| Summary: | The health benefits of <i>Dracocephalum heterophyllum</i> are widely reported in traditional Tibetan medicines, but the reported chemical composition is limited, probably due to difficulties in separating and purifying compounds. In this study, antioxidative phenylethanoids were isolated from an extract of <i>Dracocephalum heterophyllum</i> using medium- and high-pressure liquid chromatography, coupled with on-line HPLC–1,1-diphenyl-2-picrylhydrazyl recognition. Firstly, crude samples (1.3 kg) of <i>Dracocephalum heterophyllum</i> were pretreated via silica gel medium-pressure liquid chromatography to yield 994.0 g of Fr2, of which 10.8 g was then pretreated via MCI GEL<sup>®</sup>CHP20P medium-pressure liquid chromatography. The resulting Fr23 and Fr25 were further separated and purified using high-pressure liquid chromatography, and yielded 8.08 mg of Fr2391, 9.76 mg of Fr2551, 16.09 mg of Fr2581, and 8.75 mg of Fr2582. Furthermore, analysis of the purity and structures of the phenylethanoids suggested that Fr2391, Fr2551, Fr2581, and Fr2582 corresponded to decaffeoylverbascoside, rosmarinic acid, acteoside, and 2′-O-acetylplantamajoside, respectively, with all being over 95% pure. Finally, the antioxidant potential of the compounds was explored based on their ability to scavenge 1,1-diphenyl-2-picrylhydrazine, as well as through molecular docking of proteins related to antioxidant pathways. Altogether, our findings revealed that the proposed method is promising for separating pure antioxidative phenylethanoids from other natural compounds.
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