The Effects of Cooked Whole Asparagus (Asparagus Officinalis L.) and Its Purified Bioactive, Rutin (PDFDrive)

The Effects of Cooked Whole Asparagus (Asparagus officinalis L.
) and its
Purified Bioactive, Rutin, on Symptoms of DSS-induced Acute Colitis and
Recovery in C57BL/6 Mice
By
Jenifer Thi Lu
A Thesis
Presented to
The University of Guelph
In partial fulfillment of requirements

for the degree of
Master of Science
in Human Health and Nutritional Sciences
Guelph, Ontario, Canada

© Jenifer Thi Lu, January 2013
ABSTRACT
THE EFFECTS OF COOKED WHOLE ASPARAGUS (ASPARAGUS OFFICINALIS L.) AND ITS
PURIFIED BIOACTIVE, RUTIN, ON SYMPTOMS OF DSS-INDUCED ACUTE COLITIS AND
RECOVERY IN C57BL/6 MICE
Jenifer Thi Lu Advisor: Dr. Lindsay Robinson

University of Guelph, 2013
This thesis explored the effects of cooked whole asparagus and its purified bioactive,
rutin, on colitis symptoms and disease progression in mice using a chemically-induced
model of colitis. This model mimics active colitis and recovery states of ulcerative colitis.
C57BL/6 mice were fed a basal diet supplemented with 2% asparagus or 0.025% rutin for
3 weeks. Colitis was induced by 2% dextran sodium sulfate in drinking water for 7 days.
Asparagus diet was determined to contain higher antioxidant capacities than rutin diet
through antioxidant assays. During active colitis, consumption of asparagus alleviated some
clinical symptoms (stool consistency, stool blood, and spleen hypertrophy) of colitis. In
recovery, asparagus-fed mice were improving in terms of regenerating crypts, surface
epithelial, and goblet cells, potentially due to its rutin content. Overall, these findings
advocate that asparagus can be therapeutic in treating symptoms during active colitis and
recovery phases of ulcerative colitis.

