A HP20 resin-based unique method was adopted to get an active fraction of the hydroalcoholic extract of <i>G. glabra</i> roots. The fraction showed potent cytotoxicity against cancer cell line and was further subjected to detailed phytochemical investigation to obtain ten biomarkers. The isolated compounds were also tested for the cytotoxicity against the C6 glioma cell line <i>in vitro</i> using MTT assay. Among the isolated compounds, glycyrrhetic acid (<b>1</b>), glabrol (<b>6</b>), and glabridin (<b>9</b>) exhibited significant cytotoxicity. The compounds showed a dose-dependent decrease in cell viability. The active compounds were subjected to molecular docking study against topoisomerase I and topoisomerase II to support the mechanism of antitumor activity.

Keywords: C6 glioma cells; Glycyrrhiza glabra; bioassay guided isolation; cytotoxicity; glycyrrhetic acid.