Preparation and structural verification of APG

Whole Clematis tangutica plants were collected from Huangnan Tibetan Autonomous Prefecture in the southeast of Qinghai Province, China, at an altitude of approximately 3500 m above sea level. A voucher specimen was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, Air Force Medical University, Xi’an, China. Air-dried powders obtained from whole Clematis tangutica plants (2 kg) were extracted with 3 L 70% EtOH under reflux for 2 h, and repeated three times (3 L × 3). Filtrates were combined and evaporated to dryness under vacuum. The residue was suspended in H₂O and then partitioned successively with petroleum ether (4 L × 3) and n-BuOH (4 L × 4). The n-BuOH extract was subjected to column chromatography on silica gel (800 g, 15 × 60 cm), and was eluted with a CHCl₃–MeOH–H₂O gradient (from 20:1:0.1 to 6:3:0.3, lower phase) to give six crude fractions based on thin layer chromatography analysis. The fraction containing APG was concentrated under reduced pressure at 60 °C to yield crude extracts, and then, was further purified on a silica gel column (200 g, 3 × 80 cm) eluted with a CHCl₃–MeOH–H₂O gradient (from 7:3:1 to 6.5:3.5:1, lower phase) to obtain pure APG (content >99.1%). The structure of APG was verified by comparing nuclear magnetic resonance (NMR) data with those reported in the literature (Hideji et al. 1981; Delazar et al. 2005).