Isolation and quantification of antimalarial N-alkylamides from flower-head derived in vitro callus cultures of Spilanthes paniculata

This is the pioneer work reporting on simple procedure for synchronized determination and quantification of two biologically active N-alkylamides, (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (spilanthol) and (2E,4Z)-N-isobutyl-2,4-undecadiene-8,10-diynamide (UDA), using in vitro callus cultures from flower-heads of Spilanthes paniculata. The extracts were purified using preparative thin layer chromatography (TLC) and finest separation of compounds was optimized using high performance liquid chromatography (HPLC). Eventually, N-alkylamides were validated by mass spectrometry. Linearity curve with its regression coefficients (R²) obtained for both these alkylamides was 0.99. While spilanthol was quantified using tentative standard dodeca-2(E),4(E)-dienoic acid due to the non-availability of commercial standard and the precision of a developed method was evaluated in terms of relative standard deviation by measuring inter- and intra-days variation 3.52% and 1.74%, respectively. Similarly, calibration curve was obtained for the compound UDA isolated from flower-head explants from field grown parental plant with its inter- and intra-day RSD values as 4.33% and 3.61%, respectively. With this protocol, a very high yield of 2.23 mg/g of spilanthol and 4.30 mg/g dry weight (DW) of UDA, was obtained, simultaneously, from callus cultures. Flower-heads from parent plants, used as control, showed negligible amount of spilanthol and quantity of UDA was marginally higher than that in callus cultures. The highly stable biotherapeutic spilanthol and UDA with m/z 222 and m/z 230, respectively, showed retardation of malaria parasite development through blockage at ring stage of erythrocytic schizogony and ultimately lead to parasite death. The effect on parasite was additive. This study signifies the utility of in vitro cell cultures for therapeutic compound production, throughout the year, at higher yield for down-stream applications.