Antioxidant and Antiproliferative Activity of Flavonoids from Ontario Grown Onions by Pressurized Low Polarity Water Technology By Cynthya Maria Manohar A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Applied Science in Engineering Guelph, Ontario, Canada © Cynthya Maria Manohar, January 2017 ABSTRACT Antioxidant and Antiproliferative Activity of Flavonoids from Ontario Grown Onions by Pressurized Low Polarity Water Technology Cynthya Maria Manohar Advisor: University of Guelph Professor S. Neethirajan Naturally occurring plant flavonoids are in great demand in both nutra-pharmaceutical and biomedical industries. Flavonoids are a group of phytochemicals that have shown various potential health benefits and are found abundantly in onion. The present study is to screen Ontario grown onion varieties, namely Stanley, Ruby Ring, Safrane, Fortress and Lasalle, and test their antioxidant and antiproliferative capacities. Pressurized low polarity water technology is an eco-friendly technique used for extracting polyphenolic compounds from these onion varieties, followed by quantification and analysis using High performance liquid chromatography. The antioxidant activities in the test samples were measured using various antioxidant assays. Ruby Ring - a red onion variety - exhibited high phenolic and antioxidant activities when compared to the other yellow onion varieties tested. The antiproliferative activity of onion extracts against Caco-2 cancer cells were assessed through cytotoxic, apoptosis and anti-migratory assays. Stanley - a yellow onion variety - showed the highest anticancer activity against Caco-2 cells. Since there was an increase in both radical scavenging activity and total phenolic compounds by Ruby Ring, this red onion variety may be chosen as a preferred variety for developing functional food products. Keywords: Ontario onion extracts, Flavonoids, Antioxidant activity, Caco-2 cells, Antiproliferative, Apoptosis, Polyphenols. ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Suresh Neethirajan for all his support throughout this research work. I would also like to thank Dr. Suresh Neethirajan for all his advice and knowledge, and for his motivational talk and positive outlook. I would also like to thank Dr. Suresh for allowing me to attend various conferences across Canada. I would like to thank him for giving me this wonderful opportunity to work in his lab. I thank all the research members of Bionano Laboratory at the University of Guelph, for providing me with help and support at all times. Thanks to Dr. John Shi and Dr. Xue Jun for their wonderful guidance and support throughout this research work. Finally, a very big thanks to my wonderful parents who have been incredibly supportive of all my decisions. I would like to dedicate this thesis to my parents for all their motivation, love and interest that has moulded me to be who I am today. iii TABLE OF CONTENTS 1. INTRODUCTION….………………………………………………………….……………1 1.1. Onions……………………………………………………………………….……...…......2 1.2. Antioxidants and Flavonoids………………………………………….…….………….....4 1.3. Structural Activity of flavonoids……………………………...…….………………….....6 1.4. Need for Antioxidants………………………………………….……………………….....9 1.5. How Antioxidants Inhibits Free radicals………………………….……………………....9 1.6. Effects of Flavonoids on Cancer…………………………………………………………10 1.7. Metabolism and Bioavailability of Flavonoids .……...……………………….…..…......11 1.8. Pressurized Low Polarity Water (PLPW) Extraction Technique…………………..…….12 1.9. Colorectal Cancer…………………...……………………………………..……….……15 1.9.1. Flavonoids on cancer cells……………………………………………………………..16 1.10. Caco-2 Cell Line...……………………………………………………………………...17 1.11. Experimental Methods Used for Studying Antioxidants and Anti-proliferative activity………………………………………………………………………….…………….18 1.11.1. Antioxidant assays……………………………………...………….…………………18 1.11.2. Cell Culture……………………………………………………...….………………...19 1.11.2.1. Cell Imaging Technique......…………………………………………....…………...19 1.11.2.2. Cytation 5 Cell Imaging Multi-Mode Reader…………….……………..……..…....20 1.12. Objectives of the Current Research………………..…………………....……………...21 2. MATERIALS AND METHODS……………………………………..……………………22 2.1. Onion Samples…………………………………………………………….……………..22 2.2. Chemicals and Reagents…………..…………………………………….……………….23 2.3. Plant Material…………………………………………………………….………………24 2.4. Pressurized Low Polarity Water Extraction…….………………………….…………….24 2.5. High Performance Liquid Chromatography (HPLC) Analysis of Phenolic compounds..25 2.6. Quantification of Flavonoid Standard………………………………………………...….26 2.7. Total Polyphenols………………………………………………………………………..27 2.8. Total Flavonoid assay…………………………………………………………………....27 iv 2.9. Antioxidant assays……………………………………………………………………….28 2.9.1. DPPH Radical Scavenging Activity assay (2,2-diphenyl-1-picryl-hydrazyl-hydrate). 