Countercurrent separation (CCS) is an advanced liquid–liquid chromatographic separation technique that is performed by instruments such as high-speed countercurrent chromatography (HSCCC) and centrifugal partition chromatography (CPC). It has been widely used for the separation of high abundance compounds. However, the identification of low abundance compounds, such as mammalian steroid hormones, from natural sources is still a challenging task.

Researchers from Xishuangbanna Tropical Botanical Garden (XTBG) prepared a mixture of human steroid hormone reference compounds for the development of a countercurrent separation (CCS) enrichment strategy.

They employed the thin-layer chromatography (TLC)-based generally useful estimate of solvent systems (GUESS) method along with partitioning experiments to develop a process for the enrichment of these low abundance compounds with CCS.

The liquid/liquid partition coefficients of progesterone were selectively used to target this compound in a plant extract. Once a solvent system had been selected, the search for mammalian steroid hormones in plants was initiated by searching for progesterone and other steroid hormones in Vitex negundo extract.

In addition to demonstrating the first use of HSCCC to identify progesterone in plant material, this methodology offers a proof of concept to identify other steroid compounds in plant material. This method provides a CCS-driven strategy to mine plant sources for low abundance compounds.

Indeed, the HSCCC application led to the identification of estriol in V. negundo for the first time.