Development of a fast and efficient separation method of secondary metabolites from Rhodiola rosea roots by high performance countercurrent chromatography

Extracts of the dried roots of Rhodiola rosea L. (rose root) are traditionally used for their adaptogenic properties, more precisely for their anti-depressive, anti-fatigue, anxiolytic, cardioprotective, CNS stimulating, neuroprotective, and nootropic effects [ 1 ],[ 2 ]. Rhodiola rosea has also been shown to increase the lifespan of model organisms such as Drosophila melanogaster [ 3 ]. Avoiding the problem of irreversible adsorption to stationary phases, high performance counter current chromatography (HPCCC) is a highly suited separation method providing higher resolution rates compared to conventional chromatographic techniques [ 4 ]. To gain access to the full spectrum of a wide range of diverse metabolites in a standardized rose root dry extract obtained by 70% ethanol extraction, a fast and efficient HPCCC method was developed. For the newly established normal phase HPCCC method a two-phase solvent system consisting of ethyl acetate, n -butanol and water with a gradient ranging from 27.26/20.69/52.05 to 29.01/19.19/51.80, respectively, was used. Thirteen fractions were obtained based on their TLC pattern. Their dereplication was performed by UPLC-ESI-MS analysis, which led to the identification of phenylethanoids, phenylpropanoids, several phenolic compounds and their glycosides in enrichments ranging from ~50 to 70% within one fractionation step out of the subjected crude extract. The herein established HPCCC-based fractionation method enabled the preparative enrichment of major and minor constituents in one separation step, which was used for efficient further orthogonal chromatographic isolation of eight rose root metabolites for the determination of their not fully captured bioactivity spectrum.