Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr.

The study revealed, for the first time, accumulation of spilanthol, an antiseptic alkylamide, in in vitro cultures of Spilanthes acmella Murr., a medicinal plant of immense commercial value. To achieve this, in vitro shoots were regenerated via direct organogenesis from leaf-disc explants of Spilanthes. Shoots were induced in the presence of N6-benzylaminopurine (BAP) alone or in combination with either α-naphthalene acetic acid (NAA) or Indole-3-acetic acid (IAA) in Murashige and Skoog medium. The best treatment for shoot regeneration was MS + BAP (5.0 μM) + IAA (5.0 μM), which promoted adventitious shoot proliferation in >82% cultures with an average of 5.3 shoots per explant. Regenerated shoots rooted spontaneously with a frequency of 100% on half strength MS medium (major salts reduced to half strength) containing 50 g l−1 sucrose. The plantlets were acclimatized successfully with 90% survival rate. Additionally, ploidy stability of the regenerated plants was assessed by flow cytometry which showed that all investigated plants had the similar ploidy as that of the mother plant. For spilanthol identification, peaks eluted from HPLC were analyzed by mass spectrometry with its characteristic fragmentation pattern. For quantification studies, calibration curve was generated, which revealed a higher amount of spilanthol content (3294.36 ± 12.4 μg/g DW) in the leaves of in vitro plants compare to those of in vivo plants (2703.66 ± 9.6 μg/g DW of spilanthol). An efficient multiplication frequency, ploidy stability and enhanced spilanthol accumulation ensure the efficacy of the protocol developed for this industrially important medicinal plant.

► This is first report wherein in vitro cultures were utilized for spilanthol production, an alkylamide used in cosmetics.
► Spilanthol peaks, eluted from HPLC, were analyzed by mass spectrometry with its characteristic fragmentation pattern.
► A HPLC protocol was established for quantitative estimation of spilanthol for its easy reproducibility.
► Analysis of in vitro grown plants and dedifferentiated callus revealed the presence of spilanthol in all the samples.
► Significantly higher amount of spilanthol was found in in vitro leaves compare to those taken from field grown mother plants.