The strain of C militaris was stored at the collection bank of
South China Normal University (Guangzhou, China).The
stock culture was maintained on potato-dextrose-agar slants
and stored at 4°C for subculture.The liquid seed was
inoculated with mycelium mat from a stock slant and grew
in a 250-mL flask containing 100 mL of D-fructose salts
medium (1% D-fructose, 1.5% peptone, 0.1% MgSO4•7H2O, and
0.1% KH2PO4) with initial pH 7.0 at 24°C on a shaker at
180 rpm for 9 days. Cordycepin was prepared as follows: The
cultured broth from the shake flask of C militaris was
concentrated and filtered to separate mycelial biomass and
liquid medium. The collected filtrates were vacuum concentrated to approximately one-fifth of the original volume.
Proteins and polysaccharides were removed from the concentrated filtrate by using absolute ethanol. The entire
remaining content was vacuum concentrated and vacuum
freeze dried. The obtained dry powder was used to separate
cordycepin with macroporous DM 101 resin method, as
described by Ni et al [19]. The collected eluates were vacuum
concentrated to approximately one-fourth of the original
volume. The concentrated eluates were vacuum freeze dries
and then the cordycepin crystalline was obtained. The highperformance liquid chromatography system (Dalian Elite
Analytical Instrument, Ltd, Dalian, China) was used for
determination of cordycepin. The main compositions of the
cordycepin crystalline were analyzed. The content of
cordycepin, protein, and total sugar in cordycepin crystalline
is 61.55%, 11.15%, and 8.51%, respectively.