ACKNOWLEDGEMENTS
I am very grateful to have been given a wonderful opportunity in working in this
OMAFRA-funded Asparagus Project. Over these past two years, not only was I able
experience the life of a graduate student, but was also given the chance to meet and work
with a team of very knowledgeable, hard-working, and inspiring individuals from various
departments and fields. I would like start off with thanking my co-advisor, Dr. Krista
Power, who was the first person to introduce me to scientific research in 2009. It may
sound cliché, but “thank you” is simply not enough to explain my gratitude. She goes above
and beyond as an advisor and mentor, giving her personal time and efforts to help her
students succeed. I would also like to thank my advisor, Dr. Lindsay Robinson, and my
committee members, Dr. Rong Cao and Dr. David Wolyn, for all their respective expertise,
encouragement, and helpful advice, especially with writing this thesis. And thank you to Dr.
Alison Duncan who accepted to chair my defense.
Individuals at the Guelph Food Research Center have also contributed plenty to my
thesis project as well as made my time there extra meaningful and pleasant. I really
enjoyed all the planned events, such as the yearly Christmas Potlucks and summer BBQs. I
would like to acknowledge the contribution of Dr. Ronghua Liu for all her help and
expertise in HPLC, and Wendy Wu for her comforting presence and assistance in the lab.
And I am grateful to Claire Zhang, Leila Zarepoor, Emily Padhi, and Kelly Wang, for not only
their help within the lab, but also for being there for me as caring and lighthearted friends.
I would like to thank staff at the Department of HHNS, particularly Ann Stride, Andra
Williams, and Anne Lovett-Hutchinson, for their welcoming presences and graduate studies
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support and advice. And also special thanks to staff at the Department of Plant Agriculture,
specifically Verena Kulak and Jean Wolting for their quick assistance in purchasing orders.
My utmost appreciation goes to my family. Mom and dad: thank you for your
unconditional love, support, and encouragement, and for all the sacrifices you have made to
get me where I am today. I look up to both of you and hope that one day I can be as good as
a parent as you two are. Also, a thank you to my adorable younger sisters, Kathleen and
Caroline Lu, for being the best sisters in the world; you two can make my worries go away
in an instant. I would also like to thank Yen-Hsun Chen, my confidant and pillar of strength,
who believed in me at times when I did not. And lastly, thank you to my amazing roommate
and loving friend, Daisy (Ruyan) Dai, who made sure I was never too stressed and was well
fed every day (by her wonderful cooking skills!). I love you all dearly.
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Table of Contents
Chapter 1 : Literature Review .......................................................................................................................... 1
1.1 Inflammatory Bowel Disease and Ulcerative Colitis ....................................................................... 2
1.1.1 Overview ........................................................................................................................................ 2
1.1.2 Epidemiology ................................................................................................................................ 2
1.2 Risk Factors of IBD ....................................................................................................................................... 3
1.2.1 Genetic Susceptibility ................................................................................................................ 4
1.2.2 Immune System and Microflora ............................................................................................ 5
1.2.3 Environmental Factors ............................................................................................................. 6
1.3 Animal Models of IBD.................................................................................................................................. 7
1.3.1 Overview ........................................................................................................................................ 7
1.3.2 Dextran Sodium Sulfate Model of Colitis ........................................................................... 8
1.4 Etiology .......................................................................................................................................................... 14
1.4.1 Inflammatory Mediators in Intestinal Immunity ........................................................ 14
1.4.2 Reactive Oxygen Species and Oxidative Stress............................................................. 18
1.5 Complications with Ulcerative Colitis ............................................................................................... 19
1.6 Therapy ......................................................................................................................................................... 20
1.6.1 Conventional Treatments ..................................................................................................... 20
1.6.2 Complementary and Alternative Treatments ............................................................... 21
1.6.3 Natural Dietary Interventions............................................................................................. 23
1.7 Phenolic Compounds and Health ........................................................................................................ 24
1.7.1 Nomenclature and Overview............................................................................................... 24
1.7.2 Flavonoids .................................................................................................................................. 25
1.7.3 Pure Phenolic Compounds in Experimental IBD ......................................................... 26
1.8 Rutin and Quercetin.................................................................................................................................. 26
1.8.1 Overview ..................................................................................................................................... 26
1.8.2 Rutin and Quercetin in vivo and in vitro.......................................................................... 28
1.9 Asparagus (Asparagus officinalis L.)................................................................................................... 35
1.9.1 Overview ..................................................................................................................................... 35
1.9.2 Phenolic Compounds in Asparagus .................................................................................. 36
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Chapter 2 : Study Rationale, Objectives, and Hypotheses .................................................................. 38
2.1 Asparagus (Asparagus officinalis L.)................................................................................................... 39
2.2 Hypothesis.................................................................................................................................................... 40
2.3 Objectives ..................................................................................................................................................... 41
Chapter 3 : Chemical Characterization of Asparagus Flour and Assessing the Suitability of
Supplementing Asparagus into C57BL/6 Mice Diets ........................................................................... 42
3.1 Introduction ................................................................................................................................................. 43
3.2 Experiment 1: Asparagus Flour Preparation and Rutin Analysis .......................................... 44
3.2.1 Materials and Methods ....................................................................................................................... 44
3.2.1.2 Analysis of Rutin Concentration in Asparagus Flour ................................................. 45
3.2.1.3 Macronutrients and Soluble Fibre in Asparagus Flour ......................................... 46
3.3 Experiment 2: Determining the Suitability of Supplementing Asparagus into C57BL/6
Mice Diets ..................................................................................................................................................... 48
3.3.1 Materials and Methods .......................................................................................................... 48
3.3.2 Results and Discussion .......................................................................................................... 51
3.4 Summary ....................................................................................................................................................... 55
Chapter 4 : The Effects of Rutin on Acute Colitis Symptoms and Disease Progression in DSS
exposed C57BL/6 Mice .................................................................................................................................... 57
4.1 Introduction ................................................................................................................................................. 58
4.2 Materials and Methods ............................................................................................................................ 60
4.2.1 Diet Preparation ....................................................................................................................... 60
4.2.2 Mice ............................................................................................................................................... 61
4.2.3 Study Design .............................................................................................................................. 61
4.2.4 Colonic MPO Assay .................................................................................................................. 64
4.2.5 Colon Histopathology ............................................................................................................. 65
4.2.6 Statistical Analyses .................................................................................................................. 67
4.3 Results............................................................................................................................................................ 68
4.3.1 Determination of Rutin in Diets ......................................................................................... 68
4.3.2 Pre-DSS Food Intake and BW .............................................................................................. 69
4.3.3 Food and Water Intake During Acute Colitis ................................................................ 70
4.3.4 DAI During Acute Colitis ....................................................................................................... 71
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4.3.5 Organ Size Parameters During Acute Colitis ................................................................. 73
4.3.6 Colonic MPO Activity During Acute Colitis .................................................................... 76
4.3.7 Colon Histopathology During Acute Colitis ................................................................... 77
4.4 Discussion..................................................................................................................................................... 78
Chapter 5 : The Effects of Asparagus and its Purified Bioactive, Rutin, on DSS-induced Acute
Colitis and Recovery in C57BL/6 Mice ...................................................................................................... 84
5.1 Introduction ................................................................................................................................................. 85
5.2 Experiment 1: Asparagus Flour Preparation and Rutin Analysis ........................................... 86
5.2.2 Results and Discussion .......................................................................................................... 87
5.3 Experiment 2: Determining the Effects of Asparagus and Purified Rutin on DSS-induced
Colitis and Recovery in C57BL/6 Mice .............................................................................................. 88
5.3.2 Results .......................................................................................................................................... 97
5.3.3 Part 1: Acute Colitis Results ................................................................................................. 99
5.3.4 Part 2: Colitis Recovery Results ....................................................................................... 104
5.3.5 Discussion ................................................................................................................................. 113
Chapter 6 : Thesis Overview, Future Directions, and Implications .............................................. 118
6.1 Thesis Overview ....................................................................................................................................... 120
6.2 Future Directions ..................................................................................................................................... 122
6.2.1 Asparagus Characterization............................................................................................... 122
6.2.2 Bioavailability and Bioaccessibility ................................................................................ 123
6.2.3 Mechanisms of Action of Asparagus on Colon Health ............................................. 124
6.3 Implications ............................................................................................................................................... 127
Chapter 7 : References ................................................................................................................................... 129
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List of Tables
TABLE 1-1: A SUMMARY OF SOME COMMONLY STUDIED TH1 CYTOKINES ........................................................ 17
TABLE 1-2: QUERCETIN AND RUTIN IN EXPERIMENTAL COLITIS IN VIVO AND IN VITRO .................................. 31
TABLE 1-3: PROXIMATE ANALYSIS OF ONE SERVING (1 CUP) OF RAW ASPARAGUS [222] ............................. 35
TABLE 3-1: MACRONUTRIENT COMPOSITION OF ASPARAGUS FLOUR ................................................................ 48
TABLE 3-2: COMPOSITION OF EXPERIMENTAL DIETS (BD AND 12% ASPARAGUS)........................................ 50
TABLE 4-1: COMPOSITION OF EXPERIMENTAL DIETS (BD AND 0.1% RUTIN) ................................................ 60
TABLE 4-2: DAI SCORING SYSTEM ........................................................................................................................ 63
TABLE 4-3: HISTOLOGICAL GRADING OF COLON EROSION DURING DSS-INDUCED ACUTE COLITIS ................ 66
TABLE 4-4: HISTOLOGICAL GRADING OF GOBLET CELL DEPLETION DURING DSS-INDUCED COLITIS ............ 67
TABLE 5-1: MACRONUTRIENT COMPOSITION OF ASPARAGUS FLOUR ................................................................ 88
TABLE 5-2: COMPOSITION OF EXPERIMENTAL DIETS (BD, 2% ASPARAGUS, AND 0.025% RUTIN) ............ 90
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List of Figures
FIGURE 1-1: CHEMICAL STRUCTURES OF PHENOLIC COMPOUNDS ..................................................................... 25
FIGURE 3-1: ASPARAGUS PREPARATION .............................................................................................................. 45
FIGURE 3-2: RUTIN CONCENTRATION IN ASPARAGUS FLOUR ............................................................................. 47
FIGURE 3-3: DETERMINATION OF RUTIN IN BASAL DIET (BD) AND 12% ASPARAGUS DIET ......................... 52
FIGURE 3-4: FOOD INTAKE AND BODY WEIGHT (BW) MEASURED OVER 21 DAYS .......................................... 54
FIGURE 3-5: DETERMINATION OF RUTIN IN 3, 6, AND 12% ASPARAGUS DIETS .............................................. 55
FIGURE 4-1: EXPERIMENTAL DESIGN ................................................................................................................... 63
FIGURE 4-2: METHOD OF COLLECTING DISTAL COLON SECTIONS ...................................................................... 64
FIGURE 4-3: HISTOLOGICAL GRADING OF COLON EROSION IN DSS-INDUCED ACUTE COLITIS ........................ 66
FIGURE 4-4: HISTOLOGICAL GRADING OF GOBLET CELL DEPLETION IN DSS-INDUCED ACUTE COLITIS......... 67
FIGURE 4-5: DETERMINATION OF RUTIN IN BASAL DIET (BD), AND 0.025, 0.05, AND 0.1% RUTIN DIETS
.......................................................................................................................................................................... 69
FIGURE 4-6: PRE-DSS FOOD INTAKE AND BW ................................................................................................... 70
FIGURE 4-7: FOOD AND WATER INTAKE DURING DSS-INDUCED ACUTE COLITIS ............................................. 71
FIGURE 4-8: EFFECTS OF RUTIN DIETS (0.025%, 0.05%, AND 0.1%) ON DAI CLINICAL SYMPTOMS OF
DSS-INDUCED ACUTE COLITIS ....................................................................................................................... 73
FIGURE 4-9: EFFECTS OF RUTIN DIETS ON ORGAN SIZE PARAMETERS IN DSS-INDUCED ACUTE COLITIS ...... 75
FIGURE 4-10: EFFECTS OF RUTIN DIETS ON DISTAL COLON MYELOPEROXIDASE (MPO) DURING DSS-
INDUCED ACUTE COLITIS................................................................................................................................. 76
FIGURE 4-11: EFFECTS OF RUTIN DIETS ON DISTAL COLON HISTOPATHOLOGY ............................................... 77
FIGURE 5-1: ANALYSIS OF RUTIN CONCENTRATION IN ASPARAGUS FLOUR ...................................................... 87
FIGURE 5-2: EXPERIMENTAL DESIGN.................................................................................................................... 93
FIGURE 5-3: HISTOLOGICAL GRADING OF COLON EROSION DURING RECOVERY ............................................... 94
FIGURE 5-4: HISTOLOGICAL GRADING OF GOBLET CELL DEPLETION DURING RECOVERY ................................ 95
FIGURE 5-5: DETERMINATION OF RUTIN IN BD, 2% ASPARAGUS, AND 0.025% RUTIN DIETS .................... 97
FIGURE 5-6: ANTIOXIDANT ACTIVITY OF BD, 2% ASPARAGUS, AND 0.025% RUTIN DIETS ......................... 98
FIGURE 5-7: PRE-DSS FOOD INTAKE AND BW ................................................................................................... 99
FIGURE 5-8: FOOD AND WATER INTAKE DURING DSS-INDUCED ACUTE COLITIS ........................................... 100
FIGURE 5-9: EFFECTS OF 2% ASPARAGUS AND 0.025% RUTIN DIETS ON DAI DURING DSS-INDUCED ACUTE
COLITIS ........................................................................................................................................................... 101
FIGURE 5-10: EFFECTS OF 2% ASPARAGUS AND 0.025% RUTIN DIETS ON ORGAN SIZE PARAMETERS
DURING DSS-INDUCED ACUTE COLITIS ....................................................................................................... 102
FIGURE 5-11: EFFECTS OF 2% ASPARAGUS AND 0.025% RUTIN DIETS ON MYELOPEROXIDASE (MPO)
ACTIVITY DURING ACUTE COLITIS ................................................................................................................ 103
FIGURE 5-12: HISTOPATHOLOGY OF DISTAL COLON EROSION AND GOBLET CELL DEPLETION DURING ACUTE
COLITIS ........................................................................................................................................................... 104
FIGURE 5-13: FOOD AND WATER INTAKE DURING COLITIS RECOVERY ........................................................... 105
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FIGURE 5-14: IMPACT OF 2% ASPARAGUS AND 0.025% RUTIN DIETS ON SURVIVAL DURING COLITIS
RECOVERY IN MICE ........................................................................................................................................ 106
FIGURE 5-15: EFFECTS OF DIETS ON DAI DURING COLITIS RECOVERY ........................................................... 107
FIGURE 5-16: EFFECTS OF 2% ASPARAGUS AND 0.025% RUTIN DIETS ON ORGAN SIZE PARAMETERS
DURING COLITIS RECOVERY .......................................................................................................................... 108
FIGURE 5-17: EFFECTS OF 2% ASPARAGUS AND 0.025% RUTIN DIETS ON MYELOPEROXIDASE (MPO)
ACTIVITY DURING COLITIS RECOVERY ......................................................................................................... 109
FIGURE 5-18: HISTOPATHOLOGY OF DISTAL COLON EROSION DURING COLITIS RECOVERY .......................... 110
FIGURE 5-19: HISTOPATHOLOGY OF DISTAL COLON GOBLET CELL DEPLETION DURING COLITIS RECOVERY
........................................................................................................................................................................ 111
FIGURE 5-20: SERUM TH1 CYTOKINE MULTIPLEX ANALYSIS IN COLITIS RECOVERY...................................... 112
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Abbreviations
5-ASA 5-aminosalicylic acid KO Knockout
AAE Acetic acid equivalent LPS lipopolysaccharide
AP Alkaline phosphatase MAPK Mitogen-activated protein kinase
APC Antigen-presenting cells mdr1a-/- Multidrug resistance gene-deficient
BD Basal diet MeOH Methanol
BMDM Bone marrow-derived MPO Myeloperoxidase
macrophages
BW Body weight MW Molecular weight
c.i. Colitis induction MZ Monozygotic twins
CAC Colitis-associated colorectal NF-κB Nuclear factor kappa B
cancer
CAM Complementary and alternative NFDL NF-κB-dependent luciferase
medicine
CD Crohn’s disease NK Natural killer cells
DAI Disease activity index OMAFRA Ontario Ministry of Agriculture,
Food and Rural Affairs
DC Dendritic cells ORAC Oxygen radical absorbance capacity
DSS Dextran sodium sulfate PBS Phosphate buffered saline
DW Dry weight PUFAs Polyunsaturated fatty acids
DZ Dizygotic twins QOL Quality of life
EtOH Ethanol RCT Randomized controlled trials
FOS Fructo-oligosaccharides ROS Reactive oxygen species
FRAP Ferric reducing/antioxidant RT Room temperature
power assay
GBF Germinated barley foodstuff SA-PE Streptavidin-phycoerythrin
GIT Gastrointestinal tract SCFA Short-chain fatty acids
GSH Glutathione SCID Severe combined immunodeficiency
GST Glutathione S-transferase SOD Superoxide dismutase
GPX Glutathione peroxidase SPF Specific-pathogen-free
HPLC High-performance liquid TE Trolox equivalent
chromatography
HUVECs Human umbilical vein TH T helper cell
endothelial cells
IBD Inflammatory bowel disease TJ Tight junctions
IECs Intestinal epithelial cells TLR Toll-like receptor
IFN-γ Interferon gamma TNBS 2,4,6-trinitrobenzene sulfonic acid
iNOS Inducible nitric oxide synthase TNF-α Tumor necrosis factor alpha
IL Interleukin UC Ulcerative colitis
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Chapter 1 : Literature Review
1
1.1 Inflammatory Bowel Disease and Ulcerative Colitis
1.1.1 Overview
Inflammatory bowel disease (IBD) is a spontaneous chronic, relapsing, and
remitting disorder of the gastrointestinal tract (GIT) that comprises two major subtypes:
ulcerative colitis (UC) and Crohn’s disease (CD) [1]. Both conditions have significant
impacts on the quality of life (QOL) of patients, producing similar disease symptoms such
as abdominal pain, bleeding, diarrhea, and weight loss. However, UC and CD are
dramatically distinct with regards to disease location, morphology, and histopathology. In
UC, intestinal inflammation is restricted to the large intestine (colon), whereas CD can
encompass any part of GIT, but involves mostly the distal small intestine (ileum) and the
proximal colon [2, 3]. Morphologically, inflammation in UC affects only the mucosal layer of
the intestinal wall, but with CD, it can involve all layers of the intestinal wall. UC and CD do
share many inflammatory similarities such as epithelial barrier dysfunction, but
histologically, UC usually consists of more neutrophil infiltration, goblet cell mucin
depletion, and prominent crypt abscesses, while CD consists of skip lesions, non-
necrotizing granulomas, and pseudopolyps [4]. Although most patients with IBD can lead
productive lives with the use of medications and surgery, IBD is considered a lifetime
disease that has substantial personal burden with increased morbidity [3].
1.1.2 Epidemiology
IBD has been traditionally considered a disease of developed Western countries [5].
However, IBD is now emerging as a global disease as the incidence and prevalence of the
2
disorder is increasing with time and in various regions around the world [6, 7]. In Europe
and North America, the annual incidence rates for UC ranges from 0.6 to 24.3 cases, and
from 2.2 to 19.2 cases per 100,000, respectively [5, 6], while values are markedly
decreased in Asia and the Middle East with 0.1 to 6.3 cases per 100,000 [5, 6]. With regards
to prevalence rates for UC, North America has the highest with 7,000 to 46,000 newly
diagnosed cases each year [5]. Although the incidence and prevalence rates in Western
countries such as North America and Europe are still quite high, the rates are starting to
stabilize in those high-incidence populations, while continuing to increase in low-incidence
nations such as those in southern and eastern Europe [8-10] and Asia [11-15]. For instance,
time trend statistics from four Asian studies [14] demonstrated that there was: a 5-fold
increase in UC incidence amongst Japanese patients within 20 years [16]; a 7-fold increase
within 10 years in Seoul, Korea [11]; and a 6-fold increase in Hong Kong Chinese within 20
years [17]. Overall, the burden of UC appears to be increasing over time as developing
nations become increasingly industrialized [5, 18], which stresses the need for improved
therapeutic and preventative treatments.
1.2 Risk Factors of IBD
As a multifactorial disorder, the development of UC is influenced by several factors
including genetic susceptibility of the host, the host immune system, and other
environmental factors [2, 19-21].
3
1.2.1 Genetic Susceptibility
Population-based studies suggest that genetic susceptibility plays a role in the
pathogenesis of IBD, where both CD and UC are clustered as related polygenic disorders
[22]. About 20% of people with UC have a close relative with IBD [23], and the risk of
getting IBD increases 2 to 13 times for offspring of patients who have IBD. For patients who
have UC, their offspring would have an occurrence of 6.26%, and for CD patients, the
occurrence would be 9.2% [24]. However, the cause of IBD cannot be attributed to only
genetics as monozygotic (MZ; genetically identical) and dizygotic (DZ) twin studies have
shown low concordance rates. Combined data from a Swedish [25], British [26], and a
Danish [27] twin study indicated that the concordance rates for UC in MZ and DZ twins
were 15% and 4%, respectively [28].
There has also been substantial progress in the last 2 years in characterizing the
susceptibility genes involved in IBD using genome-wide association scanning in large
studies of cases and controls [29-31]. CD and UC have been confirmed in 99 susceptibility
loci/genes, 71 associated with CD and 47 with UC [32, 33]. There are multiple genes
involved in the interleukin (IL)-23/Th17 signalling pathway, including STAT3 and IL-12B,
which have been noted to be important in regulating intestinal immune homeostasis [34].
Another signaling pathway that appears to be playing a major role in IBD pathogenesis is
the IL-10 pathway [35, 36]. IL-10 is a key anti-inflammatory cytokine that helps induce and
maintain tolerance toward commensal bacteria [36]. Case-control studies have provided
evidence that IL-10 polymorphisms are significantly associated with IBD occurrence [37-
39]. Gene targeting animal studies using IL-10 gene-deficient mice have discovered that
unless these mice were kept in germ-free conditions, they would develop spontaneous
4
enterocolitis with mucosal hyperplasia, increased numbers of crypt cells, and inflammatory
cell response, to just name a few [40, 41].
1.2.2 Immune System and Microflora
It has been speculated that IBD arises from an inappropriate activation of the
immune system to the colonic microflora in part due to immunoregulatory defects of the
colonic mucosa [42]. Human GIT is colonized with an immense community of symbionts
and commensals that have vital roles in immune functioning, nutrient processing, and
other host activities [43]. In a healthy, non-diseased gut, these symbiotic and commensal
bacteria live peacefully with their host, but in a diseased state or in a genetically
susceptible individual, the relationship between indigenous gut microbes and their hosts
can shift from commensalism toward pathogenicity [43]. One genetic variant that has been
identified in CD patients, NOD2, has been implicated in altering the immune system so that
it initiates an amplified response to bacteria, causing inflammation [44]. A recent study
showed that NOD2 mutation in CD aggravated nuclear factor kappa B (NF-κB) activity and
IL-1β processing, suggesting initiation and/or promotion of mucosal inflammation [45].
Conversely, UC has been associated with a mutation in the Toll-like receptor (TLR)-4 gene
in humans [46], which is needed to detect lipopolysaccharide (LPS) contained in the cell
walls of Gram-negative bacteria in order to activate the innate immune system [47]. Thus,
carriers with this TLR-4 mutation would be at an increased risk for Gram-negative
infections and an activated mucosal immune system.
A continuous stimulation of the mucosal immune system can lead to uncontrolled
inflammation and subsequent defects to the intestinal epithelial barrier function,
5
particularly an increase in permeability of the intestinal epithelial cells (IECs) [48, 49]. This
barrier is needed to protect against luminal antigens and microorganisms from entering
the body [50]. Without this, commensal and pathogenic bacteria can cross over and illicit a
perpetual cycle of inflammation with innate and adaptive immune cells [51].
1.2.3 Environmental Factors
The significant growth in the incidence of UC during the last half century,
particularly with developing countries with a history of low prevalence, may indicate that
environmental risk factors are playing a major role in disease development [5, 19].
Emigration [52-54], industrialization, and urbanization may explain the increase in UC
incidence as changes to occupational status, diet, and lifestyle habits have all been
implicated as potential environmental risk factors for UC [5, 18]. For example, UC is more
prevalent in higher socioeconomic groups, affecting more white than blue-collar workers.
Workers who have occupations outdoors are less prone to developing UC, whereas
sedentary indoor workers have an increased risk [55].
The current role of dietary components and habits on the development and
progression of UC is not conclusive. The most consistent association noted in dietary
studies has been the connection between an increased risk of UC with high intakes of total
fats, omega-6 fatty acids, and red meat, and a decreased risk of UC with high vegetable and
fruit intake [56]. A recent case-control study also reported that a low consumption of fibre
with a high consumption of refined sugar were significantly associated with the
development of UC [57].
Interestingly, it has been widely publicized that there is an inverse relationship
between UC and cigarette smoking [58-60]; however, a meta-analysis showed that former
6
cigarette smokers had a 70% greater risk of developing UC compared to those who never
smoked [61]. The mechanisms underlying this protective effect are currently unknown, but
smoking can alter a wide range of both innate and adaptive immune functions [62]. For
instance, in clinical trials, smoking has reduced mucosal cytokine production [63],
decreased distal bowel permeability [64], and increased colonic mucus production [65].
Environmental factors are undoubtedly involved with the pathogenesis of UC, and it
is probable that the complex interactions between each factor may be responsible for the
rising incidence worldwide.
1.3 Animal Models of IBD
To fully understand the molecular events that may occur during colonic
inflammation, reliable and reproducible models of colitis are necessary. To test various
hypotheses regarding the etiology of IBD, numerous experimental models of colitis have
become available in the last decade [66]. Progress in understanding the mechanisms
involved in IBD has increased due to these models. Presently, experimental models of
colitis can be characterized as genetic [gene knockout (KO), transgenic], immunological
(adoptive cell transfer), or chemical-based [67].
1.3.1 Overview
Gene KO models involve ‘knocking-out’ or making a specific gene or DNA region
inoperative, in order to understand the roles and functions of that specific deleted gene or
DNA compared to a wildtype control. Some well-studied gene KO models of IBD include IL-
10 KO and IL-2 KO/IL-2 receptor KO mice, which have been found to develop spontaneous
7
colitis after 3 months of age [67, 68]. Transgenic models are also used to investigate the
role and functions of specific genes or DNA. Yet, transgenic models usually use mice that
are genetically engineered to overexpress a certain gene, such as with the IL-7 transgenic
mice; mice overexpressing IL-7 were discovered to develop spontaneous acute colitis
within 1-3 weeks of age [68]. Immunological models comprise of transferring specific host
cell populations, often T cells, to severe combined immunodeficiency (SCID) mice to illicit
colonic inflammation. These models allow for the study of immunopathology and immune
regulation in the intestine [69]. Lastly, the most common chemicals used to induce colitis in
rodents include dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzene sulfonic acid
(TNBS), which are administered orally and rectally, respectively [67, 68]. All of these
experimental models can produce diverse ulceration patterns at different locations in the
GIT. For instance, the DSS model closely resembles UC since ulcerations occur in the colon,
whereas TNBS mimics CD since ulcerations occur in the upper intestinal tract [70]. While
none of these experimental models can fully replicate human IBD, they are still quite useful
in developing novel therapeutic strategies and elucidating the etiology of IBD. For the
purpose of this thesis, the DSS-induced colitis model will only be used and discussed.
1.3.2 Dextran Sodium Sulfate Model of Colitis
To date, DSS is one of the most common chemical compounds to induce colitis in
animals. In the DSS model, colitis is induced by adding DSS into the animals’ drinking water
[71, 72]. Acute or chronic colitis can be induced depending on the concentration, duration,
and frequency of DSS administration. To induce acute colitis, the amount of DSS that is
added to drinking water is usually between 2 and 5%, and it is given to the animals daily
8
for short periods (4-14 days) [71, 73]. Meanwhile, to induce chronic colitis, a low
concentration of DSS, such as 1%, is given continuously for long periods (e.g. 100 days [74]
or even up to 6 months [75]), or through cycles of relatively high doses of DSS and normal
drinking water [71, 73]. The following sections will only focus on acute colitis induced by
DSS.
1.3.2.1 DSS Overview
DSS is a polyanionic derivative of dextran, a complex polymer of straight and
branched chains of glucose. Dextran is synthesized from sucrose by specific lactic-acid
bacteria, such as Leuconostoc spp and Streptococcus spp [76]. When dextran is esterified
with chlorosulphonic acid, it produces DSS, where the sulphur content is about 17%, which
is equivalent to approximately two sulfate groups per glucosyl residue of the dextran
molecule [71].
IBD is known to be a complex multifactorial disease, which is not reproducible in
any cell culture system. Therefore experimental models of IBD are most crucially needed to
investigate the pathophysiologies. Since 1985, when the first model of DSS was used to
induce colitis in hamsters [77], the model has been extensively used over the years with
other animals. Now the DSS model is widely accepted as a highly reproducible and simple-
to-induce model that is applicable to various animals (e.g. mice, rats, hamsters) [71, 78].
The model is also inexpensive, may either induce acute or chronic inflammation, and is
perceived as a representative model of experimental colitis as it mimics symptoms of UC,
clinically and histologically [70, 71, 79, 80]. However, the severity of DSS-induced
inflammation is dependent on the animal strain, DSS molecular weight (MW), and degree of
9
sulfation, and as previously mentioned, the concentration or dose, and frequency and
duration of exposure of DSS [78].
Mice can show different susceptibilities and responsiveness to DSS-induced colitis,
and this may be due to the genetics (strain and gender) and microbiological aspects of the
animal. A study analyzing DSS-induced colitis (3.5% DSS of 36-45 kDa MW for 5 days) in
nine strains of mice discovered major differences in DSS responsiveness among inbred
strains [81]. C3H/HeJ, NOD/Ltj, NOD-scid inbred strains were very susceptible to DSS-
induced lesions, while 129/SvPas and DBA/2J inbred mice showed less susceptibility [81].
Other studies have found that BALB/c mice, which are often used in DSS-colitis studies,
were also less susceptible to DSS compared to IQI/Jic [82] or C57BL/6 mice [83]. A
significant gender bias had also been found within the nine mouse strains exposed to DSS;
male mice had increased histological colon scores of severity and hyperplasia compared to
female mice [81]. There are also differences in the recovery process from inflammatory
damage, as Melgar et al. (2005) have shown that after DSS withdrawal (3% DSS for
C57BL/6 and 5% DSS for BALB/C for 5 days), BALB/c mice were able to partly recover
from the colitis insult within one week, while C57BL/6 mice progressed into chronic colitis
[83]. This indicates that genetic factors are contributing to DSS susceptibility and are
involved in the regulation of the mucosa in either limiting the inflammatory response
and/or resisting inflammatory damage.
The MW of DSS is also important in determining the severity of colitis that will be
induced [84]. DSS is highly variable in terms of MW, ranging from 5 kDa to up to 1,400 kDa
[78]. Administration of DSS at three different MW (i.e. 5, 40, and 500 kDa) produced
various degrees of colitis severity that was also location-dependent in BALB/c mice [84].
10
DSS of 5 kDa produced only sporadic lesions in the cecum and upper colon. Meanwhile, DSS
of 40 kDa produced more severe colitis than 5 kDa, affecting the middle and distal third of
the large bowel. And lastly, 500 kDa produced no lesions in the large bowel of BALB/c
mice; it was determined that this MW was too large, thereby preventing its passage
through the mucosal membrane to exert its effects [84].
1.3.2.2 Clinical, Histological, and Molecular Features of DSS-induced Colitis
The clinical and histological symptoms of DSS-induced colitis are most reflective of
UC. In the acute phase of DSS-induced colitis, weight loss, increase in stool consistency
(diarrhea), presence of bloody stool, reduced physical activity, and a decrease in food and
water intake are initially observed [71, 78]. These clinical symptoms can be seen within 2
days post-colitis induction [78], and the inflammation is entirely established within 7-10
days [81].
Histologically, DSS-induced acute colitis affects mainly the mucosa of the colon, but
can spread to the submucosa and muscularis mucosa [71]. The typical histological
characteristics include: crypt distortion; epithelial cell loss; increased mucosal edema and
permeability; immune cell infiltration (e.g. neutrophils, macrophages, and lymphocytes)
into the lamina propria and submucosa; goblet cell loss and decreased mucin production;
presence of ulcers; cryptitis; and crypt abscesses [71, 78, 83, 85]. Cryptitis and crypt
abscesses are common features of human IBD, and they involve transepithelial migration of
neutrophils into mucosal epithelial lining and crypt lumen, respectively [83]. Crypt loss and
subsequent crypt shortening occur around day 2 or 3 of DSS intake, and are thus
considered the earliest histological signs of colitis, even before the development of
inflammation [71, 85]. And by day 5 post-administration of DSS, erosion and inflammation
11
are observed [85]. It has been found that the removal of DSS results in recovery, including a
restitution of normal colonic architecture [81, 86], yet the extent of recovery is dependent
on mouse strain [83].
Molecularly, there have been various biomarkers detected with DSS-induced colitis,
which are also associated in human UC. For instance, myeloperoxidase (MPO) is a
mammalian enzyme that is stored in neutrophils and macrophages, which are recruited
during inflammatory processes. Thus, MPO activity is an indicator of the degree of
neutrophil infiltration and has been positively correlated with worsened disease state in
IBD patients [87], and also positively correlated with an increased exposure to DSS [88].
Furthermore, the expression of proinflammatory cytokines (e.g. IL-10, IL-12, interferon
(IFN)-ɣ, tumor necrosis factor (TNF)-α, and IL-1β) are also significantly increased in the
colon of IBD patients [89], and with DSS induction in mice [88].
Overall, the DSS model of acute colitis is an effective model for UC as it reliably
produces clinical, histological, and molecular features that are representatively observed in
UC patients. Therefore, this model is deemed suitable for investigating the pathogenesis
and therapeutic options for colitis.
1.3.2.3 Pathogenesis of DSS-induced Colitis
The mechanisms through which DSS initiates colitis are presently unknown, but
there have been several proposed hypotheses involving an increase in gut permeability
(e.g. mucus layer, tight junctions (TJ), epithelial cells). The increase in gut permeability
would allow the permeation of luminal bacteria and large molecules, such as DSS.
First, due to the structure of DSS, it is likely that DSS can directly alter the mucus
structure within the large intestine. There are two mucus layers, made up of a gel-forming
12
mucin called MUC2, which are found firmly attached to the epithelium surface [90]. These
layers are there to prevent luminal bacteria from crossing over into our bodies. It was
found through an in vitro study that DSS can cause quick changes to the mucus layers,
increasing permeability to bacteria [91]. The glycans on MUC2 mucin from human colon
carries multiple negative charges on sulfate residues [92]. Mucin sulfation is deemed
important for mucus function as mice lacking the Nas1 sulfotransporter that is needed for
sulfation of mucins, have increased susceptibility to colitis [93]. So it is probable that
sulfated DSS can be readily soluble in the sulfated MUC2 mucin network, thereby allowing
the sulfates in the DSS to destabilize the interactions that maintain the organization of the
mucin network.
Second, DSS can directly alter gut permeability by interacting with TJ proteins.
Studies indicate that TJ proteins, such as zona occludens-1, were reduced by DSS as early as
day 1 and increased gut permeability by day 3, which were both observed before colonic
inflammation [85, 94].
Lastly, DSS may be changing gut permeability by directly acting as a toxin to colonic
epithelial cells. In vivo studies have reported an increase in epithelial cell apoptosis with
DSS-induced colitis [95, 96]. Vetuschi et al. (2002) found that a 7 day colitis induction with
DSS in Sprague-Dawley rats increased epithelial apoptotic index by 20-fold compared to
healthy controls. Araki et al. (2010) also reported a 5-fold increase in epithelial apoptosis
in DSS-induced BALB/c mice compared to controls [95]. They also found that there was a
significant reduction (about 2-fold) in cell proliferation, thereby causing defects in the
integrity of the epithelial barrier [95]. Therefore, it appears that DSS can directly affect the
balance between apoptosis and proliferation of colonic epithelial cells.
13
In light of the above, changes in gut permeability from DSS interfering and causing
injury to the mucus layers, TJs, and epithelial cells, would allow the permeation of luminal
bacteria and content into colonic mucosa. An activated inflammatory response, further
damage to epithelial cells, and basal crypts would all likely ensue due to this permeation.
Similar to other experimental models of IBD, the DSS-induced colitis model does not fully
reflect human UC, but the proposed hypotheses of DSS pathogenesis is well correlated with
what is known with human IBD. Research has found that UC patients also had reduced
adherent mucus [97], changes in TJ protein expression [49], and increased epithelial cell
apoptosis [98]. Overall, this demonstrates that the DSS-induced colitis model is not only
reliable, reproducible, and simple to use, but it is also the most current representative
experimental model of human UC, clinically, histologically, molecularly, and pathogenically.
1.4 Etiology
The exact etiology of IBD remains unknown; however, with the vast amount of
experimental models that are currently available, as previously discussed, it is possible to
test various hypotheses [66]. Progress in understanding the mechanisms involved in IBD
have risen due to these models. For the purpose of this thesis, the role of inflammatory
mediators and oxidative stress in IBD pathogenesis will only be discussed.
1.4.1 Inflammatory Mediators in Intestinal Immunity
The epithelium, containing a dense network of innate and adaptive immune cells, is
central in monitoring the integrity and health of the mucosa by eliciting appropriate
responses to invading bacteria and antigens. Amongst the antigen-presenting cells (APCs),
14
dendritic cells (DC) are the most commonly found subtype in the intestinal lamina propria
[99]. Once activated by interaction with an antigen or injury, they initiate the adaptive
immune response by migrating to lymph nodes where they interact with T and B cells. T
helper cell (TH) gets activated by activated DC and they differentiate into either TH1 or TH2
cells. TH1 or TH2 cells then migrate to the site of injury to secrete specific small signalling
proteins called cytokines, which help mediate the inflammatory pathway [100].
Cytokines can be categorized into either pro- or anti-inflammatory
(immunosuppressor), or both (immunomodulatory), depending on the way they influence
inflammation [101] (Table 1-1). TH1 cells are involved with macrophages and are marked
by an upregulation of TNF-α, IL-1β, IFN-ɣ, IL-6, and IL-17, while TH2 cells are mainly
involved with B cells and increases in IL-4, IL-5, and IL-13 secretions [102]. These
increases in cytokine release can initiate the recruitment of other immune cells, such as
natural killer (NK) cells, neutrophils, and macrophages. These immune cells in turn
increase the levels of cytokines, amplifying the process of inflammatory defense. For
instance, NK cells can secrete IFN-ɣ and TNF-α [103], and neutrophils can release IL-1β, IL-
10, IL-6, and TNF-α [104]. Macrophages, in particular, participate in an autoregulatory
loop, where they become activated by certain cytokines, such as IFN-ɣ and TNF-α, and then
become deactivated by producing IL-10 [105].
Research over the past decade has proposed that dysfunctions of the intestinal
immune system and cross-reactivity against host epithelial cells may be one of the
etiologies of IBD. A failure to control normally protective cell-mediated responses, as
described above, can result in sustained activation of the mucosal immune system and
uncontrolled overproduction of pro-inflammatory cytokines and mediators. As previously
15
mentioned, patients with IBD have been found to have increased polymorphisms, such as
with IL-10 [37-39] and the TLR-4 gene [46], compared to healthy controls. Data from
different genetically engineered or immune-manipulated animal models of colitis have also
supported this hypothesis. For instance, studies involving mice with targeted deletions of
IL-2 and IL-10 have resulted in spontaneous and chronic colitis [106]. These findings have
resulted in the emergence of novel drug and dietary therapies for IBD which target specific
cytokines or receptors in the inflammatory cascade, including TNF-α and IFN-ɣ [102, 107].
16
Table 1-1: A summary of some commonly studied TH1 cytokines
Cytokine Activities References
(Secreted by)
IL-10 Anti-inflammatory/immunosuppressor: [104, 108,
(neutrophils, T cells, B - Inhibits antigen presentation and 109]
cells, NK cells) suppresses release of pro-inflammatory
cytokines (e.g. IFN-ɣ, TNF-α)
IL-1β Pro-inflammatory: [104, 110]
(neutrophils, resident - Immune defense against infection, ↑
macrophages, DC, transmigration of leukocytes (e.g.
monocytes) neutrophils)
- Resets hypothalamus thermoregulatory
center, leading to ↑ in body temperature
TNF-α Pro-inflammatory: [104, 111]
(neutrophils, APCs, NK - Stimulates IL-1β and IL-6 secretion, inhibits
cells) apoptosis, ↑ fibroblast and procoagulant
proliferation
- Activates neutrophils, macrophages, B cells,
↑ IFN-ɣ by T cells
IL-6 Pro- and anti-inflammatory [104, 112,
(neutrophils, T cells, (immunomodulatory): 113]
resident macrophages) - Induces B cell differentiation, T cell
proliferation and differentiation, ↑ IL-2
- Mediator in fever and acute phase
responses
- Can control pro-inflammatory cytokine
levels (e.g. TNF-α)
1L-17A Pro- and anti-inflammatory [114-116]
(TH cells) (immunomodulatory):
- Recruits immune cells to peripheral tissues
(needs NF-κB activation)
- Induces TNF-α, IL-6, and IL-1β
IFN-ɣ Pro- and anti-inflammatory [117]
(NK cells) (immunomodulatory):
- Antiviral and antiproliferative
- Activates neutrophils, NK cells, and vascular
endothelial cells, promotes T and B cell
differentiation
17
1.4.2 Reactive Oxygen Species and Oxidative Stress
Another proposed mechanism of IBD pathogenesis is through oxidative stress
caused by an imbalance of prooxidant and antioxidant pathways involving reactive oxygen
species (ROS) [118]. ROS are chemically reactive molecules containing oxygen that are
normally produced as byproducts of normal metabolism [119]. Direct measurements of
ROS are problematic due to their short half-lives; however, present techniques estimate
oxidative status by observing key antioxidant enzymes, such as glutathione (GSH),
superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) [119].
Furthermore, assays for antioxidant capacity, such as the oxygen radical absorbance
capacity (ORAC) and ferric reducing/antioxidant power assay (FRAP), can provide a
general assessment of antioxidant status [120].
It has been noted that there is increased free radical DNA damage and decreased
plasma antioxidant defences in both UC and CD [121]. Rodent models have been used to
evaluate the status of ROS during colitis. Typical observations included an increased
upregulation of ROS-inducing enzymes, such as lipid peroxidase, inducible nitric oxide
synthase (iNOS), MPO, and GPX; and a downregulation of antioxidant enzymes, such as
SOD, GSH, catalase, and glutathione S-transferase (GST) [122-124]. Esworthy et al. (2001)
has also provided a causal link between ROS in UC pathogenesis; they showed that mice
lacking the antioxidant GPX developed symptoms similar to UC even in the absence of any
chemical insults [124]. Recent studies have proposed that oxidative stress may be a
component by which cytokine gene expression is upregulated through the activation of NF-
κB [125].
18
Aside from having imbalances in the prooxidant and antioxidant pathways, an
imbalance with inflammatory mediators in intestinal immunity, as previously discussed,
may also contribute to the increased production of ROS in UC. One known characteristic of
UC is the infiltration of neutrophils and immune cells into inflamed mucosa [4]. These
immune cells are not only secreting cytokines, chemokines, and adhesion molecules to help
mediate the inflammatory process, but they also secrete ROS that increases epithelial
permeability [126]. Activated neutrophils are also known to generate superoxide radicals,
hydrogen peroxide, and MPO, which all have been involved with tissue damage [127].
Currently, it is unknown whether oxidative stress occurs prior to immune cell
recruitment or the infiltration of immune cells into inflamed tissue causes an increase
oxidative stress. Yet, it is recognized that there are homeostatic imbalances in either or
both of the pathways involved in oxidative stress and intestinal immunity. Overall, this
highlights the need for additional research to investigate potential treatments in targeting
both ROS and inflammatory mediators.
1.5 Complications with Ulcerative Colitis
Aside from a decreased QOL and personal and financial burdens [3], there are
additional health complications for UC patients due to the relapsing and remitting nature of
the disease. Through an epidemiological study, Langholz et al. (1994) found that 25% of UC
patients underwent remission within the first 3-7 years after diagnosis; 57% had recurring
relapses; and that the cumulative probability of having a relapse was 90% after 25 years of
follow-up [128].
19
The first complication with UC is the need for a coloproctectomy caused by the acute
severe colitis, which occurs in 20-25% of patients after 10-25 years [128, 129]. Long-term
problems after surgery include mortality, with an average annual rate of 0.7%, and
morbidity, such as reoperations and disturbance of sexual function [130].
The second and most fatal complication is the onset of colitis-associated colorectal
cancer (CAC) due to persistent, chronic inflammation [131]. Within 30 years of IBD onset,
more than 20% of patients will develop CAC and more than 50% will die from it because
the disease is very difficult to treat [132].
The final complication of UC is the development of extra-intestinal manifestations.
Patients with active UC can acquire disorders involving the joints (23%), skin (15%),
mouth and eye (each 4%) [133, 134]. Other complications include osteoporosis (3%), liver
disease (5%), and peptic ulcers (10%) [134-136].
1.6 Therapy
In the absence of a cure, therapy is directed towards alleviating disease symptoms
and/or maintaining remission (i.e. free from symptoms) to achieve an improved QOL.
Although disease-related mortality is low, morbidity is high since most UC patients are
continuously on medications, but when those fail, surgery (colectomy) is often required.
1.6.1 Conventional Treatments
Currently, conventional medications for UC include aminosalicylates,
corticosteroids, antibiotics, and immunomodulators. Sulfasalazine, and its derivative,
mesalazine or 5-aminosalicylic acid (5-ASA; mesalamine) are the most commonly used
forms of aminosalicylates. These have been used as first-line therapies for many years
20
during active phases and for the induction and maintenance of remission [137]. They have
been proposed to act upon epithelial cells by various mechanisms to regulate the release of
cytokines [138] and ROS [139]. Corticosteroids, such as prednisone and hydrocortisone,
are usually used for acute episodes of UC. Their mechanisms of action include inhibiting
certain cytokines, and inflammatory pathways such as the arachidonic acid cascade [140].
Unlike aminosalicylates and corticosteroids, antibiotics such as metronidazole and
vancomycin have been less effective in UC patients [141]. Immunomodulators are a new
class of drugs that are presently being investigated for UC patients to avoid long-term
usage of corticosteroids. Immunosuppressive agents, azathioprine and 6-mercaptopurine,
inhibits the proliferation of T and B lymphocytes by causing chromosomal breaks [142,
143].
Although most of these drugs are deemed safe to use, there are potential adverse
side effects with certain individuals. For instance, side effects from using 5-ASA can occur
in 10-45% of people, depending on the dose used, and symptoms may range from diarrhea
to pancreatitis [144]. Moreover, a prolonged use of steroids can increase the risk of
osteoporosis [145], impair wound healing, and cause hyperglycemia [146]. Adverse effects
of metronidazole include peripheral neuropathy, nausea, and neutropenia [142], while
immunomodulators can cause bone marrow suppression, pancreatitis, and/or minor
allergies characterized by abdominal pain, rash, and fever [143].
1.6.2 Complementary and Alternative Treatments
UC patients are increasingly using complementary and alternative medicine (CAM),
which include traditional practices such as acupuncture, as they believe they are less toxic
21
than conventional drugs, Chinese herbal medicine, aromatherapy, and/or reflexology
[147]. CAM overall appears to be more popular in Western populations than European
countries with currently up to 50% using some form of CAM [148]. Yet, the usage of CAM is
still quite high with European and Asian patients as herbal therapy seems to be the most
commonly reported therapy for IBD [149, 150].
Traditional Chinese medicine is a broad range of practices that has been a tradition
for more than 2,000 years. There have been various randomized controlled trials (RCT)
regarding certain herbal medicines in the treatment of UC, such as Kui jie qing [151], aloe
vera gel [152], wheat grass juice [153], and germinated barley food (GBF) stuff [154]. All of
these RCT reported positive results; for instance, 72% of patients treated with Kui jie qing
(alum, Halloysite, Calamine, Indigo naturalis, and plum-blossom tongue-pointing pills)
enemas were considered cured compared to 9% of controls who took sulfasalazine, oral
prednisolone, and prednisolone enemas [147]. It also appears that there are greater effects
when CAM and conventional therapy are given together than when either of the treatments
is administered separately [155]. For example, patients treated with only GBF, which
consisted of dietary fibre and glutamine-rich protein, had worse clinical disease activity
compared to conventional therapy alone, but when GBF and 5-ASA were combined, rectal
bleeding and diarrhea were reduced significantly in UC patients [154].
Although CAMs may work for some or many individuals over conventional means, it
is still worth noting that they are not always safe as most herbal remedies are unregulated
[156]. Unpurified herbal extracts contain a vast array of biologically active compounds,
which may have either beneficial or adverse effects. There have been some reports of
significant poisoning effects of some herbs and even severe side effects involving herb-drug
22
interactions [157-159]. Adulterations have also been discovered in herbal remedies, such
as the deliberate inclusion of prescription medicines (e.g. corticosteroids) [160], and/or
other toxic contaminants like lead, mercury, and arsenic to induce a desired effect [161].
Therefore, a licensing authority similar in conventional medicine is urgently needed for
herbal preparations in order to enforce quality control and regulate the production of safe
CAM products for UC therapy.
1.6.3 Natural Dietary Interventions
Over the past decade, the use of dietary interventions in achieving UC remission has
increasingly become an area of great interest as they have been found to provide effective
therapy without the adverse effects as seen with conventional drugs and CAM [162-165].
Some natural dietary components that have been studied extensively are prebiotics,
polyphenols, and essential polyunsaturated fatty acids (PUFAs). Prebiotics are non-
digestible dietary fibre (e.g. psyllium, inulin, germinated barley) that are consumed by
endogenous protective bacteria to stimulate their growth. The beneficial effects of
prebiotics are due to the metabolites or short-chain fatty acids (SCFAs) produced from
fermentation by colonic bacteria [166]. SCFAs are important in upholding colonic barrier
integrity and function by serving as an energy source for enterocytes [167]. Moreover,
SCFAs, such as butyrate, have been found to reduce colitis and anti-inflammatory cytokines,
IL-1β, TNF-α, and IL-17 [168-171]. Conversely, phenolic compounds, such as curcumin,
quercetin, rutin, and resveratrol, can exert anti-inflammatory effects by modulating NF-κB
[172-174] and mitogen-activated protein kinases (MAPKs) [175] signalling pathways and
levels of pro-inflammatory markers [174, 176] in animals with induced colitis. The role of
23
phenolics in UC will be discussed further in the succeeding sections. Lastly, omega-3 PUFAs
such as eicosapentaenoic acid and docosahexaenoic acid have also been found to alleviate a
number of inflammatory diseases including UC [177-180]. Proposed mechanisms of PUFAs
may also be through the reduction of NF-κB activity and expression of pro-inflammatory
cytokines [178, 181].
Overall, the current usage of conventional medical treatments by UC patients in the
form of anti-inflammatory (e.g. 5-ASA) and immunosuppressive (e.g. corticosteroids) drugs
[182], and the unconventional therapies involving CAM, all derive adverse side effects.
Nutrition is now proposed to be a potential candidate in IBD management due to the
evidence purported in the last decade with anti-inflammation and certain food
components, such as PUFAs and prebiotics. Therefore, diet-based approaches may play a
pivotal role in the clinical care of all patients with UC and hold promise for both preventing
and treating the disease.
1.7 Phenolic Compounds and Health
1.7.1 Nomenclature and Overview
As previously mentioned, phenolic compounds are one class of natural dietary
interventions in alleviating symptoms of UC. Phenolic compounds are naturally occurring
plant molecules found abundantly in fruits and vegetables, with currently more than 8,000
phenolic structures still to be discovered [183]. The base structure of a phenolic compound
comprises an aromatic or benzene ring with a carboxyl group attached (Figure 1-1A) [184].
There are many classes of phenolics and they are all defined by the nature of their carbon
24
backbone and the functional derivatives (esters, methyl esters, glycosides, etc.) that are
attached [185].
Figure 1-1: Chemical structures of phenolic compounds

