Discovery and characterization of selected novel natural neuroprotectants from the fruits of alpinia oxyphylla for Parkinson's disease

Abstract

Parkinson’s disease (PD) is an increasingly severe threat to public health as the aging population increases, with still no effective treatment. This thesis discusses the development of a new integrative approach, using multivariate data analysis (MVDA) of chemical profiles and bioassays, to identify, search for, and characterize potential neuroprotective compounds from the fruits of Alpinia oxyphylla—a promising traditional Chinese medicine for the treatment of PD. The primary objective of this study was to use experimental neurodegenerative disease models to identify novel compounds with in vitro and in vivo neuroprotective activities from the fruits of A. oxyphylla; elucidation of synergistic interactions between various identified bioactive constituents for the neuroprotection was a secondary goal for subsequent investigations. Fractions of the ethanolic extract of the fruits of A. oxyphylla (AOE) were subjected to initial chemical profiling analysis using liquid chromatography/mass spectrometry, and neuroprotective activity screening with a 1-methyl-4-phenylpyridinium (MPP+)–induced primary cerebellar granule neurons (CGNs) damage model (Chapter 2). MVDA was employed to correlate the chemical profiles with the neuroprotective activities, resulting in the identification of prospective candidate compounds. The top three bioactive botanical metabolites were identified as the novel compound oxyphylla A [(R)-4-(2-hydroxy-5-methylphenyl)-5-methylhexanoic acid], chrysin, and teuhetenone A. Oxyphylla A was further evaluated as a promising novel PD therapeutic candidate; its potential was evidenced through its neuroprotective effects against MPP+-induced primary CGNs death and through alleviating 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)–induced dopaminergic neuron loss and behavioral impairment in mice. The molecular mechanisms underlying the neuroprotective effect of oxyphylla A were unraveled using multidimensional liquid chromatography/tandem mass spectrometry-based quantitative shotgun proteomics analysis and labeling with isobaric tags for relative and absolute quantitation (iTRAQ); among 1277 quantified proteins out of 2649 non-redundant identified proteins, 73 proteins were significantly regulated. Downstream bioinformatics analyses suggested that the neuroprotective effects exhibited by oxyphylla A could be mediated by Rho-associated protein kinase 2 and thioredoxin. The synergistic neuroprotective effects of two known bioactive polyphenols—chrysin and protocatechuic acid (PCA)—identified from AOE was systematically elucidated (Chapter 3). In vivo studies demonstrated enhanced neuroprotective effects of PCA and chrysin, when used in combination, toward limiting MPTP-induced dopaminergic neuron loss and behavioral impairment in mice. The molecular mechanisms underlying these collective cytoprotective effects were also examined through quantitative shotgun proteomics analysis of 6-hyrdroxydopamine–treated PC12 cells, shortlisting 12 regulated protein candidates out of 2233 non-redundant identified proteins. Among them, two proteins—nucleolin and heme oxygenase 1—were identified as potential biomarkers in the cellular redox pathway; their differential expression profiles were further validated through Western blotting. PCA and chrysin confer neuroprotection through activation of an antioxidant response mediated by NRF2/HO-1 and through inhibition of inflammatory signaling via NF-κB, respectively. A downstream target of NF-κB, inducible nitric oxide synthase, was also found to have decreased expression.