Research paper Screening and identification of neuroprotective compounds relevant to Alzheimer's disease from medicinal plants of S. Tomé e Príncipe Antonio Currais a,n , Chandramouli Chiruta a , Marie Goujon-Svrzic a , Gustavo Costa b,c , Tânia Santos b,c , Maria Teresa Batista b,c , Jorge Paiva d , Maria do Céu Madureira d , Pamela Maher a Q1 a The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA b Center for Pharmaceutical Studies, Faculty Q2 of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal c Center for Neurosciences and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal d Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, 3004-516 Coimbra, Portugal article info Article history: Received 7 February 2014 Received in revised form 5 June 2014 Accepted 18 June 2014 Keywords: Aging Alzheimer Dementia Inflammation Neurodegenerative disorders Traditional medicine Africa abstract Ethnopharmacological relevance: Alzheimer's disease (AD) neuropathology is strongly associated with the activation of inflammatory pathways, and long-term use of anti-inflammatory drugs reduces the risk of developing the disease. In S. Tomé e Príncipe (STP), several medicinal plants are used both for their positive effects in the nervous system (treatment of mental disorders, analgesics) and their anti- inflammatory properties. The goal of this study was to determine whether a phenotypic, cell-based screening approach can be applied to selected plants from STP (Voacanga africana, Tarenna nitiduloides, Sacosperma paniculatum, Psychotria principensis, Psychotria subobliqua) in order to identify natural compounds with multiple biological activities of interest for AD therapeutics. Materials and methods: Plant hydroethanolic extracts were prepared and tested in a panel of phenotypic screening assays that reflect multiple neurotoxicity pathways relevant to AD—oxytosis in hippocampal nerve cells, in vitro ischemia, intracellular amyloid toxicity, inhibition of microglial inflammation and nerve cell differentiation. HPLC fractions from the extract that performed the best in all of the assays were tested in the oxytosis assay, our primary screen, and the most protective fraction was analyzed by mass spectrometry. The predominant compound was purified, its identity confirmed by ESI mass spectrometry and NMR, and then tested in all of the screening assays to determine its efficacy. Results: An extract from the bark of Voacanga africana was more protective than any other plant extract in all of the assays (EC 50 s r2.4 mg/mL). The HPLC fraction from the extract that was most protective against oxytosis contained the alkaloid voacamine (MW¼704.90) as the predominant compound. Purified voacamine was very protective at low doses in all of the assays (EC 50 s r3.4 mM). Conclusion: These findings validate the use of our phenotypic screening, cell-based assays to identify potential compounds to treat AD from plant extracts with ethnopharmacological relevance. Our study identifies the alkaloid voacamine as a major compound in Voacanga africana with potent neuroprotective activities in these assays. & 2014 Published by Elsevier Ireland Ltd. 1. Introduction Alzheimer's disease (AD) is the most common form of demen- tia in the elderly. It is characterized by the presence of senile plaques, neurofibrillary tangles and neuronal loss associated with other age-related detrimental events such as increased oxidative stress, reduced energy metabolism and inflammation (Schubert and Maher, 2012). Therefore, AD is multi-factorial in the sense that there are a large number of mechanisms that can contribute to the disease and, specifically, nerve cell death. Many, if not most, of these mechanisms can be reproduced in cell culture assays. The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology http://dx.doi.org/10.1016/j.jep.2014.06.046 0378-8741/& 2014 Published by Elsevier Ireland Ltd. Abbreviations: Aβ, amyloid beta peptide; AD, Alzheimer's disease; APP, amyloid precursor protein; ATP, adenosine triphosphate; BBB, blood–brain barrier; CLogP, lipophilicity; CNS, Central nervous system; DMEM, Dulbecco's modified Eagle's medium; ESI, Electrospray ionization; FCS, fetal calf serum; GSH, glutathione; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; HPLC, High-performance liquid chromatography; HT22, mouse hippocampal nerve cells; IAA, iodoacetic acid; LPS, lipopolysaccharide; MC65, human nerve cells; MTT, 3-(4, 5- dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; MW, molecular weight; N9, mouse microglial cells; NGF, nerve growth factor; NMR, nuclear magnetic resonance; Opti-MEM, Opti-minimal essential media; PC12, rat pheochromo- cytoma cells; ppm, parts per million; STP, S. Tomé e Príncipe; TLC, thin layer chromatography; TMS, tetramethylsilane; tPSA, topological polar surface area n Corresponding author. Tel.: þ1 858 453 4100x1480; fax: þ1 858 535 9062. E-mail address: acurrais@salk.edu (A. Currais). Please cite this article as: Currais, A., et al., Screening and identification of neuroprotective compounds relevant to Alzheimer's disease from medicinal plants of S. Tomé e Príncipe. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.046i Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