The above shows the chemical base structure of (A) a phenolic compound, and chemical
structures of (B) rutin, and (C) quercetin. Modified from [184, 186, 187].
1.7.2 Flavonoids
One of the most common phenolic classes is the flavonoid family; almost all plant
tissues are able to synthesize flavonoids. Presently, there are more than 4,000 naturally
occurring flavonoids that have been identified. Flavonoids are most often found in nature
as glycosides (a compound bound to a sugar or carbohydrate moiety), but can occur as
aglycones (non-sugar compound) in woody tissues and particularly from food processing
25
effects [188, 189]. Their base structure consists of two aromatic rings linked by three
carbons that are usually in an oxygenated C ring [188].
Flavonoids can be additionally classified into six groups depending on their
heterocycle C ring: flavones, flavanones, flavonols, isoflavones, flavanols (catechins), and
anthocyanidins [188]. Human consumption of all flavonoids is about a few hundred
milligrams [190] to 650 mg/day [191]. The most commonly consumed flavonoids are the
flavonols and flavones. Some common flavonols include quercetin, kaempferol, and
myricetin found in onions, apples, broccoli, and tea, to name a few, whereas the most
commonly consumed flavones are luteolin and apigenin found in celery, spinach, and green
pepper [192].
1.7.3 Pure Phenolic Compounds in Experimental IBD
From a vast amount of in vitro and in vivo evidence, phenolic compounds have been
acknowledged to be antioxidant and anti-inflammatory, and therefore have been proposed
to be an alternative approach to preventing or treating inflammatory diseases, including
IBD [193]. Quercetin, and its glycoside, rutin, have been the most investigated phenolic
compounds in relation to IBD (Table 1-2), and therefore will be discussed in this thesis.
1.8 Rutin and Quercetin
1.8.1 Overview
Rutin (C27H30O16; 610.517 g/mol; Figure 1-1B), also called rutoside, quercetin-3-
rutinoside, and sophorin, is a flavonoid glycoside found in asparagus, buckwheat, fruit
rinds, and the leaves and petioles of Rheum species [194-196]. Rutin has been found to be
26
chemically stable in both phosphate buffer and hydrochloric acid conditions (mimicking
the small intestine and stomach environments, respectively) [197]. When ingested, rutin
travels intact to the large intestine, where it is then rapidly hydrolyzed to its aglycone
quercetin (C15H10O7; 302.236 g/mol; Figure 1-1C) by β-glucosidase activity of colonic
microflora, such as Bacteroides distasonis, B. uniformis, and B. ovatus [198, 199].
Quercetin itself is a flavonol and is also widely distributed in nature and can be
found in foods such as apples, onions, and red grapes [200-203]. The total average intake of
flavonols (quercetin, myricetin, kaempferol) and flavones (luteolin, apigenin) was observed
to be 23 mg/day, with about 70% from quercetin [204].
In terms of bioavailability, rutin has been found to be absorbed more slowly than
quercetin in Wistar rats according to observed concentrations of quercetin metabolites in
plasma [205]. Carbonaro and Grant (2005) also found the same results but in rats of the
Hooded Lister strain; although uptake rates were similar for quercetin and rutin during the
first 5 minutes of exposure to the compounds, the maximum percentage of flavonoid that
was taken up by the gut over the following 35 minutes was significantly higher for
quercetin (60%) than for rutin (45%) [206]. It has been speculated that the absorption of
rutin by colonic epithelial cells is delayed in comparison with quercetin because the sugar
moiety on the C ring of rutin must be hydrolyzed by microflora in the large intestine prior
to absorption [207]. Moreover, in two studies observing the pharmacokinetics of quercetin
and rutin in healthy volunteers, it was found that quercetin was actively absorbed from the
small intestine and possibly even in the stomach, while the majority of rutin can travel
intact to the distal parts of the small intestine or the colon to be deglycosylated and then
absorbed [208, 209]. This has also been confirmed in animal studies where they did not see
27
any deglycosylation of rutin in the upper intestine, but rapid deglycosylation in the large
intestine [205, 210]. Thus, it may be hypothesized that quercetin and rutin would have
different anti-inflammatory effects in diseases where the occurrence of inflammation is
location-dependent along the GIT, such as in UC and CD.
1.8.2 Rutin and Quercetin in vivo and in vitro
1.8.2.1 Rutin
Rutin is proposed to be anti-inflammatory and anti-oxidative as it has been shown
to significantly reduce colitis severity in several chemically-induced colitis rodent models
(Table 1-2). One example is a study by Kwon et al. (2005), who used a DSS-induced colitis
model with female ICR mice and fed them a diet containing either 0.001, 0.01 or 0.1% rutin
or quercetin for 2 weeks. After 2 weeks, they found that dietary rutin, even at a low dose
(0.01%) attenuated colorectum shortening and IL-1β production. In addition, oral
administration of 0.1% rutin 3 days after DSS treatment significantly reversed colitis, as
shown by suppression of both colorectum shortening and IL-1β production by 43 and 52%,
respectively, compared to the control [176]. Another notable finding was from the study by
Kim et al. (2005), who found that an oral administration of rutin [10 mg/Kg body weight
(BW)] ameliorated TNBS-induced colitis in rats, reduced MPO activity to about 55% of the
control and was just as effective as sulfasalazine (30 mg/Kg BW), a common colon-specific
drug for the treatment of IBD [210].
The effects of rutin have been reported to act through suppressing pro-
inflammatory cytokine production in mice, such as IL-1β and IL-6 [176], reducing MPO
activity [210-212], and alleviating oxidative stress in the colon by counteracting GSH
28
depletion [212, 213], which is an essential antioxidant; a depletion of GSH has been seen to
cause a cascade of events resulting in cell death [211]. Moreover, pre-treatment of rutin
(25-100 µM) can dose-dependently inhibit the expression of NF-κB and TNF-α production
in LPS-stimulated human umbilical vein endothelial cells (HUVECs) [214].
1.8.2.2 Quercetin
Compared to rutin, there have only been three animal experimental colitis studies
concerning quercetin, in which only one study found beneficial results (Table 1-2). In the
first study, a rectal administration of 25 M (300 L) of quercetin was found to be just as
effective as 20 mM (300 L) 5-ASA, a common IBD-treating drug, in healing the damaged
colon [210]. However, the other two studies found no significant effects with DSS-induced
colitis severity after giving quercetin orally [172, 176]. The conflicting results may be due
to a bioaccessibility issue; most of the quercetin may not have reached the colon as they
could have been absorbed in the small intestine [176]. As discussed previously, quercetin
has been found to be actively absorbed from the small intestine and possibly even in the
stomach, while the majority of rutin can travel intact to the distal parts of the small
intestine or the colon to be deglycosylated and then absorbed [208, 209]. This may explain
why quercetin has not been seen to ameliorate DSS-induced experimental UC in mice when
quercetin was ingested orally [172, 176], but intracolonic administration of quercetin was
able to heal the colonic damage, as well as lower MPO activity, dose-dependently in rats
[210].
In vitro models suggest that quercetin may be exerting its effects through inhibiting
TNF-α and NF-κB-mediated pathways, including suppressing pro-inflammatory cytokines,
such as IL-6, IL-8, and IL-1β [172, 210, 215, 216].

