The genus Helleborus with about 20 species belongs to the family Ranunculaceae. Helleborus foetidus is an evergreen perennial plant native to Europe. Although anemonin, a steroidal glycoside, and a few phenolic compounds including a quercetin glycoside were isola ted from H. foetidus, there has been no systematic chemical work concerning the secondary metabolites of the plant. Therefore, we conducted a phytochemical examination of H. foetidus and evaluated the cytotoxicity and apoptosis-inducing activity of the isolated compounds.
The whole plants of Helleborus foetidus were extracted with MeOH. The concentrated MeOH extract was passed through a Diaion HP-20 column, successively eluted with 30%MeOH, 50%MeOH, MeOH, EtOH, and EtOAc. The 50%MeOH and MeOH eluate fractions showed cytotoxicity against HL-60 leukemia cells with IC50 values of 0.15 and 0.23 μg/mL, respectively. Then, 50%MeOH eluate fraction was subjected to column chromatography on silica gel and octadecylsilanized silica gel, giving compounds 1–5.
The structures of the new bufadienolides (1 and 2) were determined by spectroscopic analysis. Compounds 3–5 were identified as 11α-hydroxyscilliglaucoside (3), desacetylscillicyanosid (4), and hellebortin A (5), respectively, based on their physical and spectroscopic data.
Compounds 1–5 and the aglycones (4a and 5a) were evaluated for their cytotoxicity against HL-60 cells. All compounds exhibited cytotoxicity against HL-60 cells with IC50 values ranging from 0.0076 to 3.6 μM.
Compound 1 is a new bufadienolide with cytotoxicity against HL-60 cells with an IC50 value of 0.035 μM. HL-60 cells treated with 1 displayed the typical characteristics of apoptosis, such as the nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3. Furthermore, 1 was shown to induce the intracellular events associated with the mitochondrial-dependent apoptotic pathway, as was evident from the loss of the mitochondrial membrane potential and release of cytochrome c to the cytosol.
