Galanthamine production in tissue culture and metabolomic study on Amaryllidaceae alkaloids in Narcissus pseudonarcissus cv. Carlton

Abstract

The Amaryllidaceae alkaloids e.g. galanthamine (Gal), lycorine and narciclasine are noted for their pharmaceutical properties. Gal is a long acting, selective, and competitive acetylcholinesterase inhibitor approved for the treatment of early to mid-stage Alzheimer's diseases. The biosynthesis of alkaloids by plants using in vitro systems has been considered as a tool for drug discovery and production since total chemical synthesis is not economic. The biosynthetic pathways, especially for Gal, are starting to be understood, but still far from complete. This study is emphasised on the yield from whole plant and developing cell culture systems for optimized alkaloid production.In vitro cultures of Narcissus pseudonarcissus (cv. Carlton) initiated from twin-scale explants cultured on Murashige and Skoog (MS) agar medium fortified with different concentrations of growth regulators, were screened for their ability to produce alkaloids through GC–MS (gas chromatography-mass spectrophotometry). Callus obtained mainly from the MS medium containing high concentration of auxins i.e. 20 mg/l NAA (naphthalene acetic acid), while media with low auxin (4 mg/l NAA) and MS basal medium gave bulblets with both white and green shoots. Regenerated bulblets developed from callus found in both high and low auxin MS media. Tissue culture derived materials (callus and bulblets) and field grown samples (bulb, basal plate and leaves) from 'Carlton' were analyzed for galanthamine content. The highest amount of galanthamine was obtained from basal plate tissue followed by bulb tissues, leaves, bulblets with green shoots, bulblets with white shoots and regenerated bulblets. Trace amount of galanthamine was found in callus.An NMR (nuclear magnetic resonance) based metabolomic analysis showed that the relative concentrations of compounds involved in phenylalanine and tyrosine metabolism, the initial stage biosynthetic pathways that yield the Amaryllidaceae alkaloids, were higher in field samples than in vitro samples which support the GC–MS findings of high Gal production in field samples.