On-line coupling of dynamic microwave-assisted extraction with high-speed counter-current chromatography for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim.

An on-line method based upon dynamic microwave-assisted extraction (DMAE) coupled with high-speed counter-current chromatography (HSCCC) was developed for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim. The DMAE parameters were optimized by means of the Box–Behnken design. The maximum extraction yield was achieved using 30:1 ml/g of liquid–solid ratio, 10 ml/min of solvent flow rate and 200 W of microwave power. The crude extracts were then separated by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (7:3:5:5, v/v/v/v). 13.0 mg of nevadensin was isolated from 15.0 g original sample by HSCCC with five times sample injection in 12 h, and the isolation yield of nevadensin was 0.87 mg/g. The average purity of nevadensin was higher than 98.0%. The chemical structure of collected fraction was identified by HPLC, ESI-MS and 1H NMR. The results indicated that this on-line method was effective and fast for high-throughput isolation of nevadensin from L. pauciflorus Maxim.