Cordycepin standard

After 5 days of light culture, C. militaris mycelium was harvested, and then cordycepin content was determined for the next analysis. Specifically, the mycelia was lyophilized, vacuum freeze-dried to a constant weight, and ground to powder. Two grams of dry powder were weighted from each sample to perform High Performance Liquid Chromatography (HPLC) detection. HPLC assay was conducted on Waters Alliance e2695 HPLC (Milford, MA, USA) using a UV detector set at 260 nm equipped with a ZorbaxSB-C18 column (4.6 × 250 mm, 5 μm). The analysis conditions were as follows: mobile phase, 85% ultra-pure water/methanol (85:15, v/v), and the elution rate was 1.5 ml min⁻¹; injection volume, 20 μl. A standard cordycepin curve was generated using 0.02–0.25 μg/ml cordycepin standard (Sigma-Aldrich, Burlington, MA, USA). The cordycepin yield was calculated using the detected peak area according to the standard curve. The cordycepin concentration of mycelia presented in our study was calculated by normalizing in the equal biomass.

After fermentation, the mycelia were lyophilized and ground to powder. Two grams of dry powder was weighted from each sample to perform HPLC detection. HPLC assay was performed on Model LC2000 Liquid Chromatography System (TECHCOMP, Shanghai, China) equipped with an ULTIMATE AQ-C18 HPLC Column (4.6 × 250 mm, 5-Micron; Welch, Shanghai, China). The analysis conditions were as follows: mobile phase, 85% ultra-pure water and 15% methanol (v/v); flow rate, 1 ml/min; detection wavelength, 260 nm; injection volume, 20 μl. A standard cordycepin curve was generated using 0.02–0.25 μg/ml cordycepin standard (Sigma-Aldrich, United States). The cordycepin yield was calculated using the detected peak area according to the standard curve. The cordycepin concentration of mycelia presented in the study was calculated by normalizing in the equal biomass.