29
In summary, with the vast amount of evidence purporting the anti-inflammatory
and anti-oxidative activities of rutin and quercetin in vivo and in vitro, the roles of these
phenolic compounds as therapeutic tools in managing colonic inflammation is very
promising. The ability of rutin and quercetin to improve symptoms of colitis, reduce MPO
activity, and modulate TNF-α and NF-κB signalling and levels of pro-inflammatory
cytokines, encourages the use of these natural compounds clinically. These results also
promote future research to explore the effects of consuming whole foods rich in rutin or
quercetin, such as asparagus, in experimental colitis.
30
Table 1-2: Quercetin and rutin in experimental colitis in vivo and in vitro
In vivo
Phenolic Study Design Animal Model Results Reference

Compound
300 µL (10, 25, 50, and Male Sprague- Reduced colitis severity (colonic damage with [210]
100 µM) in phosphate Dawley rats, 15 regards to inflammation and ulceration
buffered saline (PBS) mg/0.3 mL/rat TNBS severity) in a dose-dependent manner, lower
rectally, 1 day after colitis (rectally) MPO activity.
induction (c.i.), 6 d
Quercetin 1 mg/Kg/d, 5 d after 5% Female Wistar rats, No significant reduction in colitis severity. [172]
DSS c.i. 5% DSS, 5 d, then 2%
DSS for 10 d
0.001 (60 µg/d), 0.01 (0.6 Female ICR mice, 5% No significant effect on histological signs of [176]
mg/d), or 0.1% (6 mg/d), DSS, 1 wk colitis severity.
14 d prior or 3 d after c.i.
10 mg/Kg/d solution Male Wistar rats, 42 Pellets coated with alginate/chitosan and rectal [211]
rectally or orally, or 10 mg/Kg TNBS rutin solution showed the best efficacy in
mg/Kg/d pellets coated (rectally) colonic healing clinically and histologically (i.e.
with caffeic erosion of mucosa), and decreased colon
Rutin acid/HPMC/alginic acid; weight/BW ratio and MPO activity.
sodium
alginate/HPMC/zinc
acetate; or sodium
alginate/chitosan, 5 d
31
0.1% rutin in AIN-76A Male multidrug No significant effect on histological signs of [217]
diet, 15 or 18 wk resistance gene- colitis severity.
deficient (mdr1a-/-)
mice
10 mg/Kg/d, 1 day after Male Sprague- Significantly healed the damaged colon and [210]
c.i., 6 d Dawley rats, 15 reduced MPO activity to about 55% of control.
mg/0.3 mL/rat TNBS
(rectally)
0.001 (60 µg/d), 0.01 (0.6 Female ICR mice, 5% Reduced colitis severity (BW loss and [176]
mg/d), or 0.1% (6 mg/d), DSS, 1 wk histologically), attenuated DSS-induced colon
14 d prior or 3 d after c.i. shortening, decreased IL-1β mRNA and protein
expression, and IL-6 mRNA expression in a
dose-dependent manner.
Acute colitis: 5, 10, 25, 50, Female Wistar rats, Acute colitis: 10 and 25 mg/Kg increased mean [212]
100 mg/Kg in dH2O, 48, 10 mg TNBS food intake, reduced macroscopic colonic
24, and 1 hr prior c.i., and (rectally) damage (i.e. colonic mucosa) after 48 hr; 10
24 hr after c.i. mg/Kg significantly had a lower loss of BW;
GSH contents were increased by 66.6%, 46.5%
Chronic colitis: 10 and 25 and 53.4% at doses of 5, 10, and 25 mg/Kg; 25
mg/Kg/d in dH2O, 24 hr mg/Kg reduced MPO and alkaline phosphatase
after c.i. for 1 wk or 2 wks (AP) activity.
Chronic colitis: After 1 or 2 wks, 10 and 25

mg/Kg reduced colonic damage score (i.e. ulcer
lesions, hyperemia, thickening of bowel wall),
MPO activity and increased GSH content; 2 wks
32
treatment reduced AP activity and inhibition of
LTB4 synthesis.
10, 25, 100 mg/Kg, 48, 24, Female Wistar rats, 25 and 100 mg/Kg reduced histologic injury [213]
and 1 hr prior c.i. 4% acetic acid (v/v) and prevented increase in AP activity, and 25
(rectally) mg/Kg counteracted GSH depletion.
In vitro
Phenolic Study Design Cell Model Results Reference

Compound
Mode-K cells stimulated Primary IEC were 100 µmol/L inhibited TNF-induced IFN-ɣ- [215]
with TNF in absence or isolated from the inducible protein IP-10 and macrophage MIP-2
presence of 100 µmol/L ileum of gene expressions at both 40 and 44 µmol/L.
quercetin TNFDαRE/WT mice Molecularly, quercetin inhibited akt
(develop phosphorylation and NF-κB binding to the IP-
spontaneous 10 and MIP-2 gene promoters, therefore
experimental ileitis) further blocking cofactor recruitment and HAT
to grow Mode-K cells activity at the chromatin of these promoters.
Quercetin
TNF-α with presence or Human colon Significantly inhibited TNF-α-mediated NFDL [210]
absence of 5, 25, 50, or epithelial cell lines expression by attenuating IL-8 upregulation,
100 µM, 16 h post- HT-29, HCT116, and dose-dependently.
transfection SW620, transfected
with NF-κB-
dependent luciferase
(NFDL) plasmid
33
Presence or absence of 1, Bone marrow- Pre-treatment dose-dependently inhibited [172]
10, or 50 µM for 1 hr, then derived secretion of TNF-α and IL-1β, and the
stimulated with LPS (50 macrophages phosphorylation of the IκB-α protein (hence
ng/mL) for 12 and 24 hr (BMDM) inhibiting activation of NF-κB pathway).
Neutrophils incubated Blood from healthy LPS induced IL-6 expression of neutrophils, but [216]
with or without LPS (100 volunteers; after pre-treatment of neutrophils with
ng/mL) for 6 hr. Some neutrophils isolated quercetin for 30 min, the effects of LPS on
neutrophils pretreated using 3% dextran increasing IL-6 (mRNA and level) were
with quercetin (40 µM) for sedimentation and eliminated.
30 min and then 100 density gradient
ng/mL LPS for 3 hr. centrifugation
Pretreatment with 0, 2.5, HUVECs Significantly decreased NF-κB activation, and [214]
5, 25, 50, or 100 µM for 6 TNF-α production, dose-dependently (25-100
hr, then stimulated with µM).
LPS (100 ng/mL) for 3 hr
TNF-α with presence or Human colon No significant inhibition of TNF-α-mediated [210]

