The phytoestrogenic isoflavones from Trifolium pratense L. (Red clover) protects human cortical neurons from glutamate toxicity

The endogenous steroid estrogen has been shown to affect neuronal growth, differentiation and survival. Genistein, daidzein and other isoflavones have been shown to mimic the pharmacological actions of the gonadal steroid estrogen with which they have structural similarities. Several studies have looked at the effect of isoflavones in the brain. In the present study, human cortical cell line HCN 1-A maintained in culture was used to test the neuroprotective efficacy of a natural mixture of phytoestrogenic isoflavones (genistein, daidzein, biochanin A and formononetin) from Red clover against glutamate toxicity. Neuronal viability was determined by MTT or trypan blue test and neuronal membrane damage was quantitatively measured by lactate dehydrogenase (LDH).The results obtained indicate that exposure of HCN 1-A cell cultures to glutamate resulted in concentration-dependent decreases in neuron viability. Concentration of glutamate ranging from 0.01 to 5 mM was toxic to these cultures. A 24-h pretreatment with 0.5, 1 and 2 μg/ml isoflavones enriched fraction (IEF) significantly increased cell survival and significantly decreased cellular lactate dehydrogenase release from differenziated cortical neurons, indicating that neurons treated with isoflavones were protected from the cell death induced by glutamate exposure. Moreover, the pretreatment with IEF prevented the morphological disruption caused by glutamate as shown by microscopical inspection. These findings indicate that IEF has a neuroprotective effect in human cortical neurons and that this effect might be resulted from his antioxidant and estrogenic actions.