Preparative isolation and purification of 12 main antioxidants from the roots of Polygonum multiflorum Thunb. using high-speed countercurrent chromatography and preparative HPLC guided by 1,1'-diphenyl-2-picrylhydrazyl-HPLC.

A hyphenated strategy by off-line coupling of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography, high-speed countercurrent chromatography, and preparative high-performance liquid chromatography was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the targeted guidance of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography experiment, 12 compounds were identified as potential antioxidants and readily isolated by high-speed counter-current chromatography and preparative high-performance liquid chromatography. Ultraviolet spectroscopy, mass spectrometry, and 1 H NMR spectroscopy were employed to identify their structures, which were assigned as gallic acid (1, 6.2 mg, 98.28%), catechin (2, 8.8 mg, 90.69%), epicatechin (3, 4.1 mg, 96.71%), polydatin (4, 5.3 mg, 94.91%), 2,3,5,4'-tetrahydroxy stilbene-2-Ο-β-D-glucoside (5, 20.2 mg, 95.23%), piceatannol (6, 5.3 mg, 96.85%), rutin (7, 5.4 mg, 97.92%), resveratrol (8, 5.2 mg, 96.94%), isorhapontigenin (9, 11.4 mg, 94.81%), hyperoside (10, 9.7 mg, 98.52%), rhein (11, 4.9 mg, 97.46%), and emodin (12, 8.2 mg, 95.74%). Notably, compounds 6 and 9 were isolated from Polygonum multiflorum for the first time. In addition, antioxidant activity of compounds 1-12 were evaluated, and compounds 1-8 and 10 exhibited stronger antioxidant activity than ascorbic acid (positive control). These results indicated that the proposed method is a highly efficient strategy to screen and isolate antioxidants from complex natural products.