 24 h, and the obtained extract was evaporated in vacuo. The resulting residue was subjected to ODS column chromatography (Cosmosil 75C18-PREP; Nacalai Tesque, Kyoto, Japan), using 20%, 40%, 50%, 60%, 70%, and 80% methanol. Because 3 was eluted in both the 50% and 60% methanol fractions, the two fractions were combined, evaporated to dryness, and subjected to silica gel column chromatography, using mixtures of acetone and hexane (20%, 30%, 40%, 50%, and 60% acetone; 1.0 L each). Because compound 3 was detected in both the 30 and 40% acetone fractions, the two fractions were concentrated, and the resulting residue (35.8 mg) was dissolved in methanol and then subjected to preparative HPLC. Conditions were as follows: column, Cosmosil 5C₁₈-AR-II 10 mm × 250 mm; solvents, water (A) and acetonitrile (B); elution, 40% B/(A+B); flow rate, 7.0 mL/min, detection, 280 nm; column temperature, 40 °C. Compound 3 was eluted at 31.1 min.
Compound 3 (N-cinnamoyl-(1H-indol-3-yl)methylamine) 2.6 mg, HR MS (positive ESI): m/z 277.1331 [M+H]⁺ (calcd. for C₁₈H₁₇ON₂, m/z 277.1341); UV-Vis (acetonitrile-water containing 0.1% formic acid): λ_max 220 and 280 nm; ¹H- and ¹³C-NMR data are shown in Table 1.

U. lactuca powder (500× g) was extracted twice at 40 °C for 4 h with 70% methanol (2 L) using an extractor (KSNP B1130-240L, Kyungseo, Incheon, Korea). The methanolic extract was concentrated under reduced pressure, followed by adsorption onto Diaion HP-20 resin (Mitsubishi Chemical Co., Tokyo, Japan) with stirring for 3 h. After filling the adsorbed resin into the column, the column was washed with distilled water and sequentially eluted with 2 L of 30%, 70%, and 100% methanol. The 30% methanol fraction was concentrated and sequentially eluted with 0–100% methanol using ODS flash column chromatography (Cosmosil 75C18-prep, Nacalai Tesque, Tokyo, Japan). Methanol (30%) fraction was concentrated and sequentially eluted with 50% methanol using Sephadex LH-20 column chromatography (GE Healthcare Life Sciences, Uppsala, Sweden). The three fractions were eluted using Sephadex LH-20 column chromatography.