Antioxidant and Antiproliferative Activity of Flavonoids from Ontario Grown Onions by Pressurized

108 Pages·2017·2.7 MB·English

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.

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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
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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
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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

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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
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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
Figure 12. The reducing power activity expressed as mM ascorbic acid equivalent tested for
the five different Ontario grown onion varieties namely Lasalle (LAS), Fortress (FOR), Ruby
Ring (RR), Stanley (STA) and Safrane (SAF). The results were collected for three different
experiments and were calculated as mean ±SD, n= 5; p<0.05……………………………….52
Figure 13. Total Antioxidant Capacity (TAC) assay expressed as mM trolox equivalent for the
five different Ontario grown onion varieties Lasalle (LAS), Fortress (FOR), Ruby Ring (RR),
Stanley (STA) and Safrane (SAF). The results done were collected for three different
experiments and calculated as mean ±SD, n= 5; p<0.05.……………………………..….…..54
Figure 14. Lipid peroxidation malondialdehyde (MDA) (nmol/g) forOntario grown onion
varieties namely Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley (STA) and Safrane
(SAF). The results done were collected for three different experiments and calculated as mean
±SD, n= 5; p<0.05……...……………………..………………………………………………55
Figure 15. The oxygen radical absorbance capacity (ORAC) (µM trolox equivalent) for the
five Ontario grown onion varieties: Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley
(STA) and Safrane (SAF) (mean ±SD, n= 5); p<0.05.…………………………………….....58
Figure 16. A) Linear correlation between total phenolic content (TPC) and percentage (%) of
antioxidant activity (ABTS scavenged effect) B) Linear correlations between total flavonoid
content (TFC) and percentage (%) of antioxidant activity (ABTS scavenged effect) C) Linear
correlation between TPC and percentage (%) of antioxidant activity (FRAP activity) D)
Correlation between TFC and percentage (%) of antioxidant activity (FRAP activity) E) Linear
correlation between TPC and ORAC (oxygen radical antioxidant capacity) F) Correlation
between TFC and oxygen radical absorbance capacity (ORAC activity)…………….……….62
Figure 17. Inhibition of Caco-2 human colorectal adenocarcinoma cancer cells treated with the
fiver varieties of onion extracts at a dilution factor of 10. Five-fold decrease in absorbance was
seen when compared to the untreated control wells. A strong antiproliferative effect of the
onion extract samples on caco-2 growth was observed. UNTRT: Untreated; LAS: Lasalle; RR:
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Ruby Ring; FOR: Fortress; STA: Stanley; SAF: Safrane; QUE: Quercetin; MYR: Myricetin;
KAE: Kaempferol…………………...…………………………………….………………….64
Figure 18. Cytotoxic effect of the Ontario grown onion varieties on caco-2 cell viability
expressed as a ratio relative to the maximum theoretical LDH release. UNTRT: Untreated;
LAS: Lasalle; RR: Ruby Ring; FOR: Fortress; STA: Stanley; SAF: Safrane; QUE: Quercetin;
MYR: Myricetin; KAE: Kaempferol..……………………………………………..…………66
Figure 19. A statistically significant increase was observed in the number of apoptotic cells
after the treatment of Caco-2 cancer cells along with the onion extract samples. There were no
significant differences noticed between the extract treatment groups. UNTRT: Untreated; LAS:
Lasalle; RR: Ruby Ring; FOR: Fortress; STA: Stanley; SAF: Safrane; QUE: Quercetin; MYR:
Myricetin……………………………………………………………………………………..68
Figure 20. Anti-migratory activity of the Ontario grown onion extracts exerted a 40 – 60%
reduction in the number of cells migrating to the cell free exclusion zone. The exclusion zone
myricetin treated wells was found to be confluent. UNTRT: Untreated; Lasalle (LAS); Ruby
Ring (RR); Fortress (FOR); Stanley (STA); Safrane (SAF); Quercetin (QUE); Myricetin
(MYR); Kaempferol (KAE)…………………………………………………………………..71
Figure 21. Images represents untreated Caco-2 cells or cells treated with Lasalle (LAS) - a
yellow onion variety flavonoid and Quercetin at 10x dilution and 100 µM respectively, after 0
and 72 h incubation period…...………………………………………………………..……...73

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LIST OF TABLES

Table 1. List of flavonoids compounds identified from the bulbs of the onion
varieties………………………………………………………………………………………36
Table 2. List of flavonoids compounds identified from the bulbs of the onion
varieties………………………………………………………………………………………36
Table 3. Concentration of the total polyphenolics present in each of the Ontario grown onion
varieties (mg/kg dried onion sample)…….…………………………………..………………37

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