Medium- and High-Pressure Integrated Chromatographic Strategies for the Isolation and Purification of Free Radical Inhibitors from Dracocephalum heterophyllum

Dracocephalum heterophyllum has been reported as a traditional Tibetan medicine with diverse therapeutic benefits for the effective treatment of various diseases. However, only a few reports on its free radical inhibitors are limited due to its complex chemical composition and difficult isolation and purification processes. In this study, five free radical inhibitors are isolated by an integrated chromatographic method from Dracocephalum heterophyllum, followed by an investigation of the in vitro antioxidant activity utilizing 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Medium-pressure liquid chromatography was used for the pretreatment of the crude extract of Dracocephalum heterophyllum and targeted separation and purification of the free radical inhibitors using high-pressure liquid chromatography; the antioxidant peaks are recognized by the separation and purification process in combination with an online HPLC-DPPH system. Five free radical inhibitors with purity higher than 95% were obtained, namely xanthotoxol, 5-hydroxy-8-methoxypsoralen, luteolin, methyl rosmarinate, and ethyl rosmarinate. Finally, DPPH assays are performed, and their IC50 values for isolated compounds are 250.39 +/- 13.32 mu M, 26.91 +/- 1.93 mu M, 66.87 +/- 14.33 mu M, 21.99 +/- 3.17 mu M, and 36.96 +/- 1.72 mu M, respectively. This method is effective in isolating free radical inhibitors from Dracocephalum heterophyllum, and it has the potential to be adopted for the isolation of antioxidants from other plants of medicinal value as well.