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      • HARVEST
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      Hormetic dietary phytochemicals from Western Canadian plants: Identification, characterization and mechanistic insights

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      KONKIN-DISSERTATION.pdf (8.463Mb)
      Date
      2015-02-26
      Author
      Konkin, David
      Type
      Thesis
      Degree Level
      Doctoral
      Metadata
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      Abstract
      Activation of mammalian stress responsive pathways by plant secondary metabolites may contribute to the protection against certain chronic diseases afforded by fruit and vegetable consumption. This work focuses on the identification of plant compounds that activate the stress-responsive enzyme quinone reductase (QR) by stabilizing the transcription factor NF-E2 related factor-2 (Nrf2). Screening methanolic extracts of plants from Western Canada for QR induction in a mouse hepatoma cell line (Hepa-1c1c7) led to the identification of twenty-one extracts capable of doubling the activity of QR. Bioassay-guided fractionation of six extracts led to the identification of novel classes of compounds with QR-inducing activity including fatty-acid derived polyacetylenes, phthalides, and cannabinoids. Studies using low molecular weight thiols and the recombinantly expressed protein Keap1, the principal negative regulator of Nrf2, supported a mechanism of QR activation involving covalent modification of Keap1 cysteines for the polyacetylenes and phthalides. Analysis of transcriptional changes in response to treatment with a panel of QR-inducing compounds provided strong support for Nrf2 activation by the polyacetylene (3S,8S)-falcarindiol and the isothiocyanate (R)-sulforaphane and weaker support for the compounds (3R,8S)-falcarindiol, 6-isovaleryl-umbelliferone (6-IVU) and (Z)-ligustilide. Additionally, transcript level analyses supported a role for the aryl-hydrocarbon receptor in QR-activation by (3R,8S)-falcarindiol, (Z)-ligustilide, (R)-sulforaphane, 6-IVU and cannabidiol and suggested that treatment with polyacetylenes with a (3R)-configuration, (Z)-ligustilide and 6-IVU causes substantial changes in the expression of genes associated with lipid homeostasis and energy metabolism. As a whole, this work provides evidence that compounds that activate QR (and Nrf2) are widely distributed in the Canadian flora. However, of these QR activators, few are active at concentrations that are expected to be achieved through dietary consumption. Nevertheless, the most exceptional compounds isolated in this work, the compounds (3S,8S)-falcarindiol and epoxyfalcarindiol are highly potent and appear to be or are expected to be specific for activating Nrf2 and thus warrant attention with respect to dietary implications and as drug candidate leads.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Biology
      Program
      Biology
      Supervisor
      Page, Jonathan E.
      Committee
      Bonham-Smith, Peta C.; Dillon, Jo-Anne R.; Krol, Ed S.
      Copyright Date
      June 2013
      URI
      http://hdl.handle.net/10388/ETD-2013-06-1066
      Subject
      Phytochemistry
      Nrf2
      Keap1
      Canadian plants
      disease prevention
      xenobiotic metabolism
      redox stress
      indirect antioxidant
      hormesis
      stress response
      quinone reductase
      polyacetylene
      phthalide
      cannabinoid
      hepa-1c1c7
      bioassay-guided fractionation
      RNA-seq
      mass spectrometry
      circular dichroism
      NMR
      liquid chromatography
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