This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.IsolationThe Ambovex solution (100 mL) was treated with 8.5 mL conc. HCl and refluxed for 2 h. After cooling, the mixture was extracted with 4 50-mL portions of chloroform. The combined extracts were evaporated to dryness, and the residue was dissolved in 10 mL acetonitrile. After filtration through a 0.45 um PTFE membrane, 100-uL aliquots were injected onto HPLC. A hederagenin standard (ChromaDex) solution (0.1 g/L in acetonitrile) was prepared and injected separately onto the HPLC column.HPLCHPLC was performed on an Agilent 1100 LC system using a Phenomenex 4.6 x 250 mm 5 micron ODS column. Gradient elution was carried out with a 2-solvent system (Eluent A: 5 % acetonitrile containing 0.1 % formic acid; Eluent B: 95 % acetonitrile containing 0.1 % formic acid) using the following gradient: 50 % B (10 min) to (2.5 %/min) 100 % B (5 min). Flow rate was 1.0 mL/min, detection was by variable wavelength detector at 205 nm. The injection volume was 100 uL. The hederagenin fractions (tR=18.25 min) from 8 runs were collected, combined, and concentrated to dryness by a gentle stream of air.NMR SpectroscopyThe samples were dissolved in 0.7 mL pyridine-d5. 1- D proton NMR spectra were acquired on a Varian Inova-500 MHz spectrometer at 298 K (25 C). Proton chemical shifts were measured relative to the most upfield pyridine-d5 singlet (delta=7.22 ppm).GC/MS AnalysisBoth hederagenin standard and hederagenin from Ambovex were per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80 C (18 h). The solvents were evaporated, and the residue was extracted with hexane. After filtration and concentration, 1 uL was injected into the GC/MS (HP 6890N GC interfaced to a 5973 MSD, using a Supelco DB-1 fused silica capillary column (30m x 0.25 mm ID) and ammonia as the ionizing gas). Temperature profile: 80 C (2 min) to (8 /min) 290 C (30 min).

National Institute of Health (NIH)
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Biotechnology Resource Grants (P41)
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University of Georgia
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