4.1. Separation of Oleanolic Acid Glucuronides

Air-dried C. officinalis flowers were extracted with boiling methanol (three times for 30 min). After adding an equal volume of water to the combined extracts, the methanol was removed by distillation in a vacuum rotary evaporator, and the aqueous residue was extracted with n-butanol; the mixture was evaporated to dryness, re-dissolved in methanol and precipitated with cold ethyl acetate. The precipitate was centrifugated at 6000 rpm for 10 min. The pellet was resuspended with the use of Potter homogenizer and centrifuged again. Glucuronides of oleanolic acid GlcUAOA were purified on a silica gel column eluted with a mixture of chloroform/methanol (95:5, 90:10) and chloroform/methanol/water (30:10:1). The fractions containing the oleanolic acid glucuronides were combined. The purity of GlcUAOA (90 %) was verified by TLC and HPLC [26]. The powder was dissolved in 96% ethanol before use.