4.5. Treatments

For treatments, BHE and its compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA) to achieve a concentration of 40 mg/mL from which further concentrations were prepared by dilution in cell growth medium. Following this, cytotoxicity screening of BHE and its compounds was performed to identify non-toxic concentrations for neuroprotection studies. Briefly 10,000 cells were seeded per well in 96-well plates and exposed to increasing concentrations (2.5, 5, and 10 µg/mL) of BHE as well as the compounds 1, 2, 4, 5, 6 and 7 (Table 2). To ensure that the amount of DMSO at the highest treatment concentration was not toxic to the cells, the control cells were treated with a similar amount of DMSO diluted in medium in the 10 µg/mL concentration. Furthermore, a quantity of MPP⁺ (Sigma-Aldrich, St Louis, MO, USA) was weighed and dissolved in un-supplemented DMEM to arrive at a concentration of 50 mM, from which the 500, 1000, 1500, 2000, and 2500 µM concentrations were prepared, and untreated cells were used as control. Treatment duration for all experiments was 24 h and the 2.5 µg/mL concentration for BHE, and its compounds and the 2000 µM MPP⁺ were chosen for further studies.

For neuroprotection studies, SH-SY5Y cells were grown in 96-well plates and pre-treated with either 2.5 µg/mL of BHE or the compounds for 2 h before adding 2000 µM MPP⁺, and treatment lasted for 24 h. Cells treated with growth medium were used as controls, and 25 µM of rutin (RT), a known neuroprotective agent, was used as a positive control.