Highly efficient, sensitive and selective molecular screening of acetylcholinesterase inhibitors of natural origin by SPE-LC/ESI-TOF-MS and novel TLC-based bioauthography

Compounds with acetylcholinesterase (AChE) inhibitory properties play an important role in therapy of mild and moderate Alzheimer's disease (AD) [1]. Among others Amaryllidaceae alkaloids with galanthamine-like skeleton possess their strong reputation. In clinical trials symptomatic improvements in some patients taking galanthamine prescribed drug were observed [2]. New findings suggest also a role of galanthamine as an adjunctive treatment in major depression [3]. In the presented paper significant improvements in screening methodology of AChE inhibitors among Amaryllidaceae alkaloids were elaborated. It comprised optimized pressurized liquid extraction (PLE) of plant materials followed by highly selective solid-phase extraction (SPE) using Oasis HLB cartridges. Pure alkaloidal fractions were analyzed by a newly developed high-performance liquid-chromatography (HPLC) on a 3µm Atlantis HILIC silica stationary phase combined with recently introduced electrospray ionization (ESI) octopole -orthogonal acceleration time-of-flight (oa TOF)-mass spectrometry (MS) with high mass accuracy (about 2 ppm) and high sensitivity [absolute limit of detection (LOD) for galanthamine was about 43 fg at signal-to-noise 13:1]. Moreover, a newly developed and validated TLC-bioautography permit galanthamine sensitivities at pg levels. In this way, more potent than galanthamine AChE inhibitor namely 1,2-dihydrogalanthamine in Narcissus jonquilla 'Pipit' extract could be found (with IC₅₀ value 0.19µM) lower of about 42% than that of galanthamine) [4].

Acknowledgments: I would like to thank Ms. Dominika Dyk for her assistance in laboratory work, and The Polish Ministry of Science and Higher Education for funding the new LC/ESI-TOF-MS system and financial support of this research project (Grant No 2 P05 F 006 29).

References: [1] Hostettmann, K. et al. (2000) Curr. Org. Chem. 4:973–1010.

[2] Assal, F. (2008)J. Am. Geriatr. Soc. 56:946–947.

[3] Elgamal, S. and MacQueen, G. (2008)J. Clin. Psychopharm. 28:357–359.

[4] Mroczek, T. (2009). J. Chromatogr. A 1216:2519–2528.