New bufadienolides from the whole plants of Helleborus foetidus and their apoptosis-inducing activity

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 isolated 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 3.6µM.

Compound 1 is a new bufadienolide with cytotoxicity against HL-60 cells with an IC₅₀ value of 0.035µM. 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.

Fig. 1