Biologically relevant secondary metabolites of Vaccinium uliginosum: bioassay-directed natural products identification of anti-neuroninflammatory agents in the Alaska bog blueberry

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McGill, Colin
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http://hdl.handle.net/11122/5006
Abstract
Dietary blueberry supplementation has demonstrated numerous health benefits including improved learning and memory in aging and neurodegenerative models, neuroprotection from ischemic events, anti-diabetic properties, and modulation of multiple inflammatory cascades. Despite previous research on antioxidant components prevalent in blueberries, no adequate explanation for a molecular mechanism for the benefits of blueberry supplementation has been proposed. Vaccinium uliginosum, the Alaska bog blueberry, possesses higher concentrations of antioxidant components than commercial varietals, and exhibits a greater oxygen radical scavenging capacity, making it an excellent candidate for the identification of biologically relevant secondary metabolites. An approach of bioassay-directed natural products identification was utilized to identify compounds in the Alaska bog blueberry responsible for the inhibition of both a magnesium-dependent neutral sphingomyelinase and NADPH oxidase in TNF-[alpha]-induced SH-SY5Y human neuroblastomas. Five relevant metabolites were identified: ß-sitosterol (1), ursolic acid (2), 3-0-(4-hydroxyphenylcarboxylic acid) 4-0-(ß-D-glucopyranosyl) gallic acid (3), malic acid (4), and 2,3-dihydroxybutane-1,2,3,4-tetracarboxylic acid (5). Neither compounds 3 or 5 had been previously described as a natural product in the literature. The identification of these compounds in the Alaska bog blueberry provides new explanations as to the benefits of blueberry consumption and offers new avenues of research for nutraceutical treatment of neuroinflammation.
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Dissertation (Ph.D.) University of Alaska Fairbanks, 2010
Table of Contents
1.0. Introduction -- 1.1. Relevance of Vaccinium uliginosum, the Alaskan bog blueberry -- 1.2. Bioassay directed fractionation for the identification of biologically relevant compounds in complex extracts -- 1.3. Review of the literature : Whole berry supplementation and the relevance of NADPH oxidase and neutral sphingomyelinase modulation -- 1.3.1. Blueberry supplementation and neuroprotection : implications in learning and memory -- 1.3.1.1. Psychomotor performance and cognition improvements in the context of neuronal signaling and oxidative insult -- 1.3.1.2. The presence of blueberry anthocyanins and polyphenols in neuronal tissue -- 1.3.1.3. Modulation of IGF-1 and activation of MAPK signaling cascades, CREB, and BDNF -- 1.3.1.4. Protection in alzheimer's models : involvement of MAPK, CREB, AChE, and GSH -- 1.3.1.5. Effects on inflammatory cytokines and ceramides -- 1.3.2. Blueberry related neuroprotection in ischemia -- 1.3.3. Anti-diabetic properties of blueberries -- 1.3.4. NADPH oxidase as a target for blueberry research -- 1.3.5. Neutral sphingomyelinase regulation of ceramide release as a target for blueberry research -- 2.0. Methods -- 2.1. Evaluation of modulation of nSMase and NOX activation by Alaska bog blueberry components -- 2.2. Materials -- 2.3. Preparation of crude blueberry extract -- 2.4. Primary fractionation of crude extract pre-treatment procedures -- 2.5. Secondary fractionation of pre-treated samples -- 2.6. Tertiary fractionation : isolation of DPG and blueberric acid -- 2.7. Tertiary fractionation : isolation of malic acid -- 2.8. Tertiary fractionation : isolation of ursolic acid -- 2.9. Isolation of ß-sitosterol -- 3.0. Results -- 3.1. Initial evidence of anti-inflammatory components in the Alaska bog blueberry : whole extract inhibition of nSMase and NOX -- 3.2. Fractionation of Alaska bog blueberry and identification of biologically relevant secondary metabolites : tracking via inhibition of nSMase and NOX -- 3.3. ß-Sitosterol : structure and analysis of NMR -- 3.4. Ursolic acid : structure and analysis of NMR -- 3.5. 3-O-(4-hydroxyphenylcarboxylic acid) 4-O-(ß-D-glucopyranosyl) gallic acid or dimeric protocatechuic acid glycoside (DPG) : structure and analysis of NMR -- 3.6. Dimeric protocatechuic acid (DPG) : discussion of MS -- 3.7. Malic acid : determination of structure via NMR -- 3.8. 2,3-dihydroxybutane-1,2,3,4-tetracarboxylic acid or blueberric acid (BBA) : structure and analysis of NMR -- 3.9. Mass spectroscopy of BBA -- 4.0. Discussion -- 4.1. Ursolic acid -- 4.2. ß-sitosterol -- 4.3. Polyphenols : DPG in context -- 4.4. Malic acid -- 4.5. Blueberric acid -- 4.6. Future directions -- 5.0. References -- 6.0. Appendices.
Date
2010-05
Type
Dissertation
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