Mgso4

S. chartreusis NRRL 3882 was cultivated in chemically defined medium [21 mM NaCl (Carl Roth, Karlsruhe, Germany), 15 mM (NH₄)₂SO₄ (Sigma-Aldrich, St. Louis, MO, USA), 8 mM MgSO₄ (VWR International, Darmstadt, Germany), 27 mM KCl (Honeywell International, Morristown, NJ, USA), 50 mM Tris (Sigma-Aldrich, St. Louis, MO, USA), 0.6 mM KH₂PO₄ (VWR International, Darmstadt, Germany), 2 mM CaCl₂ (Avantor, Radnor, PA, USA), 0.01 mM MnSO₄ (Honeywell International, Morristown, NJ, USA), 11 mM D-glucose (Thermo Fisher Scientific, Waltham, MA, USA), pH 7.5]. The pH was adjusted to 7.5 with HCl. Amino acids were supplemented at a concentration of 0.78 mM. Glutamic acid was added as mono sodium salt. Cultures were incubated for two weeks at 30 °C and 180 rpm in an Innova orbital shaker. If not indicated otherwise, all experiments were conducted three times independently.

KCl and NaCl were obtained from Sigma Aldrich (Hoeilaart, Belgium), MgSO₄ from VWR (Leuven, Belgium), and the phosphate-buffered solution (PBS) was purchased from Life Technologies (Gent, Belgium).
Eight FAs (analytical grade) were purchased either from Sigma Aldrich (Hoeilaart, Belgium) or, for Aspartame, from Alfa Aesar (Lancashire, UK), as listed in Table 1. Stock solutions and serial dilutions for each chemical were appropriately prepared in E3 (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl₂, and 0.16 mM MgSO₄) for all embryonic tests.

A batch of oil resin extract (157 g) from commercial tamanu oil (provided by “Laboratoire de Cosmétologie du Pacifique Sud” manufacture) from French Polynesia was first partitioned with EtOH (provided by Fisher chemical) and an aqueous alkaline solution of Na₂CO₃ (from VWR chemicals) (10%, v/v). Its organic fraction was washed with distilled water and then dried with MgSO₄ (provided by VWR chemicals) to give a neutral fraction (53 g) after solvent evaporation. This fraction was submitted to flash liquid chromatography on an open column with a silica gel (240–300 mesh) using a stepwise gradient from cyclohexane (provided by VWR chemicals) to EtOAc (provided by VWR chemicals), yielding 12 fractions. Fractions having similar R_f values on silica gel TLC (cyclohexane-acetone, both provided by VWR chemicals, 60:40, v/v) were combined. Fractions 7, 9 and 11 were submitted to repeated preparative HPLC using a Varian Dynamax Si column (250 × 21.4 mm id, five μm with cyclohexane-EtOAc (10:90) in isocratic eluent conditions. This chromatographic purification network led to the isolation of new compounds tamanolides E1 and E2 as a mixture (two mg) besides standard known compounds namely, calophyllolide, inophyllums (C, D, E, P), calanolides (Gut 70 and A, 12-oxo-calanolide) and tamanolides (D, P).