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 .