Enhanced in vitro production of Ruta graveolens L. coumarins and rutin by mannitol and ventilation

Abstract

Ruta graveolens shoot cultures were maintained on static medium supplemented with 0, 1, 2 and 3 % mannitol. The cultures were grown in vessels that ensured a ventilation rate of 7.44, 10.82 or 62.83 air exchanges per day (V1, V2 or V3, respectively). The growth index and fresh weight were significantly increased at 1 % mannitol and decreased with increasing mannitol concentrations, whereas the dry weight (DW) and DW % increased at higher concentrations of mannitol. Improving the culture ventilation significantly increased all of these parameters. A higher concentration of mannitol resulted in a higher proline content and percentage of coumarins and rutin, but the final accumulation of these bioactive molecules decreased. The coumarins, calculated as xanthotoxin, were increased from 8.15 to 13.60 mg g⁻¹ DW using (V1 and mannitol-free medium) and (V2 with medium enriched with 2 % mannitol), respectively. Rutin was linearly increased by raising the mannitol concentrations, achieving the highest content of 54.87 mg g⁻¹ DW using V2 and medium supplemented with 3 % mannitol. The lowest accumulation of coumarins and rutin (32, 144 mg vessel⁻¹, respectively) were found on mannitol-free medium using V1, whereas the highest rutin contents were found on medium with 1 % mannitol using V3. A GC analysis revealed the presence of five main compounds in all of the cultures, coumarin, 7-hydroxucoumarin, scopoletin, xanthotoxin and bergapten, whereas pasoralen was not detected when the cultures were maintained on mannitol-free medium, regardless of the type of vessel. Moreover, the concentrations of these compounds varied according to the mannitol concentration and ventilation.