Rutin absence of 5, 25, 50, or epithelial cell lines NFDL expression.
100 µM, 16 h post- HT-29, HCT116, and
transfection SW620, transfected
with NF-κB-
dependent luciferase
(NFDL) plasmid
34
1.9 Asparagus (Asparagus officinalis L.)
1.9.1 Overview
One vegetable that has recently attracted attention for its potential health benefits is
asparagus (Asparagus officinalis L.). Asparagus is a member of the Asparagales family and is
related to onions, leeks, and garlic [218]. The harvestable portions of asparagus are the
tops (also known as shoots), while the bottoms (also known as butts) are discarded during
processing as they are not marketable [219].
The consumption of asparagus in Canada has been steadily increasing over the
years. Specifically, the intake has nearly doubled over the past 20 years to about 0.2 Kg
asparagus per person according to Statistics Canada [220]. Asparagus is rich in numerous
macro- and micronutrients (Table 1-3), including being a good source of folate [221].
Table 1-3: Proximate analysis of one serving (1 cup) of raw asparagus [222]
Nutrient Units 1 cup (134 g)
Proximates
Water g 124.91
Energy kcal 27
Protein g 2.95
Total lipid (fat) g 0.16
Ash g 0.78
Carbohydrate, by difference g 5.20
Fibre, total dietary g 2.8
Sugars, total g 2.52
Sucrose g 0.31
Glucose (dextrose) g 0.87
Fructose g 1.34
35
One serving (1 cup) of asparagus provides approximately 70 µg/DFE of folate, which
is about one fifth of the RDA in adult males and non-pregnant and -lactating females [222,
223]. Folate has been linked with modulating the risk of developing cancers, most notably
in the colorectum [224, 225]. It has been noted that IBD patients are at a high risk for
developing folate deficiency and CAC due to the ablation of two receptor/carrier-mediated
pathways for folate transport [226]. Asparagus is also a good source of dietary fibre
(soluble and insoluble), providing 2.8 g per serving, or about 7 and 9% of the daily
adequate intake needs for adult men and women, respectively [227]. Dietary fibre from
germinated barley foodstuff (GBF) has been found to reduce colitis severity during active
or relapsing states of colitis in mouse and clinical studies [228-230]. Aside from being rich
in folate and dietary fibre, asparagus has also been found to have high antioxidant capacity
in part from the phenolic compounds embedded in its food matrix [231].
1.9.2 Phenolic Compounds in Asparagus
Among 23 commonly consumed vegetables, the highest antioxidant activity based
on dry weight was seen in asparagus [232]. In recent pharmacological studies, the main
bioactive compounds in asparagus that are responsible for antitumoral and antioxidant
activities are the phytochemicals: saponins, flavonoids, and hydroxycinnamates [233, 234].
The most abundant known flavonoid in asparagus is rutin, which has been shown to
exert antioxidant properties [235]. Maeda et al. (2005) has reported that rutin represents
60-80% of the total phenolic content of purple and green asparagus extracts [231]. It was
observed that rutin in green spears of 12 asparagus hybrid lines yielded levels ranging
from 0.015 to 0.45% of fresh weight. The level of rutin varies with asparagus genotype as
36
well as the tissue location. For instance, rutin has been found to be more abundant at the
upper portions of the spears, while the bottoms of asparagus contain a very small quantity
of rutin (less than 0.01% of fresh weight from three lines of sampled asparagus) [236].
As previously discussed, phenolic compounds and phenolic-rich foods can be
prospective natural dietary interventions for managing and treating IBD because of their
anti-inflammatory properties (Table 1-2). Therefore, rutin-rich asparagus can be a
potential candidate for alleviating active symptoms and maintaining remission in IBD.
37
Chapter 2 : Study Rationale, Objectives, and Hypotheses
38
2.1 Asparagus (Asparagus officinalis L.)
As discussed in Chapter 1, UC, characterized by relapsing and remitting
inflammation of the colon, is becoming quite prevalent worldwide. The disease not only
reduces the QOL of UC patients, but it also results in additional health complications,
including an increase in CAC risk. Current conventional therapies, such as aminosalicylates,
immunomodulators, and antibiotics, are effective in controlling disease symptoms, but are
also associated with severe side effects. Therefore, the use and research of natural dietary
interventions in relieving symptoms and achieving UC remission has garnered a great deal
of attention in the last decade. A body of evidence has advocated that natural dietary
treatments, such as prebiotics, SCFAs, and phenolic compounds, can effectually reduce
intestinal inflammation in UC.
Asparagus is a potential natural dietary treatment for UC patients as it is rich in
micronutrients and macronutrients, dietary fibre, and phenolic compounds. Dietary fibres
are known as prebiotics, and the SCFAs that are produced as a result of bacterial
fermentation, have been shown to reduce colitis and anti-inflammatory cytokines. SCFAs
also have a vital role in maintaining colonic barrier integrity and function by serving as an
energy source for enterocytes. Asparagus is also known for its phenolic content, including
high concentrations of rutin. Many pre-clinical in vitro and in vivo models of experimental
IBD have demonstrated that pure phenolic compounds have anti-inflammatory and anti-
oxidative properties. Therefore, with asparagus containing high amounts of phenolic
compounds, it is a prospective treatment for alleviating symptoms and/or delaying UC
progression.
39
Although there have been numerous in vitro and in vivo studies purporting the anti-
inflammatory and antioxidant effects of rutin, and its respective aglycone quercetin, there
have been no studies observing the association between rutin-rich asparagus intake and
intestinal inflammation in vivo. In fact, there have been no studies conducted using
asparagus in diets of healthy or diseased rodents. Additionally, no studies observing the
effects of rutin in DSS-induced colitis in the C57BL/6 mouse strain have been conducted.
From previous research, the strain of animal used is a significant factor in influencing the
severity of DSS-induced colitis. Thus, this thesis will involve three studies: a pilot feeding
trial to determine if mice will consume diet supplemented with asparagus; a rutin-dose
response study to determine if rutin effectively reduces colitis in the C57BL/6 mouse
strain; and finally, a DSS-induced colitis study comparing the effectiveness of asparagus
and purified rutin during acute colitis and recovery in reducing colitis symptoms and
disease progression.
2.2 Hypothesis
The overall thesis hypothesis is that asparagus, as a rich source of rutin, will reduce
UC symptoms and progression in mice through anti-inflammatory and anti-oxidative
mechanisms.
Specifically, it is hypothesized that:
1) C57BL/6 mice will consume a diet supplemented with cooked asparagus flour
2) Rutin will reduce colitis symptoms and disease progression in the C57BL/6 mouse
strain exposed to DSS
3) Dietary asparagus will reduce colitis symptoms and disease progression similar to rutin
in C57BL/6 mice exposed to DSS during acute colitis and recovery.

40
2.3 Objectives
The thesis objectives are to:
Study 1:
1. Characterize macronutrients, including dietary fibre, and concentration of rutin in
cooked asparagus flour.
2. Determine if C57BL/6 mice will consume diet supplemented with cooked asparagus
flour.
Study 2:
Determine if rutin will alleviate DSS-induced acute colitis symptoms and disease
progression (mimicking active colitis/relapse in UC) in C57BL/6 mice.
Study 3:
1. Determine if dietary cooked asparagus will reduce DSS-induced acute colitis
symptoms and disease progression in C57BL/6 mice.
2. Investigate the effects of dietary cooked asparagus and equivalent levels of purified
rutin during colitis recovery in C57BL/6 mice.
41
Chapter 3 : Chemical Characterization of Asparagus Flour and
Assessing the Suitability of Supplementing Asparagus into
C57BL/6 Mice Diets
42
3.1 Introduction
One vegetable that has attracted attention for its prospective health benefits is
asparagus (Asparagus officinalis L.), a member of the Asparagales family. The consumption
of asparagus in Canada has been steadily increasing over the last few years. Specifically, the
consumption nearly doubled over the past 20 years to about 0.2 Kg asparagus per person
according to Statistics Canada in 2008 [220]. Asparagus is rich in numerous macro- and
micronutrients, including being a good source of folate [221].
Among 23 commonly consumed vegetables, including beans, broccoli and cabbage,
the highest antioxidant activity based on dry weight was observed in asparagus [232]. The
main bioactive compounds in asparagus that are responsible for antitumoral and
antioxidant activities are the phytochemicals: saponins, flavonoids, and hydroxycinnamates
[233, 234]. Asparagus particularly contains a high concentration of rutin, a flavonoid,
which attributes to about 60-80% of its total phenolic content [231]. Due to the anti-
inflammatory properties of rutin and quercetin in various in vivo [176, 210-213] and in
vitro [172, 210, 214-216] colitis models, rutin-rich asparagus may also exert anti-
inflammatory and anti-oxidative activities in IBD. Yet, no study has reported the use of
asparagus in rodent diets. Therefore, this first study was conducted as a pilot trial to
characterize asparagus flour in terms of rutin and macronutrient content and determine
the palatability of diets supplemented with asparagus flour fed to C57BL/6 mice.
43
3.2 Experiment 1: Asparagus Flour Preparation and Rutin Analysis
3.2.1 Materials and Methods
3.2.1.1 Preparation of Asparagus Flour
A batch (51 Kg) of Guelph Millennium asparagus spears (8 mm diameter, about 22
cm long) was provided by the University of Guelph/Ontario Ministry of Agriculture, Food
and Rural Affairs (OMAFRA) Research Program. They were harvested at Dalton White
farms in Otterville, Ontario, and collected during the last week of May 2011. The asparagus
was washed in cold distilled water, patted dry with paper towels, and then the upper
portions were collected by cutting the top 7 cm of the spear. Only the asparagus tops were
used in the experiment as they contain the highest concentration of rutin compared to the
bottom portions of the spear [196]. The asparagus spear tops were steamed on cooking
trays for 5 minutes (Figure 3-1A and B), and then freeze-dried (-50oC; vacuum 0.5 mmHG;
48 hours) in a VirTis freeze dryer at the Guelph Food and Technology Centre pilot plant.
The freeze-dried asparagus was then ground into flour first with a Leeson Explosion Proof
Industrial blender for 1 minute, sieved (1,680 µM), and then further ground with a
Black&Decker® SmartGrind™ CBG100S stainless steel coffee grinder for 1 minute, followed
by a final sieving (600 µM). The final asparagus flour was stored in aluminum foil bags at
-80oC until further use (Figure 3-1C).
44
Figure 3-1: Asparagus preparation
Washed Guelph Millennium asparagus tops were (A) placed on stainless steel trays and
then (B) placed in a steamer for 5 minutes. (C) Freeze-dried cooked asparagus tops was
ground into flour and stored in aluminum foil bags at -80oC until further use.
3.2.1.2 Analysis of Rutin Concentration in Asparagus Flour
3.2.1.2.1 Phenolic Compounds from Asparagus Extraction Conditions
Phenolic compounds were extracted from 0.5 g of freeze-dried asparagus flour with
80% methanol (MeOH). Each sample in 15 mL sterile polypropylene tubes was combined
with 10 mL of 80% MeOH and then put on a Roto-Shake Genie rotator (Scientific Industries,
Inc., USA) overnight at a rotating speed of 7. Tubes were centrifuged at 24oC in IEC Centra
CL3R Refrigerated centrifuge (Thermo Electron Corporation, USA) at 3,000 rpm (24,451 g)
for 5 minutes, and supernatants were transferred to their respective labelled 50 mL sterile
polypropylene tube. The asparagus pellets were resuspended in 6 mL of 80% MeOH, and
then put on the rotator for 1 hour. Tubes were then centrifuged again at 3,000 rpm (24,451
g) for 5 minutes, and supernatants were transferred to their respective labelled 50 mL
sterile polypropylene tube. Resuspension in 80% MeOH was repeated once more, and then
the volume of each supernatant in its 50 mL tube was topped-up to 25 mL with 80% MeOH.
From each 50 mL tube, 3 mL was obtained and filtered (0.45 µM) with a syringe into a
brown vial for high-performance liquid chromatography (HPLC) analysis.
45
3.2.1.2.2 Detection and Quantification of Rutin and Quercetin by HPLC
The HPLC system (Agilent Technology 1100 Series, Palo Alto, Ca, 1999) used to
analyze the asparagus flour phenolic extract was equipped with a quaternary pump, an
inline degasser, a thermostatic autosampler, and a DAD detector (detects signals from 190
nm to 600 nm). A Phenomenex Phenosphere 5 µ ODS (2) 80 A column (150 X 4.60 mm)
was used for the separation. The binary mobile phase consisted of 0.2% acetic acid (Solvent
A) and MeOH (Solvent B). The column was equilibrated with 30% Solvent B, and a gradient
program was used as follows: 30 to 70% Solvent B in 10 minutes, and 70 to 100% Solvent
B in 0.5 minutes. Maintenance of 100% Solvent B occurred for 3 minutes and then brought
back to 30% Solvent B for 4 minutes. The flow rate was 1.0 mL/min. The injection volume
was 10 µL for all samples and standards. The detector was set at 360 nm for the
chromatogram output; and the absorption spectra of each peak were also monitored and
stored. Rutin and quercetin in the samples were identified by comparing their retention
times and UV absorbance spectra with the standards. Quantification was carried out by
integration of the peak areas at 360 nm. Response was linear between the tested range
from 0.00625 to 0.3 mg/mL, and the linear regression R2 was greater than 0.9999. Rutin
and quercetin standards were purchased from Sigma (Sigma Chemicals, USA), and all
samples were analyzed in triplicate.
3.2.1.3 Macronutrients and Soluble Fibre in Asparagus Flour
Asparagus flour macronutrients (carbohydrate, fat, protein), total dietary and
soluble fibre, ash, and moisture content were analyzed by Maxxam Analytics (Mississauga,
ON).
46
3.2.2 Results and Discussion
The concentration of rutin in asparagus flour was determined by HPLC to be 8.36
mg rutin/g asparagus dry weight (DW). Quercetin was not detected, indicating that rutin
was not metabolized during steaming, freeze-drying, and grinding of the asparagus (Figure
3-2).
Figure 3-2: Rutin concentration in asparagus flour

Rutin was extracted from 0.5 g of asparagus flour (in triplicate) through a series of MeOH
washes. A final 10 µL volume of sample and standards (rutin and quercetin) were used to
detect rutin by HPLC (360 nm). All peaks were identified by comparison of retention times
and UV absorbance spectra with the commercial standards.
Proximate analysis of asparagus flour showed that asparagus is high in protein and
fibre, with more than one third being soluble fibre (Table 3-1). Asparagus flour is also low
in dietary fat and available carbohydrates, as well as rich in minerals, as indicated by its
high ash content.
47
Table 3-1: Macronutrient composition of asparagus flour
g/100 g DW
Fat 5.244
Protein 49.03
Available carbohydrates 10.5
Total Fibre 23.2
Soluble fibre 7.6
Insoluble fibre 15.6
Ash 8.8
Moisture 3.3
3.3 Experiment 2: Determining the Suitability of Supplementing

Asparagus into C57BL/6 Mice Diets
3.3.1.1 Preparation of Asparagus Diet for Mice
Since asparagus had not previously been supplemented into animal diets it was not
known what level in the diet would be palatable for mice. Therefore, the asparagus
concentration in diet was initially chosen to be equivalent to the level of purified rutin
known to be effective in reducing colitis in mice. Kwon et al. (2005) previously fed ICR mice
a diet containing 0.001, 0.01 or 0.1% rutin for 2 weeks, and found that 0.1% rutin was the
most effective in reducing colitis symptoms and disease progression [176]. Therefore,
supplementing a mouse diet with a level of asparagus resulting in 0.1% rutin was chosen
for this first feeding trial. Since the asparagus flour contained 8.36 mg rutin/g asparagus
48
DW as found in Experiment 1, 0.1% rutin diet would be achieved by supplementing rodent
diet with 12% asparagus flour.
Experimental diets, AIN-93G basal diet (BD) and BD supplemented with 12%
asparagus flour (120 g asparagus flour/Kg BD), were prepared by Harlan Laboratories, Inc.
(Madison, WI). The AIN-93G diet formulation was modified to contain 5% corn oil instead
of soybean oil, and the total fibre content (cellulose) was increased to 7% from 5%. AIN-
93G diet levels of protein, carbohydrate, fat, and fibre were adjusted in the 12% asparagus
diet to account for these nutrients found in asparagus flour (Table 3-1). Therefore, both
diets had equivalent macronutrients and were isocaloric (Table 3-2). The diets were
packaged in plastic sandwich bags and stored in a walk-in refrigerator (about 4oC)
throughout the feeding trial.
49
Table 3-2: Composition of experimental diets (BD and 12% asparagus)
Ingredients
(g/Kg diet) AIN-93G BD 12% asparagus
Asparagus flour 0 120

