Puriflash 450

The liquid extract was first purified by flash column chromatography in hydrophilic interaction (HILIC) mode with the following parameters. A Puriflash 450 (Interchim) system with a Silica HP-50-F0012 (14 g, 2 cm × 8 cm, 50 μm) flash cartridge (flow rate = 15 mL/min) was used. The mobile phases (gradient) were acetonitrile (solvent A), and water (solvent B). Time programs were 0–3 column volume (CV) for 10–20%B, 3–5 CV for 20–30%B, 5–7 CV for 30–40%B, 7–10 CV for 40%B. All fractions were collected by an automated fractions collector and analyzed by TLC. The fractions showing an R_f value of 0.36 (8:2 acetonitrile:water, purple spot with p-anisaldehyde staining reagent) according to TLC analysis were combined and the solvents removed by rotary evaporation at 45 °C (using acetonitrile as a co-solvent). The residue was dissolved in D₂O and then analyzed by ¹H NMR (256 scans; JEOL JNM-ECZ500R/S1 operating at 500 MHz for ^1H).

The solvent of the pooled crude extracts was evaporated, followed by lyophilization of the samples. The dried powder (≈250 mg) was mixed with 15 g of Celit^® 545 (Carl Roth, Karlsruhe, Germany) and dried under forced air at atmospheric pressure. Then, the whole sample was applied to a reversed-phase silica gel vacuum flash chromatograph (Interchim, puriFlash^®450, Montluçon, France), using two superimposed Interchim puriFlash^® 32 g silica C18-IR-50C18-F0025 flash columns (particle size: 50 µm). The columns were eluted with a two-solvent gradient (solvent A: H₂O, solvent B: ACN). The starting linear gradient from 10% B to 100% B in 45 min at a flow rate of 15 mL/min was followed by an isocratic gradient of B at 100% B for a further 20 min. The UV 254 nm and UV scan 200–400 nm modes were used for the detection and final separation of 11 main peak fractions (F1-F11, Table 1), which were consequently sampled (100 µL aliquots) and diluted at a ratio of 1:50 for LC-MS/MS analysis. The target compound, HT-2-glucoside, was found in fraction F4 by eluting at 20–24 min (61 mL with yield ≈ 500 µg of HT-2-glucoside, with traces of HT-2 toxin and T-2 toxin, 5.8 µg and 0.6 µg, respectively (Table 1)). The dried fraction F4 was then investigated by nuclear magnetic resonance spectroscopy (NMR) for the presence of alpha- or beta-configurated HT-2-glucoside.

The fraction with the highest anti-allergic activity (F3: EtOH/EtOAc) was further purified to obtain major chemical components for structural identification and activity validation. One gram of the F3: EtOH/EtOAc fraction was purified by using medium-pressure liquid chromatography (MPLC; PuriFlash 450, Interchim, France) over reversed phase silica gel (PF-15C18AQ-F0080) and eluted with 3%-97% MeOH/H₂O. The resulting fractions were examined by thin layer chromatography (TLC). The fractions containing identical compounds were combined and concentrated under vacuum. The NMR spectra of the isolated compounds were recorded on a Bruker Avance NMR spectrometer operating at 300 MHz for ¹H and 75 MHz for ¹³C. High-resolution mass spectra were obtained using ESI on an Orbitrap Fusion^™ Tribrid^™ mass spectrometer (Thermo, Massachusetts, USA).

Optical rotation values were determined on a JASCO P-1010 polarimeter (Jasco, Tokyo, Japan). UV spectra were recorded on a PerkinElmer Lambda 365 UV-Vis spectrophotometer (PerkinElmer, Hopkinton, MA, USA). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data were measured on a Waters ACQUITY UPLC H-Class Q-TOF LC-MS spectrometer (Waters, Milford, MA, USA). High-performance liquid chromatography (HPLC) analysis was carried out on an ACQUITY UPLC H-Class System (quaternary solvent manager, sample manager, PDA detector, and ELS detector) using a YMC ODS (4.6 × 250 mm, 5 µm, 1 mL/min) column. MPLC was performed on a PuriFlash450 (Interchim, Los Angeles, CA, USA) with a Flash C18 cartridge (50 µm, 40 g, YMC, Kyoto, Japan). Semipreparative HPLC was performed on a Waters 2535 Quaternary gradient module with a FlexInject, 2489 UV–VIS detector and Fraction Collector Ⅲ (Waters, Milford, MA, USA). The NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer using tetramethylsilane as the internal standard (Bruker, Ettlingen, Germany). Thin-layer chromatography (TLC) analyses were performed on glass precoated with silica gel GF254 glass plates. All reagents for the analysis were purchased from Xilong Scientific Co., Ltd. (Guangdong, China).