Product enhancement of triterpenoid saponins in cell suspension cultures of Leucas aspera Spreng

Abstract

The present investigation reports the influence of signaling molecules methyl jasmonate (MeJA) and salicylic acid (SA) on enhanced production of triterpenoid saponins ursolic acid and oleanolic acid in cell suspension cultures of Leucas aspera Spreng. Callus was initiated in leaf explants of L. aspera on MS (Murashige and Skoog) medium supplemented with 2 mg L-1 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg L-1 picloram. Callus was transferred to MS liquid medium supplemented with 2 mg L-1 2,4-D and 0.2 mg L-1 picloram for the initiation of cell suspension culture. Influence of elicitors MeJA and SA on the biosynthesis of triterpenoids was investigated to assess whether elicitor-mediated production would serve as an alternative strategy for the over production of Leucas aspera triterpenoids. Elicitors were supplemented at different concentrations (50, 100 and 200 μM) in the suspension cultures for 24 days with 6 days intervals. Loss of cell viability was observed at high concentrations of elicitors. MeJA exhibited a maximum level of total saponin content (18.2 ± 1.21 mg g-1 DCW) at 100 μM concentration over a period of 18 days and is 4 folds higher than the respective non-elicited control. While SA at 50 μM concentration showed highest production of total saponins (15 ± 1.21 mg g-1 DCW) after 24 days of exposure and is 1.68 fold higher than the respective non-elicited control. There is an inverse relationship observed between saponins production to the biomass accumulation. Results of TLC (Thin layer chromatography), HPLC (High performance liquid chromatography), LC-ESI-MS (Liquid chromatography-Electron spray ionization-Mass spectrometry) and FT-IR (Fourier Transform Infrared spectroscopy) analyses confirmed the presence of triterpenoid saponins with the dominance of oleanane and ursane classes. Cell suspension cultures elicited with MeJA (100 μM) could serve as an alternate means for the enhanced production of oleanane and ursane classes of triterpenoids which bear pharmaceutical importance.