A comparative study of the antioxidant potential and metabolic profiling of lingonberry (Vaccinium vitis idaea) from Northern Manitoba and Newfoundland By Aakangchhya Bhusal A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Human Nutritional Sciences University of Manitoba Winnipeg, Manitoba Copyright © 2016 Aakangchhya Bhusal i ABSTRACT Lingonberries (Vaccinium vitis-idaea) are edible fruit that belong to the Ericaceae plant family. A growing body of evidence suggests that consumption of berries provide considerable health benefits due to their high polyphenols, antioxidants, vitamins and mineral content. Therefore, it is beneficial to evaluate the antioxidant capacity of berries in order to select lines with higher antioxidant capacity. The aim of this study was to determine the antioxidant capacity, total phenolics, total anthocyanins, anti-apoptotic effect and the metabolomic profile of lingonberries from Northern Manitoba (wild) and Newfoundland (greenhouse-grown). A total of 159 lingonberry samples were collected from Northern Manitoba (Lynn Lake and Flin Flon) and Newfoundland. Samples were freeze dried, lyophilized, ground into powder and extracted with solvent methanol. Total anthocyanins were determined by the pH differential method. Total phenolics were evaluated by the Folin-Ciocalteau’s assay, the anti-apoptotic effect was measured fluorescently and the oxygen radical absorbance capacity (ORAC) assay was utilized to evaluate the antioxidant capacity. Our study showed that Northern Manitoba-grown lingonberries contain a higher level of anthocyanins and phenolics compared to Newfoundland greenhouse-grown lingonberries. Results show that Northern Manitoba- grown lingonberries have higher antioxidant capacity compared to ones grown in Newfoundland. Findings from the study suggest that berries grown in green house conditions and extreme climatic conditions have different antioxidant capacity, anti- apoptotic effects, total phenolics and total anthocyanins; that is, more extreme climates may result in berries with higher antioxidant capacity, total phenolics and anthocyanins. ii Fig 16: Northern Manitoba berries have significantly higher ORAC value as compared to Newfoundland berries. Statistical analysis was done using unpaired t test N=40 (Newfoundland), N= 5 (Northern Manitoba); *P<0.0001. ACKNOWLEDGEMENTS I take this opportunity to thank everyone who has helped me directly and indirectly throughout my M.Sc. program. I feel very lucky and fortunate to get an opportunity to pursue my graduate studies under the supervision of my supervisors. Thank you to my supervisors Dr. Dan Brown and Dr. Chris Siow for all your support and help. Thank you for your valuable thoughts and constant guidance. Without your constant supervision and guidance completion of M.Sc. and research project would have been a distant reality. I would like to acknowledge Dr. Michel Aliani for serving as my advisory committee. Thank you for all your help and support for the metabolomics part of my study. It would have been impossible without your continuous support and guidance. I would like to extend my sincere thanks to Dr. Samir Debnath for providing green- house grown lingonberry samples from Newfoundland. The project would have been impossible without those samples. My lab work would have been impossible without the help of my lab member Cara Isaak and lab technicians Jo-Ann Stebbing and Suvira Prashar. Thank you for always being there to help and support me. Thanks for answering my every questions and helping me whenever I needed. Thank you to Shiva for helping me with the sample preparation. I would like to extend my sincere thanks to all my friends and family, my support system. Thank you to my mom and dad, Sita and Dr. Ram Chandra Bhusal, and my beautiful sisters; Apekshya and Samikchhya for always loving me. Words are merely iii enough to express my gratitude for all your love, care, encouragement and support. Thank you for always believing in me, loving me and praying for me. A special thanks to my wonderful husband and best friend, Deepak Upreti, who has helped me and supported me thoroughly. Thank you for believing me and standing by me through thick and thin. You have always been my source of strength. Finally, I express my gratitude to Growing Forward 2, Agriculture and Agri-Food Canada (AAFC) for financial support towards my graduate studies. iv DEDICATION I would like to dedicate this thesis to my family. To my mom and dad, sisters and my husband v TABLE OF CONTENTS ABSTRACT ………………………………………………………………………………II ACKNOWLEDGEMENTS ………………………………………………………………III DEDICATION …………………………………………………………………………....V TABLE OF CONTENTS ………………………………………………………………...VI LIST OF TABLES ………………………………………………………………………..IX LIST OF FIGURES ………………………………………………………………………..X LIST OF ABBREVIATIONS …………………………………………………………...XII CHAPTER I: LITERATURE REVIEW……………………………………………………….1 1.1. Lingonberry……………………………………………………………………………1 1.2. Nutritional composition of lingonberry……………………………………………….2 1.3. Potential health benefits of lingonberry……………………………………………....3 1.4. Berry phenolics……………………………………………………………………......6 1.4.1. Stilbenes………………………………………………………………….