Casein 200 132
L-Cystine 3.00 3.00
Sucrose 100 100
Corn starch 377 359
Maltodextrin 132 132
Corn oil 70.0 64.0
Cellulose 70.0 42.2
Mineral mix 35.0 35.0
Vitamin mix 10.0 10.0
Choline bitartrate 2.50 2.50
TBHQ antioxidant 0.014 0.014
Nutrients
(% kcal from)
Protein 19.2 19.3
Carbohydrate 63.2 63.1
Fat 17.6 17.6
3.3.1.2 Study Design
All protocols and animal procedures used in this study were approved by the
Animal Care and Use Committee (University of Guelph). Five week old male C57BL/6 mice
(n=39) were obtained from Charles River (Portage, MI) and were acclimatized for 1 week
ad libitum on water and BD. Mice were grouped into 3 or 4 per cage and maintained in
controlled room conditions: 23±2◦C room temperature (RT), 50±10% humidity, and 12-
hour light-dark cycle. Mice were fed BD (n=13) or asparagus diet (n=26) ad libitum for 3
weeks and BW and diet intake were measured a minimum of twice weekly.
50
3.3.1.3 Rutin and Quercetin Analysis of BD and 12% Asparagus Animal Diets
A sample of approximately 10 g of each animal diet was collected from cages after 3
days (representing the longest duration of RT storage). A sample of 0.5 g of each diet was
then analyzed by HPLC for rutin levels to verify that the BD had no levels of rutin and 12%
asparagus diet contained 0.1% rutin. Quercetin levels were also measured to ensure that
rutin was stable in the 12% asparagus diet after preparation and during storage conditions.
The rutin extraction and HPLC analyses procedures are as outlined above in section 3.2.1.2.
3.3.1.4 Statistical Analyses
All values are expressed as means ± SEM. Week 1 food intake and BW were analyzed
by unpaired t-tests (***=p<0.0001), and week 2-3 food intake and BW were analyzed by
one-way ANOVA on each day (SNK and Dunn’s post-hoc test, respectively). A difference
with p<0.05 was considered significant. All statistical analyses were performed using
Sigma Plot 12.0 (Systat Software Inc, USA).
As indicated by the HPLC profile, BD did not contain detectable levels of rutin, and
rutin was detected in the 12% asparagus diet (Figure 3-3). Rutin was also not metabolized
during preparation or storage of the 12% asparagus diet as quercetin was not detected.
51
Figure 3-3: Determination of rutin in basal diet (BD) and 12% asparagus diet
Rutin was extracted from 0.5 g of BD and 12% asparagus diet through a series of MeOH
washes. A final 10 µL volume of each sample and standards (rutin and quercetin) were
used to detect rutin and quercetin by HPLC (360 nm). All peaks were identified by
comparison of retention times and UV absorbance spectra with the commercial standards.
Each diet was run in triplicate.
During the first week of feeding, 12% asparagus diet was not well tolerated by the
mice as indicated by significant decreases in diet intake and BW compared to the BD group
(Figure 3-4A and B). An increase in stress and aggression in the asparagus-fed mice was
also observed, therefore it was concluded that the 12% level of asparagus was not
palatable by the mice, and thus reduced levels of asparagus diets were prepared to
determine if the palatability could be improved. This was achieved by preparing 3 and 6%
asparagus diets by grinding 12% asparagus diet pellets, and mixing with appropriate
amounts of BD. BD was also ground and fed to mice in the BD group to ensure equal diet
texture and consistency across groups. Mice in the 12% asparagus group were then divided
into three groups: 1) 3% asparagus (n=9); 2) 6% asparagus (n=9); and 3) 12% asparagus
52
(n=8), such that the mean initial BW of each group was not significantly different from each
other (p>0.05).
After dividing mice into their new treatment groups, diet intake and BW began to
increase (Figure 3-4C and D). By day 21, the average daily intake of mice in the 3% and 6%
asparagus groups were comparable to the BD group, while the 12% asparagus diet group
were not able to improve to BD levels. Body weight gain was not significantly different
between diet groups, but amongst the asparagus groups, although not significant
(p=0.156), mice fed 3% asparagus appeared to gain more body weight compared to 6 and
12% asparagus-fed mice.
53
A Diet Intake (week 1)
B Body Weight (week 1)
4
15
Average g/mouse/day
3
10
***
% Change
2
5
1 ***
0 ***
2 4 6 8
0
-5
Day
BD
u s
ag
ar
p
as
%
12
Diet Intake (weeks 2-3)

C 5 D Body Weight (weeks 2-3)
30
Average g/mouse/day
a a
4 a
BD
3 b 20
3% asparagus
% Change
6% asparagus
2 10
12% asparagus
1
0
9 12 15 18 21
0
-10 Day
BD
s
s
u
gu
gu
ag
ra
ra
ar
pa
pa
p
as
as
as
%
3%
6%
12
Figure 3-4: Food intake and body weight (BW) measured over 21 days
During week 1, the (A) average food intake per mouse (n=8-9) and (B) %BW change were
collected at days 3 and 6 or 7 (n=22-26), and during weeks 2 to 3, the (C) average food
intake per mouse (n=4-9), and (D) %BW change (n=8-13) were collected daily. Data points
are expressed as the mean ± SEM. Means with different letters are significantly different
from each other (p<0.05). Statistical analyses were performed using (A and B) unpaired t-
test (***=p<0.0001), (C) one-way ANOVA (SNK post-hoc test), and (D) one-way ANOVA on
each day (Dunn’s post-hoc). BD=basal diet.
To ensure that the mixing of the diets was correct, we analyzed the rutin levels in 3
and 6% asparagus diets as described in section 3.2.1.2. As shown by the HPLC profile, all
diets contained detectable levels of rutin (Figure 3-5). Through using the standard curve,
12% asparagus indeed contained 0.1% rutin (1.04 mg rutin/g pelleted diet), while rutin
levels in 6% asparagus was 0.05% rutin (0.503 mg rutin/g pelleted diet), and 3%
54
asparagus contained about 0.025% rutin (0.232 mg rutin/g pelleted diet). There was also
no breakdown of rutin during mixing since quercetin was not detected.
Figure 3-5: Determination of rutin in 3, 6, and 12% asparagus diets

Rutin was extracted from 0.5 g of 3, 6, and 12% asparagus diets through a series of MeOH
washes. A final 10 µL volume of each sample and standards (rutin and quercetin) were
used to detect rutin and quercetin by HPLC (360 nm). All peaks were identified by
Rutin concentration rutin of each diet was interpolated from the standard curve. Each diet
was run in triplicate.
3.4 Summary
This was the first study to characterize dietary fibre, macronutrient levels by
proximate analysis, and the concentration of rutin by HPLC in cooked Guelph Millennium
asparagus tops. Proximate analysis showed that Guelph Millennium asparagus contained
high levels of minerals protein and low levels of dietary fat and available carbohydrates.
Moreover, there were high amounts of dietary fibre, with more than one third from soluble
fibre. Rutin concentration was also determined to be 8.36 mg/g asparagus DW. The rutin in
the asparagus diets was also not broken down during steaming, freeze-drying, grinding,
55
and storage conditions. This study was also the first to execute a feeding trial with
asparagus-supplemented diet in C57BL/6 mice. The results showed that a dose of 12%
asparagus was not palatable to C57BL/6 mice, but 3% asparagus diet was. Initially, 12%
asparagus (equivalent to 0.1% rutin) was selected because Kwon et al. (2005) discovered
that ICR mice fed a diet containing 0.1% rutin had the greatest impact in reversing colitis
[176]. However, since 12% asparagus was not well tolerated by C57BL/6 mice, the next
step is to confirm if 0.025% rutin (equal to 3% asparagus) can effectively reduce symptoms
of DSS-induced acute colitis in C57BL/6 mice.
56
Chapter 4 : The Effects of Rutin on Acute Colitis Symptoms and
Disease Progression in DSS exposed C57BL/6 Mice
57
4.1 Introduction
Fruits and vegetables are a major source of phenolic compounds in human diets
[183]. Due to their structure, phenolics are known to have anti-oxidative and anti-
inflammatory activities that have been associated with human health benefits [237, 238].
One of the most common classes of phenolics is the flavonoid family since they are
synthesized by almost all plant tissues [188, 189]. Out of the flavonoids, the most
frequently consumed compound is rutin (C27H30O16; 610.517 g/mol), also called rutoside;
quercetin-3-rutinoside; and sophorin. Rutin is found in asparagus, buckwheat, fruit rinds,
and the leaves and petioles of Rheum species [194-196]. Upon ingestion, rutin is
metabolized by gut microflora to its aglycone, quercetin (C15H10O7; 302.236 g/mol) [198,
199]. Quercetin itself is a flavonol and is also widely distributed in nature and can be found
in foods such as apples, onions, and red grapes [200-203].
IBD, a remitting and relapsing inflammatory condition, is now emerging as a global
disease as the incidence and prevalence is increasing with time, geographically [5-7, 18].
Currently, the annual incidence rates of UC in North America is 2.2 to 19.2 cases per
100,000 [5, 6], and prevalence of IBD is known to be the highest in North America, with
about 7,000 to 46,000 newly diagnosed cases each year [5]. Existing conventional
therapies, such as aminosalicylates, corticosteroids, antibiotics, and immunomodulators,
can produce adverse side effects that can range from hyperglycemia to pancreatitis [142-
145]. Rutin and quercetin have demonstrated anti-inflammatory properties in various in
vivo [176, 210-213] and in vitro [172, 210, 214-216] models of colitis, and thus may prove
effective in alleviating inflammation during relapse in IBD patients.
58
It has been found in a colitis model by Kwon et al. (2005) that ICR mice fed a diet
containing 0.001, 0.01 or 0.1% rutin for 2 weeks dose-dependently improved colitis
symptoms [176]. Mice fed 0.1% rutin had the greatest impact, preventing colitis by the end
of the study [176]. Evidence has also shown that there are differences in susceptibility to
DSS in various mouse strains [81]. For instance, BALB/c mice, which are often used in DSS-
induced colitis studies, are less susceptible to DSS compared to IQI/Jic [82] or C57BL/6
mice [83]. Therefore, it is questionable whether 0.1% rutin would also be effective in
alleviating DSS-induced colitis symptoms and disease progression in C57BL/6 mice as it
was found to be with ICR mice [176].
In Chapter 3, it was discovered that 3% asparagus was the most palatable to
C57BL/6 mice as diet intake was comparable to BD. When looking at these three diets in
terms of rutin content, 3% asparagus would be equivalent to 0.025% rutin, while 6 and
12% asparagus would equal to 0.05 and 0.1% rutin, respectively. However, from Chapter 3,
it can be seen that 12% asparagus (0.1% rutin) was not well-tolerated by C57BL/6 mice,
meaning it would not be practical to proceed with a colitis intervention using this dose of
asparagus in C57BL/6 mice.
To date, there has not been a study examining the effects of purified rutin in
C57BL/6 mice using the DSS-induced colitis model. Therefore, the objectives of this study
are to determine if rutin can be effective in reducing acute colitis symptoms and
progression in the C57BL/6 mouse strain, and establish whether a low rutin dose (that is
achievable through an asparagus supplemented diet) is also effective.
59
4.2 Materials and Methods
4.2.1 Diet Preparation
Four experimental diets were prepared similar section 3.3.1.1. The same AIN-93G
BD as in Chapter 3 and BD supplemented with 0.1% rutin diet were used in this study
(Table 4-1). The 0.025 and 0.05% rutin diets were made by grinding 0.1% rutin diet pellets
and mixing with appropriate amounts of BD. BD was also ground and fed to mice in the BD
group to guarantee equal diet texture and consistency across groups. The rutin trihydrate
used was purchased from Sigma (Sigma Chemicals, USA) with 97% purity.
Table 4-1: Composition of experimental diets (BD and 0.1% rutin)

Ingredients
(g/Kg diet) AIN-93G BD 0.1% rutin
Rutin trihydrate 0 1.10

Casein 200 200
L-Cystine 3.00 3.00
Sucrose 100 100
Corn starch 377 376
Maltodextrin 132 132
Corn oil 70.0 70.0
Cellulose 70.0 70.0
Mineral mix 35.0 35.0
Vitamin mix 10.0 10.0
Choline bitartrate 2.50 2.50
TBHQ antioxidant 0.014 0.014
Nutrients
(% kcal from)
Protein 19.2 19.2
Carbohydrate 63.2 63.2
Fat 17.6 17.6
60
4.2.1.1 Determination of Rutin in Diets
After the rutin diets were made, rutin concentration was verified using procedures
described in section 3.2.1.2 to ensure correct mixing.
4.2.2 Mice
All protocols and animal procedures used in this study were approved by the Animal
Care and Use Committee (University of Guelph). Five week old male C57BL/6 mice (n=62)
were obtained from Charles River (Portage, MI) and were acclimatized for 1 week ad libitum
on water and BD. Mice were housed into 3 or 4 per cage under controlled room conditions:
23±2◦C RT, 50±10% humidity, and 12-hour light-dark cycle.
4.2.3 Study Design
The experimental protocol is summarized in Figure 4-1. Six week old C57BL/6 mice
(n=62) were divided into four treatment groups: 1) BD (n=26); 2) 0.025% rutin (n=12); 3)
0.05% rutin (n=12); and 4) 0.1% rutin (n=12), such that the mean BW of each group was
not significantly different from each other (p>0.05). Then each group was fed their
respective diet for 2 weeks, and during this period, diet was provided fresh daily, and food
intake and BW were recorded daily. After 2 weeks, experimental colitis was induced by
adding 2% (w/v) DSS (MP Biomedicals, USA, MW 36,000-50,000) in the drinking water for
7 days. Half of the BD group received DSS-free drinking water and served as healthy
controls. During this time the mice continued on their respective diet ad libitum.
During the DSS cycle, food and water intake were measured daily. Furthermore,
mice were monitored daily for disease activity index (DAI), which is a research tool to
quantify clinical symptoms of colitis. The DAI is an average of three scores, which includes
61
measuring BW loss, stool consistency, and stool blood. BW loss was calculated as the %
difference compared to BW at the start of the DSS cycle. Stool consistency and stool blood
scores were determined by smearing fresh fecal samples onto Hemoccult SENSA test slides
(Beckman Coulter, USA). The three scores were on a scale from 0 to 3 with 0 representing
the normal/healthy condition, and 3 representing the worst clinical outcome (Table 4-2).
Mice were euthanized prior to study completion if malocclusion was evident, BW loss
exceeded 20% of initial BW, or if a DAI score of 3 was achieved.
At the end of the DSS cycle, all mice were sacrificed by cervical dislocation from a
trained lab technician. Blood collection by cardiac puncture was also completed by the lab
technician. The entire colon and cecum were excised for macro and micro analyses of
colonic inflammation. Colon length was measured from the ceco-colonic junction to the
rectum with a ruler. The colon was cleaned of fecal material, weighed, and washed with 1X
PBS. Then 1 cm colon sections of the distal colon were collected and fixed in 10% buffered
formalin solution for later histological analysis (Figure 4-2). The remaining colon segments
were snap frozen in liquid nitrogen. Organs (spleen and cecum, with and without its
digesta) were excised, weighed, and snap-frozen in liquid nitrogen. All snap-frozen tissues
were stored in -80oC for later analyses.
62
Figure 4-1: Experimental Design
C57BL/6 mice were acclimatized on BD for 7 days and then switched over to experimental
diets for the remainder of the study. After 14 days of experimental diets, a 7-day DSS cycle
started where all mice received 2% DSS in their drinking water, except for half of the BD
group who received DSS-free water. During the 7 days, collection of diet and water intake,
and DAI and body condition of mice were monitored daily. At the end of the 7 days, all mice
were sacrificed and tissue parameters and samples were collected.
Table 4-2: DAI scoring system

