Abstract: Preparative high performance liquid chromatography with C18 and C18-HCE columns was used to prepare nucleoside compounds from Cordyceps militaris including cordycepin, adenosine and two monomeric compounds, and their structures were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS) and nuclear magnetic resonance (NMR) spectroscopy. The inhibitory effects of cordycepin, compound I and compound II on the proliferation of HepG2 liver cancer cells were comparatively evaluated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The results showed that the C18-HCE column could effectively improve the retention and selectivity of polar compounds, facilitating the separation and purification of nucleosides. The purities of adenosine, cordycepin, compound I and compound II were 90.49%, 98.11%, 96.34%, and 98.33% and their yields were 0.053%, 0.253%, 0.368%, and 0.231%, respectively. Compound II was identified as a new substance, which was similar to compound I in structure, both being nucleoside-type compounds substituted with amino acids or amino acid derivatives. Anti-tumor activity experiments showed that all compounds could significantly inhibit the proliferation of HepG2 hepatoma cells, and their effects decreased in the order: compound I > cordycepin > compound II.

Key words: amino acid derivatives; nucleosides; separation and purification; structure identification; anti-tumor