Methods for Differentiation and Phytochemical Investigation of Crataegus by Nuclear Magnetic Resonance Spectroscopy
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METHODS FOR DIFFERENTIATION AND PHYTOCHEMICAL INVESTIGATION OF CRATAEGUS BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY by Jensen Allan Lund B.Sc. (Hons.), The University of British Columbia, 2013 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) April 2016 © Jensen Allan Lund, 2016 The undersigned certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: Methods for Differentiation and Phytochemical Investigation of Crataegus by Nuclear Magnetic Spectroscopy Submitted by Jensen Allan Lund in partial fulfillment of the requirements of The degree of Master of Science . Dr. Paul Shipley, Chemistry, UBCO Supervisor, Professor (please print name and faculty/school above the line) Dr. Susan Murch, Chemistry, UBCO Supervisory Committee Member, Professor (please print name and faculty/school in the line above) Dr. Soheil Mahmoud, Biology, UBCO Supervisory Committee Member, Professor (please print name and faculty/school in the line above) Dr. Daniel Durall, Biology, UBCO University Examiner, Professor (please print name and faculty/school in the line above) External Examiner, Professor (please print name and university in the line above) April 25, 2016 (Date submitted to Grad Studies) Additional Committee Members include: Dr. Paula Brown, NRG, BCIT (Please print name and faculty/school in the line above) Abstract Crataegus (hawthorn) is a genus of flowering, fruit-bearing, small-to-medium-sized trees native to northern temperate zones. Hawthorn has an extensive ethnobotany with numerous examples of use in the food and medicine of the Chinese, North American Aboriginals, and Europeans. Modern hawthorn natural health products (NHPs) are used for cardiovascular ailments and have shown promise for adjunctive therapy in treatment of chronic heart failure. Although many NHPs containing Crataegus are licensed for sale in Canada, and there is extensive ethnobotanical use, little is known about the potentially therapeutic phytochemistry of North American hawthorns. NHP monographs allow three Crataegus spp. for use in NHPs. Regulatory requirements demand that specifications for NHPs include identity of the source material. Hence, it is important that there are methods available to differentiate species and this is the rationale for the work in this thesis. The first objective of this thesis was to explore differentiation of Crataegus spp. by nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis (MVDA) of leaf extracts. Two European and two North American species were harvested in late summer 2008 from the same test farm and used in this study. The results suggest that we may best differentiate Crataegus spp. by 1D-1H-PRESAT-NMR-acquired data that correlate to phenolic compounds. Multiple significance analysis methods were used to provide lists of potential biomarkers in the phenolics spectral region that may give insight to differential therapeutic effects of hawthorn NHPs. Machine learning was applied to chemometrics data and results correlated with and outperformed model accuracy in traditional methods of MVDA. The second objective of this thesis was to quantify flavonoids in the same samples. I found that vitexin and its derivatives were significantly more concentrated in the European leaves and iii rutin significantly more concentrated in the North American leaves. The concentrations of rutin and naringenin reported in this study are the highest reported for Crataegus. The results were validated by comparison to high performance liquid chromatography, which produced generally consistent results of mean concentrations as measured in the leaf extracts. iv Preface The literature necessary to write the traditional Chinese medicine section in Chapter 1.5.1 was translated with the assistance of Ying Liu. A version of Chapter 2 has been presented as a poster at the 12th annual Natural Health Products Research Society of Canada conference and the 2015 UBC Okanagan Biology Graduate Symposium. Lund, J., Brown, P., Shipley, P. (2015). An Evaluation of NMR Data Acquisition and Multivariate Data Modeling for Differentiation of Crataegus spp. The HPLC analysis presented in Chapter 3 is based on work conducted at BCIT’s Natural Health and Food Products Research Group laboratory in Burnaby, BC with the assistance of Dr. Paula Brown and Elizabeth Mudge. The validated method for HPLC analysis of hawthorn leaf flavonoids was developed by this group (Mudge and Brown, 2016). I was responsible for participating in extraction optimization, preparation of hawthorn leaf extracts for analysis, analysis by HPLC, and quantification of compounds based upon chromatographic data. v Table of Contents Abstract ................................................................................................................................... iii! Preface ...................................................................................................................................... v! Table of Contents ................................................................................................................... vi! List of Tables ........................................................................................................................... x! List of Figures ....................................................................................................................... xiv! List of Abbreviations ........................................................................................................... xix! Acknowledgements ............................................................................................................. xxii! Dedication ........................................................................................................................... xxiv! Chapter 1: Introduction ......................................................................................................... 1! 1.1! Botany and ecology of Crataegus ............................................................................................. 1! 1.2! Classical taxonomy and identification of Crataegus spp. ......................................................... 3! 1.3! Molecular and chemical techniques for taxonomy and species identification of Crataegus .. 12! 1.3.1! Chemotaxonomy .............................................................................................................. 16! 1.3.2! Metabolomics and its relationship to chemotaxonomy ................................................... 16! 1.3.3! Localization of specialized metabolites with respect to plant anatomy ........................... 18! 1.4! Phytochemistry of Crataegus .................................................................................................. 20! 1.5! Ethnobotany and modern natural health products (NHPs) of Crataegus ............................... 29! 1.5.1! Ethnobotanical use of Crataegus ..................................................................................... 29! 1.5.2! Modern use of Crataegus in natural health products (NHPs) and dietary supplements (DSs).... ........................................................................................................................................ 31! 1.6! Regulatory requirements of natural health products (NHPs) and dietary supplements (DSs).......................................................... ....................................................................................... 33! 1.6.1! Identification of botanical materials for use in natural health products (NHPs) and dietary supplements (DSs) ........................................................................................................... 34! 1.6.2! Evaluation of strength and potency of botanical materials for use in natural health products (NHPs) and dietary supplements (DSs) ......................................................................... 35! 1.6.3! Regulatory requirements for Crataegus natural health products (NHPs) and dietary supplements (DSs) ....................................................................................................................... 35! vi 1.7! Methods for evaluation of potency and authentication of Crataegus natural health products (NHPs) and dietary supplements (DSs) ............................................................................................ 38! 1.7.1! Evaluation of potency of Crataegus botanical materials ................................................. 39! 1.7.2! Authentication of Crataegus botanical materials by morphology and molecular methods.. ...................................................................................................................................... 40! 1.7.3! Potential alternative techniques for authentication of Crataegus botanical materials ..... 41! 1.7.4! Nuclear magnetic resonance (NMR) spectroscopy ......................................................... 43! 1.7.5! Nuclear magnetic resonance spectroscopy-based metabolomics .................................... 44! 1.7.6! Special considerations in nuclear magnetic resonance spectroscopy and its application to multivariate statistical modeling .............................................................................................. 45! 1.7.7! Quantification by nuclear magnetic resonance spectroscopy .........................................