Silica gel 60

IR spectra were recorded as ATR-FTIR spectra using a Perkin-Elmer UART TWO FT-IR-spectrometer. The UV/VIS spectra were recorded in high-purity solvents (UVASOl^®) using the JASCO double-beam photometer (V-630). NMR spectra were recorded in MeOD using a Bruker NEO-500 instrument operating at 500 and 125 MHz for ¹H and ¹³C{¹H} NMR, respectively. Spectra referencing was accomplished using the CD₂HOD solvent peak for ¹H and the CD₃OD solvent peak for ¹³C NMR spectra (δ = 3.31 and 49.0 for ¹H and ¹³C{¹H} signals, respectively). Multiplicities in ¹H NMR spectra were described using the common descriptors s (singlet), d (doublet), t (triplet), or m (multiplet). High-resolution mass spectra were obtained by EI-TOF (70 eV) using Waters Micromass instruments. Reversed-phase semi-preparative HPLC was performed on Shimadzu LC-20AP pump equipped with DGU-20A5R degassing unit, a Shimadzu SPD-M20A detector, a Shimadzu SIL-20ACHT auto-sampler and a Phenomenex Gemini C₁₈ column (10 × 250 mm, 10 μm). Data were recorded and analyzed using LabSolutions software. For column chromatography, Silica gel 60 (0.063–0.2 mm, Macherey-Nagel) was used as solid matrix. TLC was carried out on precoated Silica gel 60 plates (0.20 mm). Compounds were visualized under UV light and further by spraying with H₂SO₄–EtOH (1:9, v/v). All solvents used were of analytical grade.

TLC was performed on Kieselgel 60 F254 plates (Merck, Darmstadt, Germany); for column chromatography, Silica gel 60 (0.063–0.200 and 0.04–0.063 mm, Macherey-Nagel, Düren, Germany) was used. Compounds containing UV-absorbing groups were detected with a UV-cabinet Camag (Omicron Research, Hungerford, UK); substances with free or Boc-protected amino groups were visualized by a ninhydrin reagent. BER and its derivatives were detected using Dragendorff’s reagent.

All solvents and reagents used in the phytochemical profile analysis were of analytical grade (ACS) and purchased from Sigma-Aldrich (Saint Louis, MO, USA). Pristimerin and tingenone, isolated from Maytenus chiapensis (Celastraceae) [18 (link)], and 2,3-epoxyjuanislamin, isolated from Calea urticifolia (Asteraceae) [48 (link)], were used as comparison standards after their NMR characterization. Sephadex LH-20 used for column chromatography (CC) was supplied by Pharmacia Biotech. Silica gel 60 (particle sizes 15–40 and 63–200 μm, Macherey-Nagel) and Silica gel 60F₂₅₄ used for CC and analytical and preparative thin layer chromatography, respectively, were purchased from Panreac (Barcelona, Spain). Benznidazole from Sigma-Aldrich (St Louis, MO, USA) and miltefosine from Æterna Zentaris (Charleston, SC, USA) were used as reference drugs for trypanocidal and leishmanicidal activities, respectively.

For sample extraction,
methanol from Merck (MeOH, liquid chromatography–mass spectrometry
(LC-MS) grade, Darmstadt, Germany) and tetrahydrofuran from Sigma-Aldrich
(THF, unstabilized, Schelldorf, Germany) were used. Extraction cells
were filled up with calcined sea sand. A SpeedExtractor E-914 system
(BÜCHI Labortechnik GmbH, Essen, Germany) was used for all
extractions. Every sample was extracted with MeOH at 100 °C and
100 bar as cleanup (discarded), followed by the extraction with THF
at 185 °C and 100 bar, according to Dierkes et al.^{13 (link)} The THF extract was collected in 60 mL vials
each filled with 200 mg of silica gel 60 (70–270 mesh, Machery-Nagel,
Düren, Germany) as the collecting medium. After extraction,
THF was evaporated, and the remaining silica gel was then ground and
homogenized by a mortar.

Dic-Ac (8.5 g) was chromatographed on a column (C1) with silica gel 60 (Macherey-Nagel, Düren, Germany) providing 58 fractions. Fraction 35 (500 mg) of C1 showed a yellowish precipitate soluble in methanol and was purified in a column with Sephadex LH-20 (SIGMA-ALDRICH^®, Saint Louis, MO, USA) and MeOH:CHCl₃ solution (1:1—v:v), yielding a mixture with 3 compounds (Ac-1), (Ac-2) and (Ac-3). The chromatographic process of the Dic-Ac is shown in Scheme 1.