An efficient high-speed countercurrent chromatography method for preparative isolation of highly potent anti-cancer compound antroquinonol from Antrodia camphorata after experimental design optimized extraction.

To avoid irreversible stationary phase adsorption and tedious and time-consuming separation steps, high-speed countercurrent chromatography was employed for the preparative separation of anti-tumor compound antroquinonol from solid fermentation culture of Antrodia camphorata for the first time. A Box-Behnken experimental design, based on three parameters including liquid-to-solid ratio, extraction time, and extraction temperature, was applied to optimize the ultrasonic extraction procedure. The optimal extraction condition was set as follows: liquid-to-solid ratio: 49.57:1; extraction time: 55.76 min; extraction temperature was arranged as 44.21°C. Meanwhile, an optimized solvent system containing petroleum ether, ethyl acetate, methanol, and water (4:1:4:1, v/v/v/v) was selected for the preparative separation of antroquinonol at a flow rate of 2.0 mL/min. The yield of isolated antroquinonol was determined to be 6.0 mg from 0.67 g of ethyl acetate extracts. The isolated antroquinonol was elucidated by ultra-high-performance liquid chromatography-tandem mass spectrometry, and NMR spectroscopy, and by comparison with literature data. The purity of isolated antroquinonol was determined to be 97.12%. This study confirmed that high-speed countercurrent chromatography was powerful and cost-effective for the preparative separation of the high-potently anti-tumor compound antroquinonol from solid fermentation culture of A. camphorata.