Combiflashez prep

All reagents and solvents were
purchased from Sigma-Aldrich Co. LLC (Merck KGaA, Darmstadt, Germany)
unless indicated otherwise. Water was purified by a Millipore purification
system (Merck KGaA). NMR spectra were recorded on a Jeol ECA-500 (Jeol
Ltd., Tokyo, Japan). Solvent mixtures for chromatography are reported
as volume/volume (v/v) ratios. Reverse phase high performance liquid
chromatography (HPLC) purifications were performed on a CombiFlash
EZ Prep (Teledyne Isco, Inc.) with a RediSep Prep C18 column (20 ×
150 mm²) with Phase A/Phase B gradients [Phase A: H₂O with 0.1% formic acid; Phase B: MeOH with 0.1% formic acid]
at a flow rate of 19 mL/min. The HPLC was equipped with a UV–vis
detector and an evaporative light scattering detector (ELSD). High-resolution
mass (HRMS) analyses were performed by electrospray ionization–time
of flight (ESI-TOF) on an Agilent 6230 Accurate-Mass TOF-MS mass spectrometer.

In order to ascertain the structures of the main products, the method presented in the “Ozonation experiments” section was used to prepare a sample, except that the concentration was 100 mg/L and the ozonation was stopped after 30 min. Subsequently, the sample was evaporated in a rotavapor. After evaporation, the sample was dissolved in chloroform, and 200 mg Celite was added. The sample was evaporated and transferred to a RediSep Rf Teledyne ISCO cartridge (5 g). The chromatographic separation was performed using automated flash system (CombiFlash EZ prep, Teledyne ISCO) equipped with a Redi Sep Rf Gold HP silica column (4 g) and using ethyl acetate:petrol ether as the eluent system (gradient program). After separation, the fractions were evaporated and analyzed using ¹H NMR and ¹³C NMR experiments run on a MHz Bruker AVANCE-III NMR-system with a liquid nitrogen-cooled Prodigy BBO probe.

The isolation of active compounds of the O. vulgare ethanolic extract was carried out in two steps. First, fractions with different polarity were isolated through column chromatography. To this end, the ethanolic extract was dried and adsorbed on a silica gel (Acros Organics, silica gel for chromatography, 0.060–0.200 mm, 60 A). The silica slurry was charged onto a column packed with heptane. The eluent polarity was increased in a stepwise manner: heptane (100%), heptane-ethylacetate (50:50%), dichloromethane-methanol (60:40%) and methanol (100%). The fractions showing biological activities were further purified by preparative high pressure liquid chromatography (Prep HPLC Column: VDS optilab VDSpher 100 SIL 5 μm, 20.0 × 30 mm) in a gradient (100% heptane ➔ 100% ethylacetate) coupled with an automated fractionating system (CombiFlash® EZ Prep, Teledyne ISCO).