Screening and optimizing media constituents for enhanced production of medicinal N-alkylamide Deca-2E,6Z,8E-trienoic acid isobutylamide from dedifferentiated in vitro cell lines of Spilanthes paniculata

- An alternate strategy is developed to maximize cell biomass to preserve natural plant resource.
- The calli from leaf-disc explants produced maximum spilanthol of 2.81 mg/g DW following the statistical strategy, Placket-Burman (PB) and central composite design (CCD). While the production of spilanthol from same cell lines (before optimization) and from leaves of parent plant (control) was 1.75 mg/g DW and 0.26 mg/g DW, respectively.
- Statistical tools overcame limitations of classical methods of one dimensional approach that do not guarantee the optimal conditions.

Spilanthes paniculata is a rich source of therapeutic polyunsaturated fatty acid amides. This report established the cell cultures from leaf-disc explants of Spilanthes paniculata as an alternative method of cell biomass utilization and conservation of natural plant resources. Furthermore, a methodology to maximize the production of a prime N-alkylamide, Deca-2E,6Z,8E-trienoic acid isobutylamide (spilanthol), from in vitro cell cultures was optimized using statistical tools. Placket-Burman (PB) design was applied to identify the significant nutrient media constituents. Among the five variables tested, Murashige and Skoog (MS) major salts, sucrose, 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-benzylaminopurine (BAP), were found to have significant effect on spilanthol production. The optimal concentrations of the four variables were determined using central composite design (CCD), which is widely used response surface methodology (RSM) design. The most suitable concentration of variables for spilanthol production were, 1½ MS, 5% sucrose, 4.82 µM BAP and 1.8 µM 2,4-D. At these optimal parameters, the maximum yield of spilanthol was obtained experimentally as 2.81 mg/g DW, which was found to be very close to its predicted value of 3.72 mg/g DW. The developed mathematical model was found to fit well with the experimental data by which the higher production of metabolite was achieved as compared to non-optimized media constituents. Before optimization, the callus cultures and leaves from parental plant (control) yielded 1.75 mg/g DW and 0.26 mg/g DW spilanthol, respectively. Thus, by using statistical model, it is possible to understand the existence of interactions between variables and their effect on maximizing the production of therapeutic compounds.

368