Date on Master's Thesis/Doctoral Dissertation

12-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Anatomical Sciences and Neurobiology

Committee Chair

Litvan, Irene

Author's Keywords

Annonacin; Pawpaw; Asimina triloba; Annonaceous acetogenins; Tau; Neurotoxicity

Subject

Pawpaw; Polyketides; Neurotoxicology

Abstract

The acetogenin annonacin, from the tropical annonaceous plant Annona muricata, is a lipophilic, mitochondrial complex I inhibitor reported to be more toxic than rotenone to mesencephalic neurons. The temperate annonaceous plant Asimina tri/oba (pawpaw) is native to the Eastern United States and its products are available online. This study determined whether annonacin is in the pawpaw fruit pulp and whether it or the crude ethyl acetate (EtOAc) extract is toxic to cortical neurons and induces pathological changes in the microtubule associated protein tau. Pawpaw extract was prepared by pulp extraction with methanol and liquid-liquid partitioning with EtOAc. Annonacin was isolated from the crude EtOAc extract via column chromatography using a gradient solvent system of increasing polarity. Mass spectroscopy, nuclear magnetic resonance and infrared spectroscopy were used to compare isolated material with synthetic annonacin and a natural annonacin sample. Toxicity of isolated annonacin and the total EtOAc extract were determined in primary rat cortical neurons using the MIT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The effects of annonacin on tau phosphorylation were determined by immunocytochemistry and western blot analysis. The average concentration of annonacin in the fruit pulp was 0.07 ± 0.03 mg/g. Purified annonacin (30.07 µg/ml) and crude EtOAc extract (47.96 µg/ml) reduced relative viability of cortical neurons by 50% 48 hours post treatment. Annonacin toxicity was enhanced in the presence of crude extract. Annonacin treatment resulted in a decrease in the neuronal specific marker microtubule associated protein 2 (MAP2) and tau immunoreactivity, and tau expression was decreased on western blot. In conclusion, Asimina triloba fruit contains a high concentration of annonacin, which is toxic to cortical neurons. Based on these results, annonacin acts to reduce overall levels of tau, though it remains elusive whether or not this is a direct or indirect effect of annonacin action. Crude fruit extract also induced neurotoxicity, highlighting the need for additional studies to determine the bioavailability after consumption and potential risks of neurodegeneration associated with chronic exposure to pawpaw products.

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