Cytoprotective effect of Coreopsis tinctoria extracts and flavonoids on tBHP and cytokine-induced cell injury in pancreatic MIN6 cells

Coreopsis tinctoria flowering tops infusion is traditionally used in Portugal for treating the symptoms of diabetes. Recent studies have revealed its antihyperglycemic activity when administered for 3 weeks to a STZ-induced glucose intolerance model in the rat and glucose tolerance regain was even clearer and pancreatic function recovery was achieved when administering Coreopsis tinctoria flavonoid-rich AcOEt fraction. In this study we aimed to evaluate the protective effect of Coreopsis tinctoria flowering tops aqueous extract, AcOEt fraction and the pure compounds marein and flavanomarein, against beta-cell injury, in a mouse insulinoma cell line (MIN6) challenged with pro-oxidant tert-Butyl-Hydroperoxide (tBHP) or cytokines. The protective effects of Coreopsis tinctoria flowering tops extracts and pure compounds were evaluated through pre-incubating MIN6 cells with samples followed by treatment with tBHP (400 μM for 2 h) after which viability was determined through ATP measurements. In order to assess whether plant extracts were involved in decreasing reactive oxygen species, superoxide anion production was determined through a lucigenin-enhanced chemiluminescent method. Lastly, the direct influence of Coreopsis tinctoria extracts and main compounds on cell survival/apoptosis was determined measuring caspase 3 and 7 cleavage induced by cytokines. Coreopsis tinctoria flowering tops extracts (25–100 μg/mL) and pure compounds (200–400 μM), when pre-incubated with MIN6 cells did not present any cytotoxicity, instead they increased cell viability in a dose dependent manner when challenged with tBHP. Treatment with this pro-oxidant also showed a rise in superoxide radical anion formation in MIN6 cells. This increase was significantly reduced by treatment with superoxide dismutase enzyme (SOD) but not by pre-treatment with Coreopsis tinctoria flowering tops extracts. Caspase 3/7 activation measurements show that Coreopsis tinctoria flowering tops extracts, as well as marein and flavanomarein, significantly inhibit apoptosis. Coreopsis tinctoria extracts and pure compounds show cytoprotection that seems to be due to inhibition of the apoptotic pathway, and not through a decrease on superoxide radical production.