Score % BW loss score Stool consistency score Stool blood score
0 0-1% Normal Normal (- Hemoccult)
1 1-5% Soft, but formed + Hemoccult
1.5 --- --- Trace of blood
2 5-10% Very soft Visible blood
3 10-20% Diarrhea Rectal bleeding
63
Figure 4-2: Method of collecting distal colon sections
Colons from (A) Basal diet-fed and (B) DSS-treated mice were excised, cut once (as
indicated by the red line), and 1 cm sections of the distal colon were fixed in 10% buffered
formalin solution for later histological analyses.
4.2.4 Colonic MPO Assay
Colonic MPO levels were measured using a MPO ELISA kit (Cederlane Laboratories
Ltd, Canada) according to the manufacturer’s instructions. Frozen distal colons were
weighed then transferred into homogenization tubes containing 5 ceramic beads (2.8 mm,
MO BIO Laboratories Inc., CA) and lysis buffer (200 μL/10 mg colon). The lysis buffer
comprised of 28 μg aprotinin/mL, 1 μg leupeptin/mL, and 1 mM Phenylmethanesulfonyl
fluoride in a Tris buffer with 10% glycerin and 5 mM EDTA. Samples were homogenized
using a Powerlyzer TM (MO Bio laboratories Inc, USA) for 20 seconds at 3500 rpm.
Powerlyzer TM is a tissue homogenizer incorporated with bead-beating technology. The
homogenized samples were centrifuged at 1500 rcf (g) for 15 minutes at 4oC. Supernatants
were collected and then centrifuged twice more to ensure the purity of the sample.
64
Supernatants were collected and stored in -80oC until MPO analysis. Diluted colon protein
lysates were analyzed for MPO levels as per kit instructions. MPO concentration (ng/mL)
was read with a plate reader (BioTek PowerWave XS2, USA) at 450 nm. Final MPO
concentrations were corrected for dilution factor and wet tissue colon weight, and then log
transformed [log units (u)/g].
4.2.5 Colon Histopathology
At sacrifice, 1 cm distal colon was excised and fixed in 10% formalin for 24 hours,
and then transferred to 70% ethanol (EtOH) until paraffin embedding. Tissues were
embedded in paraffin, sectioned (5 μm), and stained with Haematoxylin & Eosin or Alcian
Blue for histological analyses of crypt erosion and goblet cell depletion, respectively. The
histological scoring system for colon erosion consisted of 5 scores with 1 showing normal
crypts and intact surface epithelial cells; scores 2 to 4 representing an increase in crypt
erosion and surface epithelial cells; and 5 indicating a total loss of crypt architecture and
surface epithelial cells (Table 4-3 and Figure 4-3). The scoring system for goblet cell
depletion also consisted of 5 scores with 1 displaying normal intact goblet cells; scores 2 to
4 showing a marked increase in goblet cell loss; and 5 with a total loss of goblet cells (Table
4-4 and Figure 4-4). Each cross section received a score from 1 to 5. All slides were scored
blindly by three different individuals, and the final scores were averaged.
65
Table 4-3: Histological grading of colon erosion during DSS-induced acute colitis
Score Description
1 Normal with intact crypts and surface epithelial cells
2 Loss of <1/3 of crypts, but surface epithelial cells are still visible
3 Loss of 2/3 of crypts, but some crypt structures and most surface epithelial
cells are still visible
4 Loss of >2/3 of crypts, but most surface epithelial cells are still visible
5 Loss of entire crypts and surface epithelial cells
Figure 4-3: Histological grading of colon erosion in DSS-induced acute colitis

Haematoxylin & Eosin-stained distal colon cross-sections were scored for erosion based on
five scores: (1) normal with intact crypts and surface epithelial cells; (2) loss of <1/3 of
crypts, but surface epithelial cells are still visible; (3) loss of 2/3 of crypts, but some crypt
structures and most surface epithelial cells are still visible; (4) loss of >2/3 of crypts, but
surface epithelial cells are still visible as indicated by the black arrow; and (5) loss of entire
crypt and surface epithelial cells (20X magnification).
66
Table 4-4: Histological grading of goblet cell depletion during DSS-induced colitis
Score Description
1 Normal with intact goblet cells
2 Loss of <1/3 of goblet cells; increase in number/size of goblet cells
3 Loss of 2/3 of goblet cells
4 Loss of >2/3 of goblet cells
5 Complete loss of goblet cells
Figure 4-4: Histological grading of goblet cell depletion in DSS-induced acute colitis
Alcian blue-stained distal colon cross-sections were scored for goblet cell depletion based
on five scores: (1) normal with intact goblet cells; (2) loss of <1/3 of goblet cells; increase
in number/size of goblet cells; (3) loss of 2/3 of goblet cells; (4) loss of >2/3 of goblet cells;
and (5) complete loss of goblet cells (20X magnification).
4.2.6 Statistical Analyses
All values are expressed as means ± SEM. Food and water intake, and DAI were
analyzed by one-way ANOVA on each day (SNK post-hoc test). For organ size parameters,
67
one-way ANOVA (SNK post-hoc test) was used for colon weight-to-length ratio and cecum
weight, while Dunn’s post-hoc test was used for spleen weight. MPO activity (with log10
transformed data), and histopathology scores (colon erosion and goblet cell depletion)
were analyzed by one-way ANOVA (Dunn’s post-hoc test). A difference with p<0.05 was
considered significant. Statistical analyses were performed using Sigma Plot 12.0 (Systat
Software Inc., USA).
4.3 Results
4.3.1 Determination of Rutin in Diets
As indicated by the HPLC profile, rutin was detected in all diets, except the BD
(Figure 4-5). Through using the standard curve, the 0.1% rutin diet contained 0.926 mg
rutin/g pelleted diet, the 0.05% rutin diet contained 0.434 mg rutin/g pelleted diet, and
0.025% rutin diet had 0.221 mg rutin/g pelleted diet. This indicates that rutin in 0.05%
rutin diet was indeed half of 0.1% rutin diet; and the rutin in 0.025% rutin diet was also
half of 0.05% rutin diet. Moreover, rutin was not metabolized during preparation or
storage of the rutin diets as quercetin was not detected.
68
Figure 4-5: Determination of rutin in basal diet (BD), and 0.025, 0.05, and 0.1% rutin
diets
Rutin was extracted from 0.5 g of 0.025, 0.05, and 0.1% rutin diets through a series of
MeOH washes. A final 10 µL volume of each sample and standards (rutin and quercetin)
were used to detect rutin and quercetin by HPLC (360 nm). All peaks were identified by
Rutin concentration of each diet was determined by standard curve. Each diet was run in
triplicate.
4.3.2 Pre-DSS Food Intake and BW
After 14 days of feeding, there were no significant differences between groups with
regards to food intake and BW gain (Figure 4-6).
69
A Diet Intake B
Body Weight
4.5 28
4.0 26 BD
g/day/mouse
0.025% rutin
3.5 24 0.05% rutin
g
3.0 0.1% rutin
22
2.5 20
2.0 18
2 4 6 8 10 12 14 2 4 6 8 10 12 14
Day Day
Figure 4-6: Pre-DSS food intake and BW

The (A) average daily intake per mouse (n=3-9) and (B) % change in BW (n=12-26) was
measured over 14 days. Data points are expressed as the mean ± SEM.
4.3.3 Food and Water Intake During Acute Colitis
During the 7 day DSS cycle, DSS caused a significant reduction in diet and water
intake. By the end of DSS cycle, there were no significant differences among DSS-treated
groups with diet (Figure 4-7A) and water intake (Figure 4-7B).
70
A Diet Intake B Water Intake
5 5
BD
4 4 a a BD+DSS
a
mL/day/mouse
g/day/mouse
0.025% rutin+DSS
3 3 0.05% rutin+DSS
ab ab
0.1% rutin+DSS
2 2 b
b
b
1 1
0 0
2 4 6 8 2 4 6 8
Day Day
Figure 4-7: Food and water intake during DSS-induced acute colitis
The (A) average daily diet intake per mouse (n=3-5) and (B) daily water intake per mouse
(n=3-5) was measured over 7 days during 2% DSS-induced acute colitis. Data points are
expressed as the mean ± SEM. Means with different letters within the same day are
significantly different from each other (p<0.05). Within each day, some letters are
representing more than one treatment group. Statistical analyses were performed using
one-way ANOVA (SNK post-hoc test) on each day. BD=basal diet, DSS=dextran sodium
sulfate.
4.3.4 DAI During Acute Colitis
The clinical symptoms of DSS-induced colitis are most reflective of UC. In the acute
phase of DSS-induced colitis, weight loss, diarrhea, and the presence of bloody stool are
initially observed [71, 78]. These clinical symptoms can be seen within 2 days post-colitis
induction [78].
DSS-induced weight loss is usually a result of reduced diet intake and an indicator of
increased inflammation [71, 78]. We observed that mice started to experience BW loss
starting from days 4 and 5 of DSS exposure in the BD+DSS and rutin-treated groups,
respectively (Figure 4-8A). By day 7, BW loss was significantly different between mice fed
0.025% rutin compared to the remaining DSS-treated groups.
71
Increase in stool consistency or diarrhea is also a common phenomenon seen in
DSS-induced colitis, similar in UC, which is caused by an increased permeability of the
intestinal cells [239] or hyperosmolarity in the lumen induced by DSS [240]. As observed in
our study, significant stool consistency changes occurred in all DSS-exposed mice by day 1,
which then worsened over time (Figure 4-8B). Collectively, rutin-treated mice attenuated
the onset of diarrhea at the end of the DSS cycle; however, this effect was not dose-
dependent.
Increased stool blood, leading to rectal bleeding, is also often observed in DSS-
induced colitis, which is usually a result of damaged crypts and surface epithelium, and
ulcer formation [71, 78]. Hemoccult positive stools were observed in all DSS-treated groups
within 1 day of DSS exposure, and throughout the cycle, DSS-treated groups were not
significantly different from each other (Figure 4-8C).
When all scores were combined (DAI), 0.1% rutin-fed mice had a significantly lower
DAI score than mice in the BD+DSS group at the end of the 7-day acute colitis cycle (Figure
4-8D). Overall, rutin-fed mice had significantly lower DAI scores than the BD+DSS group,
though this effect was not dose-dependent.
72
A B Stool Consistency
Body Weight
3
10 a
a ab
a a
5 a ab b
a 2
a
% Change
a b
Score
a b b a ab b
0 bc b a
1 2 3 4 5b 6 7b
1 a a b
c ab
-5 Day c
b b b c c c
c b
-10 0 BD
0 1 2 3 4 5 6 7 BD+DSS
0.025% rutin+DSS
Day
0.05% rutin+DSS
0.1% rutin+DSS
C Stool Blood D DAI
3 2.5
a a a
a 2.0
ab
2 ab
1.5 a
a
Score
Score
a a b
a
1.0 a
1 a
a a
a 0.5
b b b b b b b b b b b b b b
0 0.0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Day Day
Figure 4-8: Effects of rutin diets (0.025%, 0.05%, and 0.1%) on DAI clinical
symptoms of DSS-induced acute colitis
During the 7-day 2% DSS cycle, mice (n=12-14) were scored daily for (A) BW loss, (B) stool
consistency, and (C) stool blood. The average of all three scores was expressed as (D) DAI.
All data are expressed as the mean ± SEM. Means with different letters within the same day
are significantly different from each other (p<0.05). Within each day, some letters are
sulfate.
4.3.5 Organ Size Parameters During Acute Colitis
Not only does DSS induce colitis symptoms (DAI), but it also affects the colon, and
extraintestinal organs, such as the cecum and spleen. It is believed that DSS shortens the
colon by damaging crypts [71, 78] and increasing colon weight by inducing a thickening of
the colon via edema and muscular hypertrophy [86]. As observed in this study, DSS
73
induced an increase in colon weight to length ratio; however, none of the rutin diets were
able to significantly attenuate this parameter (Figure 4-9A).
Cecum weight, usually known to be an indicator of dietary fibre fermentation [241],
has also been affected by DSS. DSS has been shown to inhibit fermentation [242], thereby
reducing the production of anti-inflammatory SCFAs, such as butyrate [168-171, 243]. DSS
can also increase cecum weight by directly causing inflammation to caecal tissue, which
would result in focal mucosal edema and infiltration of mononuclear or mixed inflammatory
cells [244]. In this study, there was not a significant attenuation of DSS-induced cecum weight
with rutin-fed mice (Figure 4-9B).
Lastly, DSS can induce an increase in spleen weight (i.e. hypertrophy) as observed
by several rodent studies as a response to increased colonic inflammation [83, 245-248]. In
this study, it was found that mice fed the 0.025% rutin diet had significantly reduced spleen
hypertrophy, having similar spleen weights as the mice in the BD group (Figure 4-9C).
74
A B
0.3 0.08
a a a a a
a a a
0.06
Colon (g/cm)
0.2 b
Cecum (g)
b
0.04
0.1
0.02
0.0 0.00
SS
SS
BD
SS
SS
SS
BD
SS
SS
SS
+D
+D
+D
+D
+D
+D
+D
+D
tin
tin
tin
BD
tin
in
BD
tin
t
ru
ru
ru
ru
ru
ru
%
5%
1%
1%
5%
%
05
05
02
0.
0.
02
0.
0.
0.
0.
C
0.008
0.006 a
Spleen (g/BW)
a
a
0.004 ab
b
0.002
0.000
BD
SS
SS
SS
SS
+D
+D
+D
+D
tin
BD
tin
tin
ru
ru
ru
%
1%
5%
05
0.
02
0.
0.
Figure 4-9: Effects of rutin diets on organ size parameters in DSS-induced acute
colitis
Data shows the effects of rutin diets on (A) colon weight-to-length ratio (n=11-12), and
extraintestinal organs: (B) cecum (n=11-13) and (C) spleen (corrected for BW; n=11-14)
during 7 days of 2% DSS-induced colitis. All data are expressed as the mean ± SEM. Means
with different letters are significantly different from each other (p<0.05). Statistical
analyses were performed using (A and B) one-way ANOVA (SNK post-hoc test) and (C)
one-way ANOVA (Dunn’s post-hoc test). BD=basal diet, DSS=dextran sodium sulfate.
75
4.3.6 Colonic MPO Activity During Acute Colitis
MPO activity is an indicator of the degree of neutrophil infiltration that has been
positively correlated with a worsened disease state in IBD patients [87], and is also
positively correlated with an increased exposure to DSS [88]. In this study, colonic MPO
activity was increased by DSS, but none of the rutin groups were able to attenuate this
increase (Figure 4-10). This may be attributed to the large variations seen between all
treatment groups before the data was transformed (data not shown).
5
a a a
a
4
Log10 MPO (u/g)
b
3
0
SS
BD
SS
SS
SS
+D
+D
+D
+D
tin
BD
tin
tin
ru
ru
ru
5%
1%
%
05
02
0.
0.
0.
Figure 4-10: Effects of rutin diets on distal colon myeloperoxidase (MPO) during DSS-
induced acute colitis
After 7 days of 2% DSS-induced colitis, distal colon protein lysates (n=12-14, in duplicate)
were used in a commercial ELISA kit to detect MPO activity. All data are expressed as the
mean ± SEM. Means with different letters are significantly different from each other
(p<0.05). Statistical analysis was performed using one-way ANOVA (Dunn’s post-hoc) on
log10 transformed data. BD=basal diet, DSS=dextran sodium sulfate.
76
4.3.7 Colon Histopathology During Acute Colitis
4.3.7.1 Colon Erosion and Goblet Cell Depletion
The typical histological characteristics of DSS-induced colitis include: crypt
distortion; luminal epithelial cell depletion; and loss of mucin-producing goblet cells within
crypts [71, 78, 83, 85]. It is believed to be caused by the ability of DSS to alter the mucus
layer lining the luminal epithelium [91]. DSS would then be able to act as a direct toxin to
surface epithelial cells to increase cell apoptosis [95, 96] and gut permeability [71, 85, 94],
leading to the permeation of luminal microflora and antigens to activate an inflammatory
response that can result in damaged basal crypts. As observed in our study, there was a
significant increase in colon erosion and goblet cell depletion scores with DSS, yet rutin
was not able to significantly affect this change (Figure 4-11).
A Colon Erosion B Goblet Cell Depletion
5 4 a
a
4 a a
a 3 a
a a
3
Score
Score
2
2
b
b 1
1
0 0
SS
BD
SS
SS
SS
SS
BD
SS
S
DS
S
+D
+D
+D
+D
+D
+D
+D
tin
tin
BD
tin
tin
tin
BD
tin
ru
ru
ru
ru
ru
ru
1%
%
%
1%
%
05
5
05
0.
02
0.
02
0.
0.
0.
0.
Figure 4-11: Effects of rutin diets on distal colon histopathology

