The combined extract was concentrated in vacuo to about 100 mL and applied to an Amberchrom CG 300C open column and manufactured up in 10% methanol. The column was eluted with a methanol gradient from 20% to 100% in 20% increments and fractions collected, monitored by thin layer chromatography, and checked by liquid chromatography MS photodiode array detection for composition. Fractions obtained with 60% methanol were enriched in gypsogenic acid saponins for instance vaccaroside B , while fractions obtained with 100% methanol were enriched within the gypsogenin saponin, segetoside H . The appropriate fractions were combined and evaporated to dryness, affording segetoside H enriched and vaccaroside B enriched components. Gypsogenin The segetoside H enriched material from over was dissolved in one.5 M HCl and EtOAc and heated to 90 C for 27 h. The reaction was cooled to ambient temperature and diluted with brine . The pH was adjusted to around 5 with 1 M NaOH and citric acid, as well as the mixture was extracted with EtOAc .
Wortmannin The mixed organic extract was washed with brine , dried , and concentrated in vacuo. The residue was chromatographed on silica gel utilizing diethyl ether as eluant to afford gypsogenin being a white reliable, homogeneous by thin layer chromatography and HPLC. GC MS of trimethylsilyl derivative gypsogenin showed .95% purity. 1H NMR : d 9.65 , 5.51 , four.ten , 3.34 , one.38 , 1.thirty , 1.03 , 1.01 , 0.98 , 0.91 . Sapogenin Diacid Mixture Vaccaroside B enriched fractions were combined and evaporated to dryness. The residue was dissolved in 1 M NaOH and heated at 60 C for 5 h below a nitrogen ambiance. The reaction mixture was cooled to ambient temperature plus the pH adjusted to about five with one M citric acid. The mixture was diluted with brine and extracted with EtOAc . The mixed organic extract was washed with brine , dried , and concentrated in vacuo. The residue was chromatographed on silica gel making use of EtOAc as eluent to afford a mixture consisting predominantly of gypsogenic acid at the same time as small quantities of seco gypsogenic acid and 16 hydroxygypsogenic acid .
1H NMR : d five.twelve , three.66 , 2.75 , one.08 , 0.91 , 0.86 , 0.84 , 0.69 . Ultimate Purification of Sapogenins Final purification of every sapogenin was achieved by HPLC fractionation employing an Agilent 1100 series HPLC using a quaternary pump, diode array Veliparib ABT-888 detector monitoring at 209 nm, as well as a fraction collector. A Phenomenex Gemini C18 column was applied with an elution gradient from 22.5% CH3CN, 0.12% CH3COOH to 35% CH3CN, 0.12% CH3COOH at a flow rate of 3 mLmin21 for thirty min. 1 minute fractions were collected as well as the fractions containing detected peaks had been analyzed by GC MS for presence on the sapogenins of interest.