Silica gel 60 f254 aluminium plates

Two separate Soxhlet extractions of 40.95 g and 41.36 g of plant material were carried out with 500 mL of ethanol each for 24 h. The native extracts were evaporated to dryness on a rotary evaporator (Büchi R-100, Büchi, Flawil, St. Gallen, Switzerland). A total of 2.63 g of dry extract yielded from the first Soxhlet extraction were separated by open-column chromatography on 25 g of silica (40–63 µm, VWR) with hexane, ethyl acetate and methanol in solvent systems of rising polarity, leading to 67 fractions.
TLC analyses of fractions were carried out on Silica Gel 60 F₂₅₄ aluminium plates (Merck, Darmstadt, Hessen, Germany) with mobile phase systems hexane/ethyl acetate/methanol = 75/20/5 (v/v) for apolar fractions and hexane/ethyl acetate/methanol = 60/20/20 (v/v) for polar fractions. The staining reagent consisted of 0.5 mL of 4-anisaldehyde, 10 mL of acetic acid (both Carl Roth), 85 mL of methanol and 5 mL of sulfuric acid (Carl Roth). After spraying, the plates were heated at 105 °C for 5 min.

All solvents were purified and dried using standard methods prior to use. Commercially available reagents were used without further purification. All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringe techniques to transfer solutions. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel 60 F-254 aluminium plates. Melting points were determined on Stuart digital melting-point apparatus/SMP 30 in open capillary tubes and uncorrected. Nuclear magnetic resonance (¹H-NMR, ¹³C-NMR) spectra were recorded using an Avance Bruker 500 MHz, 125 MHz spectrometer in DMSO-d₆. Chemical shifts reported in parts per million (ppm) with TMS as an internal reference, and the coupling constants (J) expressed in hertz (Hz). Splitting patterns are denoted as follows: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublet. HRMS were determined with Agilent QTOF mass spectrometer 6540 series instrument and were performed in the ESI techniques at 70 eV.

All the chemicals and solvents were procured and utilised as such from the suppliers. Wherever necessary, anhydrous solvents were used. Thin layer chromatography (TLC) analysis was done by utilising Merck silica gel 60 F₂₅₄ aluminium plates. Stuart digital melting point apparatus (SMP 30) was used in determining the melting points of the compounds, which are uncorrected. ¹H and ¹³C NMR spectra were recorded using Bruker Avance 500 MHz and 125 MHz respectively using DMSO-d₆ as the solvent. Chemical shift values are recorded in ppm using TMS as the internal standard. HRMS were determined by Agilent QTOF mass spectrometer 6540 series instrument and were performed using ESI techniques at 70 eV.