Identification Of Metabolites Of Dysosma Versipellis （Hance） By Combination Of Chromatography Isolation And ¹³C NMR Pattern Recognition

Dysosma versipellis (Hance) is a famous traditional Chinese medicine for the treatment of snakebite, weakness, condyloma accuminata, lymphadenopathy, and tumors for thousands of years. In this work, we estalished and developed three methods to quickly and effectively separate and identify the components from Dysosma versipellis (Hance). the resluts were as follows:Firstly, we have successfully developed a novel strategy to characterize and identify the new anti-cancer flavone dimers and podophyllotoxins from Dysosma versipellis (Hance) by combination of reversed-phase liquid chromatography (RPLC) and 13C NMR pattern recognition. It only required a simple LC fractionation and following NMR map analyses. As a result,7 known compounds including 5 podophyllotoxins and 2 flavones were quickly identified by comparison between the clustered 13C NMR shifts and reported NMR data. In addition, according to the information from the 13C NMR map and HPLC analyses, six new flavone dimers podoverine D-I along with another two known compounds were successfully isolated and identified. All recognized compounds expressed potential cytotoxity to several cancer cells in vitro. RPLC fractionation-13C NMR pattern recognition strategy to rapidly separate and identify major bioactive natural products from non-targeted fraternization to targeted purification was reported firstly in our works. This is an important advancement for natural product dereplication and searching for new ones from complex Traditional Chinese Medicines or other natural resources. An integrate strategy for systematic extraction and isolation of podpophyllotoxins and flavonoids from Dysosma versipellis (Hance) was established, which involved liquid-liquid extraction,13C NMR-based pattern recognition and following targeted conical counter-current chromatography separation. Firstly, the hexane/ethyl acetate/methanol/water (HEMWat) solvent systems were used for the selective extraction of different kinds of components from the roots of Dysosma versipellis (Hance), and the extractions were pre-characterized by 13C NMR in the following step. According to the obtained results above, the extracts were then subjected to the following separation or further extraction. At last the extracts which from the system of hexane/ethyl acetate/methanol/water (HEMWat,2:8:2:8, v/v/v/v) were selected for the following separations. The extract of the lower phase, which was rich in saccharides and glycosides, was directly subjected to the counter-current chromatography (CCC). Meanwhile the extract of the upper phase, which was rich in podophyllotoxins, was extracted again to remove the inclusive grease by hexane/ethyl acetate/methanol/water (HEMWat,9:1:9:1, v/v/v/v) solvent system, following by the CCC separation of the remaining components. To purify the hit compounds, an improved novel conical CCC method was introduced and 17 compounds were obtained at last. Interesting, three flavone dimers including a new one were purified. The integrate strategy for systematic extraction and isolation by combination of liquid-liquid extraction,13C NMR-based pattern recognition and following targeted conical counter-current chromatography separation was proved an efficient method for the selective extraction and targeted separation of natural products.Four podophyllotoxin-like lignans including 4’-demethylpodophyllotoxin, a-peltatin, podophyllotoxin, β-peltatin as major cytotoxic principles of D. versipellis were successfully isolated and purified by several novel linear and step gradient counter-current chromatography methods using the systems of hexane/ethyl acetate/methanol/water (4:6:3:7 and 4:6:4:6, v/v/v/v). Compared with isocratic elution, linear and step-gradient elution can provide better resolution and save more time for the separation of podophyllotoxin and its congeners. Their cytotoxicities were further evaluated and their structures were validated by high-resolution electrospray TOF MS and nuclear magnetic resonance spectra. All components showed potent anticancer activity against human hepatoma cells HepG2. And it proved an effective method to separate the podophyllotoxins from D. versipellis by CCC.