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Oxytropis falcata Bunge, known as "the King of Herbs" in Tibetan medicine, is used for treatment of hyperpyrexia, pain, wounds, inflammation and anthrax. However, it is difficult to isolate compound with high-purity from O. falcata because of its complexity. In this work, an efficient method was successfully established for the separation of 3-hydroxy-3-methylglutaryl (HMG) flavonoid glycosides from O. falcata by 2D preparative chromatography (2D-HPLC). An ODS C18 preparative column was used for the first-dimension preparation, and the