…8 1.4.2. Tannins………………………………………………………………….…..9 1.4.3. Phenolic acids………………………………………………………….….10 1.4.4. Flavonoids……………………………….……………………………...…13 1.4.4.1. Anthocyanins……………………………………………13 1.4.4.2. Flavonols………………………………………………...15 1.4.4.3. Flavanols………………………………………………...17 1.5. Effects of cool climate on production of secondary metabolites……………….…...18 1.6. Free Radicals…………………..…………………………………………………….19 1.7. Antioxidants…………………………………………………………………………21 1.8. Oxidative stress and diseases…….………………………….………………………22 1.9. Ischemia-reperfusion………..………………………………………………….……23 vi 1.10. Some Methods to determine antioxidant capacity……………………………….....24 1.10.1. Oxygen radical absorbance capacity (ORAC)………………………...…25 1.10.2. Total Phenolic Content (TPC)…………………………………………...26 1.11. Metabolomics………………………………………………………………………26 CHAPTER II: RESEARCH PLAN…………………………………………………..…31 2.1. Knowledge gap……………………………………………………………………..32 2.2. Aim of the study……………………………………………………………………32 2.3. Hypotheses………………………………………………………………………….33 2.4. Overall Objective……………………………………………………………………33 2.5. Specific Objectives………………………………………………………………….33 Chapter III: METHODOLOGY………………………………………………………...34 3.1. Plant material………………………………………………………………………..35 3.2. Sample preparation………………………………………………………………….36 3.3. Determination of total anthocyanin…………………………………………….…...48 3.4. Determination of antioxidant capacity………………………………………………49 3.4.1. Total phenolic assay…………………………………………….…………49 3.4.2. Oxygen Radical Absorbance Capacity (ORAC) assay……………………49 3.5. Cell Culture…………………………………………………………………….……50 3.6. Ischemia reperfusion model in vitro ………………………………………………..51 3.7. Determination of caspase-3-activity……………………………………………...…51 3.8. Metabolomics-Untargeted Approach………………………………………………..52 3.8.1. Selection of sample……………………………………………………..…52 3.8.2. Sample preparation…………………………………………………….….53 3.8.2. LC-qTOF-MS conditions and Analysis…………………………………...54 CHAPTER IV: RESULTS………………………………………………………………55 4.1. Basis for selection of samples ……………………………………………………....56 4.2. Total anthocyanin content ……………………………………………………….….58 4.3. Total phenolic content……………………………………………………………….65 vii 4.4. Antioxidant activity ………………………………………………………………..69 4.5. Measurement of caspase-3-activity………………………………………………....76 4.6. Caspase-3-activity of pooled samples……………………………………………....79 4.7. Metabolomics results…………………………………………………………….....80 CHAPTER V: DISCUSSIONS………………………………………………………….82 CHAPTER VI: FUTURE DIRECTIONS AND LIMITATIONS……………….……92 REFERENCES…………………………………………………………………………...95 viii LIST OF TABLES Table 1: List of polyphenolic compounds with food sources, Basic skeleton and Number of carbon atoms……………………………………………………………………….……………...8 Table 2: Various Sources of free radicals……………………………………………………….20 Table 3: Greenhouse growing conditions for Newfoundland lingonberry……………………...35 Table 4: Average annual precipitation, altitude with GPS location sites for the harvested sites of wild Manitoba (Lynn Lake and Flin Flon) lingonberry and cultivated lingonberry from Newfoundland…………………………………………………………………………….……...36 Table 5: Background information on lingonberry samples with genotype, propagation type, growing condition and province…………………………………………………………………38 Table 6: Composition of ischemic medium……………………………………….………….…47 Table 7: Total Anthocyanin Content (ACY), Total phenolics (TP), Antioxidant Capacity (ORAC) and Total Anthocyanins/ Phenolics in lingonberry from Manitoba and Newfoundland…………………………………………………………………………………....69 Table 8: Total anthocyanins (ACY), Total phenolics (TPH) and Antioxidant capacity (ORAC) for pooled samples……………………………………………………………………………….70 Table 9: A total of 4400 entities were detected in both the samples. In positive mode, 84 entities were statistically significant between the samples from Manitoba and Newfoundland. In negative mode, 62 entities were statistically significant between the groups……………………………..79 Table 10: Bioactivity of some compounds unique to Northern Manitoba lingonberry………....80 ix Table 11: Bioactivity of some compounds unique to Newfoundland lingonberry…………..….81 LIST OF FIGURES Fig. 1: List of phenolic compounds in berries…………………………………………………….7 Fig. 2: Structure of examples of stilbenes: resveratrol, pterostilbene and piceatannol……….….9 Fig. 3: Structure of phenolic