Free radical scavenging and apoptotic effects of Cordyceps sinensis fractionated by supercritical carbon dioxide

Supercritical carbon dioxide (SC-CO₂) was used as the elution solvent for fractioning ethanolic extract (E) of Cordyceps sinensis (CS), a traditional Chinese herbal remedy, into R, F1, F2, and F3 fractions. This extractive fractionation method is amenable to large scale and is nontoxic. These four fractions were characterized in terms of total polysaccharides and cordycepin concentrations, scavenging ability of free radicals, and anti-tumor activities. Experimental results demonstrated that fractionation altered the distributions of total polysaccharides and cordycepin in fractions. Fraction R was the most active fraction to scavenge free radicals and inhibit the proliferation of carcinoma cells, followed by the fraction F1 and the extract E. The effect of scavenging on 1,1-diphenyl-2-picryl hydrazyl (DPPH) of CS extract and fractions at 2 mg/ml was R (93%), F1 (75%), E (66%), F2 (47%), and F3 (27%). The IC₅₀ (50% cell growth inhibitory concentration) of tumor cell proliferation and colony formation on human colorectal (HT-29 and HCT 116) and hepatocellular (Hep 3B and Hep G2) carcinoma cells by fraction R were around 2 μg/ml. Conversely, R did not affect the growth of normal dividing human peripheral blood mononuclear cells (PBMC) by exhibiting a large value of IC ₅₀ over 200 μg/ml. Accumulation of tumor cells at sub-G ₁ phase and the fragmentation of DNA, typical features of programmed cell death, were observed in a time and dose dependent manner. Scavenging of free radicals and anti-cancer activity (value of IC₅₀) correlated closely with the quantities of polysaccharides (Spearman's rho = 0.901 and -0.870, respectively). Taken together, our findings suggest that fraction R, obtained by SC-CO₂ fluid extractive fractionation, showed strong scavenging ability and selectively inhibited the growth of colorectal and hepatocellular cancer cells by the process of apoptosis.