Development of ultrasound-assisted mixture extraction and online extraction solution concentration coupled with countercurrent chromatography for the preparation of pure phytochemicals from Phellinus vaninii.

Herein, ultrasound-assisted mixture extraction (UAME) and online extraction solution concentration (OESC) were conducted to extract products from crops and plants. These techniques were coupled with parallel countercurrent chromatography (PCCC) and applied for continuous extraction and online isolation of chemical constituents from Phellinus vaninii. The UAME instrument comprises extraction and solution separation chambers. It provides higher extraction efficiency and fewer impurities and is suitable for processing various sample matrices. The OESC device comprises a spray nozzle, concentrating cylinder, and hot-blast air nozzle. The mechanical parameters for UAME and OESC were optimized, and the operation of online UAME and OESC coupled with PCCC was described. Raw plant materials were extracted using a two-phase extractant comprising petroleum-ethyl acetate-ethanol-water (0.5:2.0:0.5:2.0, v/v/v/v). The aqueous and organic phases were then concentrated using the OESC technique. Two CCC runs were conducted for preparatory work. After extraction and online concentration, the concentrate was pumped into the CCC for separation. During PCCC separation, continuous automated extraction and concentration were still conducted. When the first cycle of the UAME/OESC/PCCC was completed, followed by the initiation of the second cycle, and the process was continued. Six target compounds with purities exceeding 97.22% were successfully separated using the CCC solvent systems comprising n-hexane-ethyl acetate-acetonitrile-water (5.5:2.5:5.0:0.4, v/v/v/v) and n-butanol-ethanol-water (4.5:1.3:6.5, v/v/v). Compared with conventional extraction methods, the proposed UAME/OESC/PCCC method has higher efficiency, facilitates high-purity separation of analytes, and offers opportunity for automation and systematic preparation of natural products.