28 2.9.2. ABTS Radical Scavenging assay (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)…………………………………………………………………………………………..29 2.9.3. Ferric Reducing Antioxidant Power (FRAP) assay……………………..……...……....29 2.9.4. Total Antioxidant Capacity (TAC) Colorimetric assay……………………………...…30 2.9.5. Lipid Peroxidation (MDA) assay………...………………….…………………….…...30 2.9.6. Oxygen Radical Absorbance Capacity (ORAC) assay…………………..…..………...31 2.10. Cell Culture……………………………...…………………………………….….….....32 2.11. Caco-2 Cell Proliferation by MTS Colorimetric assay……………………..……..…….32 2.12. Determination of Osmotic Lysis and Cytotoxicity…………………..………………….33 2.13. Induction of Apoptosis in Human Colorectal Adenocarcinoma Cells…………...….…..33 2.14. Cell Migration…………………………………………………………………....……..34 2.15. Statistical Analysis………...…………………………………………………….…...…34 3. RESULTS AND DISCUSSION………………………………………………………...…35 3.1. Extraction and Quantification of Flavonoids by HPLC Analysis…………….…..…..….35 3.2. Total Polyphenols Content………...…………………………………………..……..…..40 3.3. Total Flavonoid Contents…………...……………………………………..…………..…43 3.4. Antioxidant assays……………………………………………….……….…………..….45 3.4.1. DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) Radical Scavenging Activity assay…...………………………………………………………………………..….………...47 3.4.2. ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) Radical Cation Decolourization…….…………………………….….……………………………………….49 3.4.3. Ferric Ion Reducing Antioxidant Power (FRAP) assay……………………..………….51 3.4.4. Total Antioxidant Capacity (TAC) assay……………………………….……………...53 3.4.5. Lipid Peroxidation Malondialdehyde (MDA) assay………………………………...…54 3.4.6. Oxygen Radical Absorbance Capacity (ORAC) assay………………………….……...56 3.5. Correlation between TPC, TFC and Total Antioxidant Activity of the onion extracts tested……………………………………………………………………………….…………59 3.6. Cell Culture Studies…………...………………………………………..……………..…63 3.6.1. Cell Proliferation by MTS assay……………………………………......………..…….63 v 3.6.2. Cytotoxicity and Cytolysis by Lactate Dehydrogenase (LDH)……….…..………...…65 3.6.3. Induction of Cell Death in Caco-2 cells by Flavonoids……………………………..…..67 3.6.4. Cell Migration assay…………………………...………………………………………70 4. CONCLUSIONS…………………………………………………………………………..73 5. REFERENCES…………………………………………………………………………….77 vi LIST OF FIGURES Figure 1. Canadian colorectal cancer statistics (2016). Source: Canadian cancer society………………………………………………………………………………………….2 Figure 2. Inhibition of free radicals by antioxidants………………………………………….10 Figure 3. Photograph of the Speed SFE NP model 7100 pressurized low polarity instrument (Applied Separation Inc., Allentown, PA, USA) used for this study at the facilities of the Guelph Food Research Centre of Agriculture and Agri-Food Canada (AAFC)………………………………………………………………..……………..……….14 Figure 4. Photograph of the Cytation 5 multimode plate reader (Biotek, Winooski, VT) used at Bionano Lab, University of Guelph…...……………………………………………………21 Figure 5. Ontario grown onion varieties A) Fortress, B) Safrane, C) Lasalle, D) Ruby Ring, and E) Stanley.…………….………………………………………...………………………..22 Figure 6. Flowchart showing the process of extracting onion powder. The four steps would be: (1) peeling, cutting and grinding, (2) smashed onions kept at -30°C in the fridge overnight, (3) freeze drying of the smashed onions, (4) fine powdered samples stored for analysis. ………..24 Figure 7. HPLC chromatogram peaks of the flavonoids and anthocyanins identified from the five varieties of Ontario grown onions namely A) Fortress B) Lasalle C) Safrane D) Stanley E) Ruby Ring at 254 nm…...…………………………………………………………………….26 Figure 8. Determined total phenolic content from the five onion varieties namely Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF). The results were calculated as mean ±SD, n= 5.………………………………………………………………..42 Figure 9. The total flavonoid content present in the five onion varieties namely Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF). The results were calculated as mean ±SD, n= 5.………………………………………………………………..44 Figure 10. The DPPH radical scavenging activity (% of inhibition) for the five different Ontario grown onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF). The data collected for three different experiments is calculated as mean ±SD, n= 5; p<0.05……...…………………………………………………………..…………48 vii Figure 11. ABTS radical scavenging activity (% inhibition) for the five Ontario grown onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane (SAF) The data collected for three different experiments is calculated as mean ±SD, n= 5; p<0.05………………………………………………………………………………………...50