INITIAL CHARACTERIZATION OF STEM BARK EXTRACTS FROM PHYLLANTHUS MUELLERIANUS AS SOURCE OF NEW NATURAL ENTITIES WITH ANTI-CHOLINESTERASE PROPERTIES

The plant kingdom is an endless source of chemically diverse bioactive compounds, which are used in traditional folk medicine for the treatment of a wide array of diseases. A previous investigation on the bioactive phytocomponents present in the methanol extract of Phyllanthus muellerianus (PMME) demonstrated an interesting activity against C. sporogenes (MIC= 100 µg/ml) and S. pyogenes (MIC= 300 µg/ml) [1], which supported the traditional use of the extract by local populations in Cameroon. Looking for activity beyond the claimed traditional use [2], PMME was evaluated on human cholinesterase enzymes, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) as selected targets for Alzheimer’s disease (AD) treatment. Indeed AChE inhibitors derived from natural products (galanthamine and rivastigmine) are currently licensed to alleviate cognitive symptoms in dementia. Due to the limited available pharmacological treatments for AD, extensive research has been directed towards the identification of other AChE inhibitors, arising from the plant kingdom. A rational basis for our investigation on the bark extracts of Phyllanthus muellerianus is related to a study carried out in 2007 by Joshi e Parle, in which the antiamnesic potential for Phyllanthus amarus in mice was demonstrated [3]. Anticholinesterase activity was in vitro evaluated by Ellman’s assay [4] using recombinant human AChE and BuChE from human serum. Due to the interesting activity found for the de-fatted PMME (% inhibition at 100 g mL-1 on hAChE = 52.6 ± 0.6, on hBuChE = 70.1 ± 3.1%, n=4), PMME was fractionated by flash chromatography affording six fractions (PMF1-6); PMF1, PMF2 and PMF4 significantly inhibited human cholinesterases, thus they were further purified by a RP-18 solid phase extraction. The bio-guided fractionation allowed the identification of three active phytocomponents, with different selectivities against the two ChEs. Two out of the three bioactives have been isolated so far: compound A from PMF1 and compound B from PMF4, both alkaloids. Compound A showed to be a hBuChE selective inhibitor (IC50 = 44.8 ± 3.0 g mL-1 and IC50 = 382 ± 15 g mL-1, for hBuChE and hAChE, respectively) while compound B showed comparable inhibitory potencies against both enzymes (IC50 = 5.45 ± 0.20 g mL-1 and IC50 = 12.6 ± 0.4 g mL-1, for hAChE and BuChE, respectively). Compared to the commercially available AD drug galanthamine, compound B is one order of magnitude less active on hAChE (galanthamine IC50 = 0.6 g mL-1), representing, however, a potential new natural scaffold to undergo towards optimization. The chemical structure of the two isolated alkaloids is under investigation by 13C and 1H-NMR. Moreover, the isolation of compound C in suitable amount for the evaluation of the biological profile and its structural elucidation has being carried out. References [1] G. Brusotti, I. Cesari, G. Frassa, P. Grisoli, C. Dacarro, G. Caccialanza, J. Ethnopharmacol., 2011, 35, 797–800. [2] R. Brisson, Etudes pygmées, SELAF n 376, Ed Peeters, 1999, Paris. [3] H. Joshi and M. Parle, African Journal of Biomedical. Research., 2007, 10, 165–173. [4] G.L. Ellman, K.D. Courtney, V. Jr. Andres, R.M. Feather-Stone, Biochem. Pharmacol., 1961, 7, 88-95