One-step simultaneous purification of three water-soluble constituents in crude extracts from Danshen by adsorption chromatography on oligo-β-cyclodextrin substituted agarose gel media

A new adsorption chromatographic method for the simultaneous purification of Danshensu, Protocatechuic aldehyde and Salvianolic acid B (Lithospermic acid B) from a crude extract from radix of Danshen (Salviae miltiorrhiza) has been developed using oligo-β-cyclodextrin immobilized to Sepharose HP agarose gel media. Sample loads of up to 2.3 mg crude extracts/ml gel gave satisfactory results. The target components were eluted using step-wise isocratic elution with increasing concentrations of acetic acid. The separated fractions were identified by LC–MS. The recoveries of Danshensu, Protocatechuic aldehyde and Salvianolic acid B were 88.3%, 90.2% and 73.6% with purities of 99%, 99%, and 98%, respectively. Isocratic elution in 6% acetic acid gave baseline separation of Danshensu and Protocatechuic aldehyde, whereas 40% acetic acid was required for purification of Salvianolic acid B. Full separation efficiency could be restored by cleaning the column with 0.5 M NaOH followed by distilled water and 30% acetic acid.