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

A hyphenated strategy by off-line coupling 1, 1'-diphenyl-2-picrylhydrazyl -HPLC, high-speed counter-current chromatography and preparative HPLC was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the target-guidance of 1, 1'-diphenyl-2-picrylhydrazyl-HPLC experiment, twelve compounds were identified as potential antioxidants and readily isolated by high-speed counter-current chromatography and preparative HPLC. 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. This article is protected by copyright. All rights reserved.