| Summary: | Free radicals, including 1,1-diphenyl-2-picrylhydrazyl, mediate oxidative stress to cause many chronic diseases (including cardiovascular diseases, diabetes and cancer). The extract of traditional Tibetan medicine <i>Lepidium latifolium</i> L. (<i>L. latifolium</i>) was reported to have free radical inhibition ability. Therefore, a system method was established to separate the ethanol extract of <i>L. latifolium</i> to prepare two main antioxidant compounds. First of all, silica gel and a medium-pressure liquid chromatography tower were used for pre-treatment of the ethanol extract of <i>L. latifolium</i> to obtain the main antioxidant active component fraction 4 through online high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl (HPLC-DPPH) assay. Then, fraction 4-1 was obtained by one-dimensional preparation using Megres C18 chromatographic column, and two active compounds with IC<sub>50</sub> values 59.9 and 71.3 μg/mL were obtained by two-dimensional preparation using Click XIon chromatographic column. Through the study of the chemical components and separation methods of <i>L. latifolium</i>, the combination of HPLC-DPPH assay and two-dimensional preparative liquid chromatography was realized, providing a reference for the separation of active compounds from <i>L. latifolium</i>.
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