Five BEO extractive fractions (BEOs) differing in chemical composition were used. Cell proliferation was determined by MTT and cell count assays. The trypan blue exclusion test and annexin V/PI staining were done to assess their cytotoxic activity. Genotoxic effects were detected by the comet assay. The cell cycle was also analysed cytofluorimetrically. Reactive oxygen species and Δψm were measured fluorimetrically. Western blotting analyses for some apoptosis-related proteins were carried out.
Treatment of SH-SY5Y cells with some types of BEOs decreased rate of cell growth by a mechanism correlated to both apoptotic and necrotic cell death. Coloured BEOs act by increasing generation of reactive oxygen species, responsible for the drop in Δψm, and modulating p38 and ERK1/2 MAPKs, p53, Bcl-2 and Bax signalling pathways. Finally, we identified bergamottin and 5-geranyloxy-7-methoxycoumarin as the bioactive molecules that play a pivotal part in the antiproliferative effects exerted by coloured BEOs.
Our study provides novel insights into the antiproliferative effects of BEO, which could be exploited in the context of multitarget pharmacological strategies.