Cytotoxic activity of water extract fractions of Hypericum androsaemum L. on colorectal cancer cells

Hypericum androsaemum L. (HA) grows wild in shadowy sites, namely in northern region of Portugal, where it is widely used as a medicinal herb. According to some authors, this species is used in popular medicinal preparations as a cholagogue, hepatoprotector, and diuretic and in kidney failure [1]. In this work, the composition of water soluble and volatile components of HA infusions was characterized and the total extract (HA-T) fractioned in order to identify the active compound(s) responsible for their cytotoxic effects. HA-T and each of its seven fractions (HA-A to HA-G) were tested against CO115 cells, at 40 µg/mL. Effects on cell viability/proliferation and apoptosis were assessed by MTT and nuclear condensation assays, respectively. The expression of intermediates of the MAPK/ERK and PI3K/AKT signalling pathways and the expression of apoptotic markers were evaluated by western blot. HA-E and F showed decreases in cell viability in relation to control. Of these fractions, only HA-F showed to slightly increase apoptosis. The effects of individual fractions did not explain the 40% apoptosis reached in response to the total and the reconstituted extract, suggesting that the synergistic effects of the extract's constituents are needed for the HA cytotoxic properties against the CO115 cells. In agreement with this, only HA-T showed significant increases in the apoptotic markers cleaved caspase-9 and cleaved PARP-1. Relative to the total extract (HA-T), only slight decreases in BRaf (HA-E), pERK (HA-E) and pAKT (HA-E and HA-F) were observed. Only HA-T reduced significantly the expression of BRaf, pErk and pAKT. These results support the hypothesis that there is more than one compound in the HA-T responsible for the HA toxicity on the colorectal cancer cells.