Supercritical fluid extraction of cordycepin and adenosine from Cordyceps kyushuensis and purification by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) was used to extract cordycepin and adenosine from Cordyceps kyushuensis. An orthogonal array design (OAD) test, L9(3)4, including pressure, temperature, the mount of modifier and the flow rate of CO2 was performed to get the optimal conditions. The process was then scaled up by 30 times with a preparative SFE system under 40 MPa, 40 °C, the mount of modifier (0.04 ml min−1) and a flow rate of CO2 (2.0 l min−1). The preparative SFE extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a selected two-phase solvent system composed of ethyl acetate–n-butyl alcohol–water at an optimized volume ratio of 1:4:5, and the collected fractions were analyzed by high-performance liquid chromatography (HPLC). Two nucleosides were obtained and yielded 8.92 mg of cordycepin, 5.94 mg of adenosine with purities of 98.5 and 99.2% from 400 mg SFE crude extraction, in one-step separation, respectively.