An in vitro model to measure Nerium oleander toxicity using Hep G2 cell line

Abstract

Introduction: Nerium oleander is an evergreen shrub belonging to the Apocynaceae family which is locally found as an ornamental shrub decorating roadsides and gardens. All parts of oleander contain cardiac glycosides such as oleandrin, folineriin, adynerin and digitoxigenin which are potentially fatal when ingested. Various factors influence the glycoside content including the season, location, part of the plant, and the colour of the flower. Cardiac glycosides adversely affect multiple organ systems, with the cardiovascular and gastrointestinal system being the most studied, however other organs including the liver are also adversely affected. This study aimed at developing an in vitro model to test the toxicity of cardiac glycosides extracted from local species of Nerium oleander on hepatic cells. Methods: Leaves and twigs belonging to white flowered and pink double flowered cultivars were collected during summer, dried, and extracted with ethanol using the Soxhlet apparatus. The toxicity of these extracts was evaluated against human hepatic tumour cells (Hep G2) via MTT colorimetric assays using concentrations ranging from 0.05 ppm to 5 ppm over a duration of 24, 48 and 72 hours.Results: Data indicates that there is a positive correlation between concentration of the extract and cell death for all concentrations of pink double flower extracts tested, likely via apoptosis. In case of white flower extracts, results were varied. Time dependent toxicity was observed for pink double flower extracts, but not for white flowered extracts. A direct comparison in cell viability between pink double flowered extracts and white flowered extracts revealed a significant difference between leaves extract, but not for the twigs extract. Generation of IC50 values demonstrated that the pink double flower leaves and twigs extracts exhibited the highest toxicity at a duration of 48 hours, where a concentration of 0.03 ppm was modelled to be fatal to 50% of the cells. Conclusion: This study established an in vitro model for assessing toxicity of Nerium oleander extracts on hepatic cells using the Hep G2 cell line, which demonstrated that the pink double flower extracts demonstrated higher toxicity than the white flower extracts, likely attributed to varying cardiac glycoside levels. This highlights the importance in local plant species variations, and the possibility of using flower coloration as an indicator for cardiac glycoside content and toxicity in Nerium oleander.