230 400 mesh silica gel

The structures of compounds were unambiguously assessed by ¹H NMR and ¹³C NMR recorded on a Bruker AC 400 or 100 (Milan, Italy), respectively, spectrometer and analyzed using the TopSpin software package (version 2.1). Chemical shifts were measured using the central peak of the solvent. Column chromatography purifications were performed under “flash” conditions using Merck 230–400 mesh silica gel. TLC was carried out on Merck silica gel 60 F254 plates, which were visualized by exposure to an aqueous solution of ceric ammonium molibdate.

The dichloromethane fraction (m = 19 g; IC₅₀ = 1.63 μg/mL) was subjected to column chromatography on 230–400 mesh silica gel (Merck, Darmstadt, Germany) eluting with a gradient of ethyl acetate in n-hexane. Ninety sub-fractions of ca 250 mL each were collected and combined into 4 major sub-fractions B1—B4 according to their TLC profiles. Sub-fraction B1 (n-hexane/EtOAc (95/5), 8.1 g) was found to contain mainly fatty substances and traces of hexane soluble compounds. Sub-fraction B2 (n-hexane/EtOAc (90/10), 2.1 g) was subjected to repeated silica gel column chromatography using a mixture of n-hexane:ethyl acetate in a gradient mode as eluent to afford the mixture of β-sitosterol (4) and stigmasterol (5) (17.6 mg). Sub-fraction B4 (n-hexane/EtOAc (65/35), 4.5 g) was fractionated on silica gel column chromatography with an isocratic solvent system of n-hexane/ethyl acetate (30/70) to afford a mixture of compounds 2 and 3 which were further purified using a sephadex LH-20 (Sigma-Aldrich, Munich, Germany) column chromatography with a mixture of CH₂Cl₂/MeOH (20/80) as eluent to afford pure betulin (2) (2.0 mg) and betulinic acid (3) (27 mg) [17 (link)–19 ]. Sub-fraction B3 was not further studied due to limited quantity.

The structures of sugar-based aromatic esters (Figure 6) were unambiguously assessed by MS, ¹H NMR, and ¹³C NMR. The ESI-MS spectra were recorded with a Waters Micromass^® ZQ™ (Waters Corporation, Milford, MA, USA) spectrometer in negative or positive mode using a nebulizing nitrogen gas at 400 L/min and a temperature of 250 °C, cone flow of 40 mL/min, capillary of 3.5 kV, and cone voltage of 60 V; only the molecular ions [M − H]⁻, [M + NH₄]⁺, [M + Na]⁺, and [M + HCOO]⁻ were given. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AC 400 or 101 (Bruker, Billerica, MA, USA) spectrometer, respectively, and were evaluated using the TopSpin 2.1 software package; chemical shifts were determined using the central peak of the solvent. Column chromatography purifications were performed under “flash” conditions using Merck 230–400 mesh silica gel (Darmstadt, Germany). Thin layer chromatography was carried out on Merck silica gel 60 F254 plates, which were visualized by exposure to ultraviolet light and to an aqueous solution of ceric ammonium molybdate.

Column chromatography was performed using 230–400 mesh silica gel (Merck, Darmstadt, Germany), 70–230 mesh silica gel (Merck) and sephadex LH-20 (Sigma–Aldrich). Thin-layer chromatography (TLC) was performed on a pre-coated aluminium sheet of silica gel 60 F254 (Merck). The spots of com­pounds were detected using UV lamps at two wavelengths (254 and 365 nm) and then fixed using a 10% sulfuric acid spray reagent, followed by heating to 373 K. The high-resolution mass spectra were recorded in positive mode using a QTOF mass spectrometer (Bruker, Germany) equip­ped with an HESI source. The spectrometer operates in posi­tive mode (mass range 100–1500, with a scan rate of 1.00 Hz), with automatic gain control to provide high accuracy mass measurements within the mass range. NMR spectra were recorded in deuterated chloro­form (CDCl₃) and/or deuterated methanol (MeOD) using a Bruker DRX 500 NMR spec­trom­eter (Bruker, Rheinstetten, Germany); the chemical shifts (δ) are given in ppm relative to tetra­methyl­silane (TMS) (Sigma–Aldrich, Germany) as the inter­nal standard.

Bis(1H-indol-3-yl)methane (compound 5) and all organic solvents that were used in this study were purchased from Sigma (Milan, Italy). Prior to use, acetonitrile was dried with molecular sieves with an effective pore diameter of 4 Å. Column chromatography purifications were performed under ‘‘flash” conditions using Merck 230–400 mesh silica gel. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel plates (silica gel 60 F254), which were visualized by exposure to ultraviolet light and an aqueous solution of cerium ammonium molybdate (CAM). ESI-MS spectra were recorded with a Waters Micromass ZQ spectrometer). EI-MS spectra were recorded with a Shimadzu QP-5000 Mass spectrometer. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AC 400 or 100, respectively, spectrometer and analyzed using the TopSpin software package. Chemical shifts were measured by using the central peak of the solvent.