Isolation and Characterization of Compounds

Compounds were isolated by means of open column chromatography (CC). Analytical TLC was carried out on precoated silica gel 60F254 plates (Merck, Darmstadt, Germany). The isolation procedures and purity of compounds were checked by thin layer chromatography (TLC), visualized by means of UV light, and sprayed with Ce(SO₄)₂ 2(NH₄)₂SO₄ 2H₂O. All ¹H- and ¹³C-, DEPT and 2D NMR experiments (COSY, HSQC and HMBC) were recorded on a Bruker AVANCE III HD 500 MHz at 500 and 125 MHz, respectively, using CDCl₃ with tetramethylsilane (TMS) as internal standard. Mass spectrometry in positive ion mode FABMS was performed using a JEOL JMX-AX505HA mass spectrometer. Optical rotations were measured on a 241 digital polarimeter at 25 °C (Perkin Elmer, Waltham, MA, USA) equipped with a sodium lamp (589 nm) and a microcell. Indomethacin (indo), dimethyl sulfoxide (DMSO), etoposide, lipopolysaccharide (LPS) from Escherichia coli serotype 055:B5, sodium nitrite (NaNO₂), phosphoric acid (H₃PO₄), N-(1-naphtyl) ethylenediamine dihydrochloride, sulfanilamide and ursolic acid were purchased from Sigma Aldrich (Mexico City, Mexico). ADVANCED DMEM/F12 (Dulbecco’s Modified Eagle Medium/Ham’s F-12), DMEM/F12 (Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12), fetal bovine serum (FBS) and Glutamine (GlutaMax) were from GIBCO (Waltham, MA USA). MycoZap^MT Plus-CL antibiotic was from Lonza (Allendale, NJ, USA). [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was from Promega Co (Fitchburg, WI, USA). IL-6 and TNF-α kit was from PEPROTECH (Mexico City, Mexico). QUANTI-Blue^MT and Zeocin were from InvivoGen (San Diego, CA, USA). Murine macrophage cell line RAW 264.7 (Tib-71^TM) was from ATCC^® (Georgetown, Washington, DC, USA) and RAW-Blue^TM was from InvivoGen. Compounds 5 and 7 were quantified using an HP Agilent Technologies 6890 gas chromatograph equipped with a MSD 5973 quadrupole mass detector (HP Agilent), equipped with a capillary column HP-5MS (length: 30 m; inside diameter: 0.25 mm; film thickness: 0.25 µM). The helium carrier gas was set to the column (1 mL per minute at constant flow). The inlet temperature was set at 250 °C while oven temperature was initially at 40 °C (held for 1 min) and increased to 280 at 10 °C/min. The mass spectrometer was operated in positive electron impact mode with ionization energy of 70 eV. Detection was performed in selective ion-monitoring (SIM) mode and peaks were identified and quantitated using target ions.