Preparative isolation of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography

• A convenient method for preparing three nucleosides from Cordyceps militaris.
• Three nucleosides are cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine.
• Three nucleosides were isolated by macroporous resin NKA-II.
• Three nucleosides were purified by recycling HSCCC at one time.

In this study, cordycepin, N6-(2-hydroxyethyl)-adenosine (HEA) and adenosine from the fruiting bodies of Cordyceps militaris were separated by using macroporous resin NKA-II adsorption. The parameters of static adsorption were tested and the optimized conditions were as follow: the total adsorption time was 12 h, 50% ethanol was used for desorption and the desorption time was 9 h. The crude sample that was prepared by macroporous resin NKA-II contained 3.4% cordycepin, 3.7% HEA and 4.9% adenosine. Then the crude sample was further purified by recycling high-speed counter-current chromatography (HSCCC) with ethyl acetate, n-butanol, 1.5% aqueous ammonium hydroxide (1:4:5, v/v/v) as the optimized two-phase solvent system. Three nucleosides including 15.6 mg of cordycepin, 16.9 mg of HEA and 23.2 mg of adenosine were obtained from 500 mg of crude sample in one-step separation. The purities of three compounds were 98.5, 98.3 and 98.0%, respectively, as determined by high performance liquid chromatography.