Bergamot (Citrus bergamia, Risso) essential oil (BEO) comprises a volatile fraction (93-96% of total) containing terpenes and oxygenated derivatives and a non volatile fraction (4-7% of total) containing coumarines and furocoumarines (psoralens). In the present study, using an experimental model of excitotoxicity in vitro, we investigated the neuroprotective potential of BEO.

For the study, cultured human SH-SY5Y neuroblastoma cells were used. BEO and its fractions were kindly provided by the Consortium of Bergamot (Reggio Calabria, Italy) Exposure of SH-SY5Y cells to the excitotoxin N-methyl-D-aspartate (NMDA; 1 mM, n= 9) induces significant cytotoxic effects (P<0.001 vs control) as evaluated by trypan blue exclusion assay and these are significantly reduced (P<0.01) by competitive (CGP 40116, 100 µM; n=3) and non-competitive (MK801, 200 nM, n= 3) antagonists of NMDA subtype of glutamate receptors. To characterize the pharmacotoxicological profile of BEO in SH-SY5Y cultures, cells were exposed to a medium containing graded dilutions (1:20,000-1:2,000; n= 6 experiments per concentration) of BEO and cell viability was assessed by trypan blue staining 24 hours later. BEO dose-dependently affected cell viability, 1:5,000 (BEO 0.02% in medium) being the first dilution able to induce a statistically significant (P<0.001) increase over control cell death whereas no significant effects on cell viability were observed by using higher dilutions (1:10,000 and 1:20,000) of BEO. Similar results were obtained by using fractions of BEO deprived of monoterpene hydrocarbons (BEO-MHF) and of psoralens (BEO-BF).

Pre-treatment of SH-SY5Y neuroblastoma cultures with BEO (1:10,000 and 1:20,000 dilutions; n=6 experiments per concentration) significantly (P<0.001) reduced cell death caused by NMDA. Protection from NMDA-induced cell death was also afforded by BEO-BF supporting the deduction that psoralens may not be implicated. Quite importantly, BEO-MHF (1:20,000-1:5,000 dilutions; n= 5 experiments per concentration) failed to rescue SH-SY5Y neuroblastoma cultures from cell death induced by NMDA (P> 0.05 vs cell death induced by NMDA given alone). The latter observation suggests that monoterpene hydrocarbons found in the volatile fraction of BEO are responsible for the neuroprotective effects of BEO.

To clarify the mechanisms involved in the neuroprotective action, we evaluated the ability of BEO to affect the accumulation of intracellular reactive oxygen species (ROS) evoked by abnormal stimulation of NMDA receptors for glutamate and studied by using 2’,7’-dichlorodihydrofluorescein diacetate (Mattson et al., 1995). A statistically (P<0.05) significant reduction of NMDA-induced ROS accumulation was obtained by pre-treating SH-SY5Y cultures with BEO (1:5,000-1:20,000 dilutions; n= 3) and BEO-BF (1:5,000-1:20,000 dilutions; n= 3) whereas BEO-MHF was ineffective. Collectively, our data demonstrate that BEO is endowed with neuroprotective characteristics that may stem from the antioxidant activity of monoterpene hydrocarbons though further experimentation is needed for this to be confirmed.

Mattson et al. (1995) Meth. Cell Biol . 46 : 187-216.