Hexane ethyl acetate

The crude extract of S. buxifolia bark was obtained at optimum extracting conditions (solvent, temperature, and time), determined in previous experiments (2.3). The crude bark extract (CE) was then dissolved in steriled distilled water in a separating funnel, equilibrated, and successively extracted with petroleum ether, hexane: ethyl acetate (85:15; v/v) (Sigma‐Aldrich, Singapore) to obtain fractions of various polarities. The fractionation protocol is shown in Figure 1; fractionation was performed in triplicate. The organic fractions (petroleum ether or hexane: ethyl acetate) were concentrated by a rotary evaporator at 40°C, followed by drying at room temperature.

The NMR spectra were acquired utilizing a Varian Gemini 2000 instrument (1H 300 MHz, 13C 75.45 MHz) or a Bruker Avance 300 (1H 300 MHz, 13C 75.45 MHz) spectrometer. Chemical shifts (δ) in ppm are given relative to Me4Si coupling constants (J) in Hz. EI mass spectra were recorded with a Micromass GC-T. Melting points were recorded with an electrothermal melting point apparatus and were uncorrected. Analytical TLC was carried out on Merck 5785 Kieselgel 60F254 fluorescent plates. Flash chromatography uses silica gel of 35–70 µm particle size. Dimethylformamide was distilled from magnesium sulfate. Phosphorus pentoxide, SnCl4, and 2-Fluoro-4-nitrobenzoic acid were purchased from Sigma-Aldrich (Darmstadt, Germany). Dichloromethane, diethyl ether, and hexane-ethyl acetate were procured from Sigma-Aldrich (Darmstadt, Germany).