Accumulation of madecassoside - a major component of centelloside - in centella (Centella asiatica (L.) Urban) cells elicited by salicylic acid

Background and Purpose: Use of elicitors is one of the most important methods to increase the production of secondary metabolites in plants. Salicylic acid is a common chemical elicitor and also a signalling molecule in the signal transduction systems, stimulating biosynthesis of enzymes. However, its influence on the accumulation of madecassoside, a valuable bioactive compound, in centella cell cultures remains uncharacterized. Materials and Methods: Callus of centella (Centella asiatica (L.) Urban) was used to establish cell suspension culture on the MS (Murashige and Skoog 1962) medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid and 3 mg/L thidiazuron. The cell biomass was harvested after 24 days of culture. Concentrations of salicylic acid from 50 to 200 mu M were added to the medium at the beginning of cell culture. The timing of 100 mu M salicylic acid addition on days 5, 10 or 15 after inoculation was also studied. Callus growth and biosynthesis of madecassoside in centella cells were characterized. Results and Conclusions: The cell fresh (dry) biomass increased from the 3rd to 24th day to a maximum of 8.1 +/- 0.4 g/flask (0.95 +/- 0.05 g/flask). The highest madecassoside concentration of 27 +/- 0.7 mg/g dry weight was also obtained on the 24th day of culture. The cell growth was inhibited with salicylic acid additions at the beginning of culture (day 0), reaching the minimum at 200 mu M salicylic acid. However, biosynthesis of madecassoside was significantly enhanced at all salicylic acid concentrations, with the maximum (82 +/- 3.5 mg/g dry weight) reached at 100 mu M salicylic acid. The growth of cells treated with 100 mu M salicylic acid was the lowest when salicylic acid was added at the beginning of the culture and the highest when the addition was postponed until day 15 (harvested on day 24). The madecassoside concentration reached as high as 114 +/- 3.1 mg/g dry weight in the treatment with 100 mu M salicylic acid added after 10 days of culture (a 4-fold increase compared with the control without salicylic acid). In general, salicylic acid is effective in enhancing biosynthesis of madecassoside in centella cells.