•Phytochemical study of folk medicinal plant D. sinensis Hemsl was performed by HPLC-ESI–MSn.•The MS fragmentation pathways of different types of previous pregnane glycosides from this plant were studied.•The structures of thirty-eight unknown pregnane glycosides, combined with six known pregnane glycosides were deduced according their ion fragmentation.In the search for novel natural products in the plant, particularly those with potential bioactivity, the MS fragmentation behavior of nine pregnane glycosides previously isolated from Dregea sinensis Hemsl was investigated by electrospray ionization-multistage tandem mass spectrometry (ESI–MSn) equipped with an ion trap analyzer. Furthermore, by high-performance liquid chromtography (HPLC) coupled to ESI–MSn, the thirty-eight unknown pregnane glycosides, combined with six known pregnane glycosides were deduced on the basis of the fragmentation patterns observed from the crude EtOAc extract of D. sinensis Hemsl. This study shows that the HPLC-ESI–MSn is an effective method to rapid and accurate online identification of pregnane glycosides from plant extracts.Download high-res image (111KB)Download full-size image

•Phytochemical study of folk medicinal plant Dregea sinensis Hemsl was performed.•Six new polyhydroxy steroidal glycosides were obtained from Dregea sinensis Hemsl.•Their chemical structures were elucidated on the basis of extensive NMR and HR-ESIMS experiments.The phytochemical investigation of the roots of Dregea sinensis Hemsl yielded six new polyhydroxy steroidal glycosides (1–6). The chemical structures have been elucidated on the basis of extensive one- and two-dimensional NMR spectroscopy, as well as by mass spectrometry. The cytotoxic activity of these compounds were evaluated in vitro while compounds 3 and 4 showed weak activity against human leukemia cells (HL-60) with IC50 values of 14.10 μM and 19.16 μM, respectively.