Distal colon cross-sections were scored for (A) colon erosion (n=10-13) and (B) goblet cell
depletion (n=11-14) after 7 days of 2% DSS-induced colitis. All data are expressed as the
mean ± SEM. Means with different letters are significantly different from each other
(p<0.05). Statistical analyses were performed using one-way ANOVA (Dunn’s post-hoc).
BD=basal diet, DSS=dextran sodium sulfate.
77
4.4 Discussion
The first objective of this study was to determine if rutin was effective in reducing
DSS-induced acute colitis symptoms and disease progression in C57BL/6 mice. After
feeding C57BL/6 mice three doses of rutin (0.025, 0.05, and 0.1%) in diet for 2 weeks prior
to and during DSS colitis induction, there were mild effects from all rutin doses in
suppressing clinical symptoms, such as stool consistency and DSS-induced spleen
hypertrophy. The second objective was to ascertain if rutin at its lowest dose (that is
achievable through an asparagus diet as observed in Chapter 3), could also decrease DSS-
induced colitis symptoms in C57BL/6 mice. It was found that 0.025% rutin was effective,
especially with significantly attenuating spleen hypertrophy. Although the beneficial effects
observed from the rutin diets were not dose-dependent, the results still established that
the beneficial effects could be attained from a low rutin dose, which can be acquired
through diet.
However, unlike Kwon et al. (2005) [176], our findings showed that consumption of
0.025, 0.05, and 0.1% rutin in diet by C57BL/6 mice did not dose-dependently attenuate
clinical symptoms. There may be mouse strain differences in terms of BW gain and diet
intake, and microflora responsible for metabolism of rutin and eliciting and/or promoting
immune-mediated colonic inflammation.
A notable difference between the C57BL/6 mice used in this study and the ICR mice
in the study by Kwon et al. (2005) was their BW prior to colitis induction and the amount of
diet intake they consumed each day. The ICR mice were on average 30 g and consumed
about 6 g of pelleted diet each day (200 g/Kg BW) [176], while the C57BL/6 mice in our
study were on average less than 20 g before colitis induction and consumed about 3 g of
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powder diet each day (150 g/Kg BW). A study by Bachmanov et al. (2002) has verified that
there are substantial strain variations in food and water intakes independent from BW
from 28 inbred strains [249]. Moreover, the ICR mice were fed rutin in MF pellets, which is
a non-purified diet, while the C57BL/6 mice in this study were fed rutin supplemented to a
purified diet, AIN-93G. Goto et al. (2010) has found different effects of feeding mice fructo-
oligosaccharides (FOS) in purified and non-purified diets in DSS-induced colitis, with the
non-purified diet having more of a protective effect. The authors speculated that this may
have been due to an increased production of SCFAs and water holding capacity in the
intestinal contents that were observed in mice fed the non-purified diet [250]. These
differences would ultimately produce varying end results, depending on how much rutin
was attained by the animal prior to and during DSS exposure.
There was also an observed aggravated BW loss within the 0.025% rutin group;
there were two mice in the same cage that lost considerable BW starting at day 5, which
explains for the large variation seen with BW loss in this group. When these two mice are
removed from the DAI data analyses, 0.025% rutin is comparable with 0.05 and 0.1% rutin
(data not shown). These two mice were not excluded because they did not have
malocclusion, but it is possible that they were not eating 0.025% rutin diet but in its place
ate cage bedding and/or exhibited coprophagia. It has been found in A/J mice that failed to
consume a synthetic low-fat diet, began to eat their own cage bedding and displayed
coprophagia in order to prevent starvation [251]. Since we monitored diet intake by cage
(n=3 or 4), and estimated the diet intake per mouse, there is no way of knowing the exact
individual intake of each mouse.
79
Another strain difference could be in microbial ecology and the ability of C57BL/6
and ICR mice to metabolize rutin. It is known that before absorption can occur, rutin has to
be hydrolyzed to its aglycone, quercetin, by β-glucosidase activity of colonic microflora,
such as Bacteroides distasonis, B. uniformis, and B. ovatus [198, 199]. Therefore, the
transformation of native phenolics, such as rutin, into their respective metabolites is
dependent on the individuals and their intestinal microflora. In humans, both metabolite
“producers” and “non-producers” have been reported [252, 253]. For instance, only one-
third to one-half of the population contains specific microbiota (that is currently
unidentified), who are able to metabolize daidzein, a soy isoflavone, to equol that has been
claimed as a potent estrogenic metabolite with great antioxidant activity [254]. In mice, gut
microbiota is related to both genetic and environmental factors. Genetically, the gut
microbiota of inbred mice, such as C57BL/6 mice [255], is more similar between animals
than those of outbred mice, such as ICR mice [256, 257]. Environmentally, when scientists
compared C57BL/6 mice bred from two different locations, they revealed significant
differences in microbial profile, but when they compared C57BL/6Sca mice raised in
separate rooms within the same breeding center, they found the profiles were not
significantly different from each other [256]. Therefore, the degree of rutin metabolism is
dependent on individual variation in the composition of the colonic microbiota.
Moreover, the composition of the colonic microflora in mice is also vital in
determining the inflammatory response involved in colitis. One of the theories of IBD
etiology is an inappropriate activation of the immune system by colonic microflora, which
could occur before or after immunoregulatory defects of the colonic mucosa [42]. It has
been reported that: 1) luminal bacteria are needed for the development of chronic
80
immune-mediated intestinal inflammation; 2) commensal bacterial species have dissimilar
pro-inflammatory capabilities, with some being more destructive than others; and 3) each
species has a different role in the inflammatory process [258]. For example, B. vulgatus has
been associated with increased active colitis. Germfree B27 TG rats colonized with six
different obligate and facultative intestinal anaerobic bacteria, including B. vulgatus,
developed much more active colitis and gastritis compared to littermates colonized with
the same selected bacteria without B. vulgatus [259]. Moreover, treatment of
metronidazole (an antibiotic that suppresses Bacteroides spp) in B27 TG rats raised under
specific-pathogen-free (SPF) environment was able to treat and prevent the onset of colitis
[258]. Thus, the severity of colonic inflammation would be dependent on colonic microflora
populations.
The most prominent result observed in this study was a reduction in DSS-induced
spleen hypertrophy with 0.025% rutin-fed mice. This increase in DSS-induced spleen
hypertrophy has also been observed in other rodent studies [83, 245-248]. The spleen is
known as a functionally diverse organ with active roles in immunosurveillance, filtering
blood, storing monocytes, and hematopoiesis [260]. It is possible that spleen enlargement
occurs when there is increased inflammation within the body, such as in the colon. Thus, an
increase in spleen weight could mean that it is performing its normal functions at an
amplified rate to: clear microorganisms and antigens from the blood stream; initiate
immune reactions; synthesize IgG, an antibody isotype that controls infections of body
tissues; and remove abnormal red blood cells [261]. The attenuation by 0.025% rutin-fed
mice could be attributed to the anti-oxidative nature of rutin and quercetin [212, 213, 262,
263]. ROS are highly reactive molecules produced by normal metabolism that can cause
81
damage to tissues [119]. During inflammation, activated immune cells are recruited to
manage inflammation, but sequentially they increase ROS production [126, 127].
Therefore, rutin and quercetin may be directly acting as a free radical scavenger,
eliminating ROS, and/or maintaining a balance in the antioxidant defense system [212, 213,
262, 263].
In previous studies, rutin was able to attenuate the increase in DSS-induced colonic
MPO levels (indicator of neutrophil infiltration) [210, 211] and colon histopathology scores
[176, 210, 212], but in this study, there were no significant attenuations seen from rutin
diets. These dissimilar results could, again, be attributed to strain and study differences.
Cecum weight has not been measured in any rodent studies observing rutin. Cecum weight,
typically known to be an indicator of dietary fibre fermentation [241], has also been
affected by DSS. DSS has been shown to inhibit fermentation [242], thereby reducing the
production of anti-inflammatory SCFAs, such as butyrate [168-171, 243]. Thus, for dietary-
fibre containing diets to counteract the inhibition of fermentation caused by DSS, there
must be an adequate level of dietary fibre to produce SCFAs. But in this study, the level of
dietary fibre in all rutin diets was the same as the BD group. Therefore, we did not see an
attenuation of DSS-induced cecum weight by any rutin groups.
In summary, this study demonstrated that rutin was effective in reducing some
markers of colitis, such as DSS-induced stool consistency, and spleen hypertrophy in
C57BL/6 mice. Yet, the outcomes were weaker compared to previously reported rutin
effects in other mouse strains, which may be accounted by strain differences in BW gain
and diet intake as well as microflora status. Although the beneficial effects of rutin in this
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study were not dose-dependent, the lowest rutin dose (0.025%) was still seen to be
effective in attenuating the onset of diarrhea and reducing spleen hypertrophy.
83
Chapter 5 : The Effects of Asparagus and its Purified Bioactive,
Rutin, on DSS-induced Acute Colitis and Recovery in C57BL/6
Mice
84
5.1 Introduction
IBD, comprising of UC, is a lifetime disease of remitting and relapsing inflammation
of the large intestine [1]. Since UC patients are often interchanging between a relapse or
remissive state, potential therapies should aim to target either of these states, or both, to
reduce the time in relapse and accelerate towards remission. Foods that are rich in natural
dietary compounds with anti-inflammatory and anti-oxidative activities are thus proposed
as potential treatments for IBD patients. For instance, the juice from Cydonia oblonga Miller
(Quince), a fruit tree cultivated in many countries, contains high amounts of flavonoids,
namely rutin, quercetin, and kaempferol. Quince juice can significantly diminish
inflammation and ulcer indices in TNBS-induced colitis in rats [264]. There are currently
limited studies observing the effects of certain whole foods or dietary components during
recovery from DSS-induced colitis. Yet, from the limited studies that have been completed,
positive results have been reported. For instance, FOS, a prebiotic that stimulates the
colonic growth of bifidobacteria to promote intestinal health, was found to produce a faster
recovery from DSS-induced colonic damage with increased crypt depth and crypt area
compared to the control in C57BL/6 mice [265].
As observed in Chapter 4, and in various rodent models, rutin can significantly
reduce experimentally-induced colitis symptoms, possibly owing to its anti-inflammatory
[176, 210-213] and anti-oxidative nature [212, 213, 262, 263]. A recent review has
established that quercetin, the aglycone of rutin, can exhibit protective effects on intestinal
tight junction (TJ) barrier function via the assembly and expression of TJ proteins [266],
such as claudin-4 [267]. Hence, it is probable that 0.025% rutin could also offer benefits
during recovery in repairing the damaged colon.

85
To date, there has not been a study examining the effects phenolics in recovery from
DSS-induced colitis nor cooked whole asparagus in acute colitis or remissive states of
experimental UC. With asparagus containing high concentrations of rutin and additional
anti-inflammatory bioactives (e.g. soluble fibre), asparagus is a potential candidate for
alleviating symptoms during colitis and remission, possibly more so than just purified
rutin. Asparagus may also be harbouring other phenolic compounds with antioxidant
potential. Therefore, in order to expand the therapeutic potential of rutin and rutin-rich
asparagus, the objective of this study is to evaluate the effects of cooked asparagus
(equivalent to 0.025% rutin) and purified 0.025% rutin in diet using both a DSS-induced
acute colitis (i.e. relapse) and recovery model in C57BL/6 mice.
5.2 Experiment 1: Asparagus Flour Preparation and Rutin Analysis
5.2.1.1 Preparation of Asparagus Flour
A batch (25 Kg) of Guelph Millennium asparagus spears (8 mm diameter, about 22
cm long) was provided by the University of Guelph/OMAFRA Research Program. They were
harvested at Dalton White farms in Otterville, Ontario, and collected during the first week
of May 2012. The procedures in preparing the asparagus flour is the same as described in
section 3.2.1.1.
5.2.1.2 Analysis of Rutin Concentration in Asparagus Flour
The procedures used in extracting rutin from asparagus flour and measuring rutin
concentrations by HPLC are described in section 3.2.1.2.
86
5.2.1.3 Macronutrients and Soluble Fibre in Asparagus Flour
Asparagus flour macronutrients (carbohydrate, fat, protein), total dietary and
soluble fibre, ash, and moisture content were analyzed by Maxxam Analytics (Mississauga,
ON).
As indicated by the HPLC profile, rutin was detected in asparagus flour (Figure 5-1).
Moreover, quercetin was not detected, indicating that rutin was not broken down during
steaming, freeze-drying, and grinding of the asparagus. Using a rutin standard curve, the
concentration of rutin in asparagus flour determined to be 12.92 mg rutin/g asparagus DW.
Figure 5-1: Analysis of rutin concentration in asparagus flour

Rutin was extracted from 0.5 g of asparagus flour through a series of MeOH washes. A final
10 µL volume of sample and standards (rutin and quercetin) were used to detect rutin by
HPLC (360 nm). All peaks were identified by comparison of retention times and UV
absorbance spectra with the commercial standards. Concentration of rutin was
interpolated from the standard curve. Each diet was run in triplicate.
87
Proximate analysis of asparagus flour revealed that asparagus is high in protein, and
low in dietary fat and available carbohydrates, as well as rich in minerals, as indicated by
its high ash content (Table 5-1). Asparagus flour is also rich in dietary fibre, with more than
one third being soluble fibre. The macronutrient composition of the asparagus flour in this
study is also comparable with the asparagus flour used in Chapter 3.
Table 5-1: Macronutrient composition of asparagus flour
g/100 g DW
Fat 5.486
Protein 45.58
Available carbohydrates 15.8
Total Fibre 21.7
Soluble fibre 7.9
Insoluble fibre 13.8
Ash 8.5
Moisture 3
5.3 Experiment 2: Determining the Effects of Asparagus and Purified

Rutin on DSS-induced Colitis and Recovery in C57BL/6 Mice
5.3.1.1 Preparation of Diets for Mice
It was found in Chapter 4 that 0.025% rutin can significantly reduce some
parameters of colitis in C57BL/6 mice. Therefore, in this study, a level of asparagus that
was equivalent to 0.025% rutin was added to rodent AIN-93G BD. Since the asparagus flour
88
was found to contain 12.92 mg rutin/g asparagus DW in Experiment 1, supplementing
rodent diet with 2% asparagus flour achieved 0.025% rutin in the diet.
Experimental diets: AIN-93G BD; BD supplemented with 2% asparagus flour (20 g
asparagus flour/Kg BD); and BD supplemented with 0.025% rutin (0.28 g rutin/Kg BD)
were prepared by Harlan Laboratories, Inc. (Madison, WI). The rutin trihydrate used was
purchased from Sigma (Sigma Chemicals, USA) with 97% purity. The AIN-93G diet
formulation was modified to contain 5% corn oil instead of soybean oil, and the total fibre
content (cellulose) was increased from 5% to 7%. AIN-93G diet levels of protein,
carbohydrate, fat, and fibre were adjusted in the 2% asparagus diet to account for the
nutrients determined in asparagus flour (Table 5-1). Therefore, all diets had equivalent
macronutrients and were isocaloric (Table 5-2). The diets were packaged in plastic
sandwich bags and stored in a walk-in refrigerator (about 4oC) throughout the
intervention.
89
Table 5-2: Composition of experimental diets (BD, 2% asparagus, and 0.025% rutin)
AIN-93G BD 2% asparagus 0.025% rutin
Ingredients
(g/Kg diet)
Rutin 0 0 0.280
trihydrate
Asparagus 0 20 0
Casein 200 189 200
L-Cystine 3.00 3.00 3.00
Sucrose 100 100 100
Corn starch 377 373 377
Maltodextrin 132 132 132
Corn oil 70.0 68.9 70.0
Cellulose 70.0 65.7 70.0
Mineral mix 35.0 35.0 35.0
Vitamin mix 10.0 10.0 10.0
Choline 2.50 2.50 2.50

bitartrate
TBHQ 0.014 0.014 0.014
antioxidant
Nutrients (%
kcal from)
Protein 19.2 19.2 19.2
Carbohydrate 63.2 63.2 63.2
Fat 17.6 17.6 17.6
After the diets were made, rutin concentration was confirmed using procedures
described in section 3.2.1.2 to ensure proper diet preparation.
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5.3.1.3 Antioxidant Activity of Diets
The remaining soluble phenolic extracts from analyzing rutin by HPLC were used in
measuring antioxidant activity of diets by ORAC and FRAP.
5.3.1.3.1 ORAC
Antioxidant activity of diet sample extracts was determined by the ORAC assay
according to procedures described by Li et al. (2011) with modifications [268]. A stock
solution of fluorescein (Sigma Chemicals, USA; 144.65 x 10-3 mM) was prepared by
dissolving 4.8 mg of fluorescein in 100 mL phosphate buffer (75 mM, pH7.4). The working
fluorescein solution (8.68 x 10-6 mM) was prepared daily by mixing 9 μL of the stock
solution with 14.991 mL of 75 mM phosphate buffer. The AAPH (Sigma-Aldrich, USA)
radical solution (153 mM) was also prepared daily by dissolving 414 mg of AAPH in 10 mL
of 75 mM phosphate buffer. Serial dilutions of Trolox (0-100 μM) were used to generate a
standard curve from which the ORAC values of the samples were generated. As the ORAC
assay is extremely sensitive, the samples were diluted appropriately before analysis to
avoid interference. In this study, soluble phenolic extracts were diluted 25X and AHBP and
BHBP were diluted 200X with phosphate buffer.
In a 96-well plate, 150 μL of fluorescein working solution and 25 μL of sample, blank
(phosphate buffer), or standards were loaded to each well. The plate was heated for 30
seconds at 37°C prior to the addition of AAPH. The fluorescence was read immediately
after the addition of AAPH and continuously every minute for 1 hour. Each sample was
analyzed in triplicate using a microplate reader (BioTek Fluorescent, USA) with
fluorescence filters containing an excitation wavelength of 485 nm and an emission
91
wavelength of 528 nm. The ORAC values, expressed as μM of Trolox equivalent (TE)/g DW
of diet, were calculated from the Trolox standard curve.
5.3.1.3.2 FRAP
The FRAP assay used for determining antioxidant activity in diet sample extracts
was based on the procedures described by Li et al. (2011) [268]. Standards (0-1,000 μM
ascorbic acid) and samples at 10 μL were added to a 96-well microplate in triplicate. In
each well, 300 μL of working FRAP regent was added. The FRAP reagent was made daily
and consisted of acetate buffer (300 mM, pH 3.6), 2,4,6-tripyridyl-s-triazine (Sigma-Aldrich,
USA; 10 mM), and FeCl3·6H2O (20 mM) at a ratio of 10:1:1 (v/v/v). After 30 minutes of
incubation at RT, the absorbances were recorded at 593 nm using a microplate reader
(BioTek, PowerWave XS2, USA). The FRAP values, expressed as μM of acetic acid equivalent
(AAE)/g DW of diet, were calculated from the ascorbic acid standard curve.
5.3.1.4 Mice
All protocols and animal procedures used in this study were approved by the Animal
Care and Use Committee (University of Guelph). Five week old male C57BL/6 mice (n=99)
were obtained from Charles River (Portage, MI) and were acclimatized for 1 week ad libitum
on water and BD. Mice were grouped into 3 or 4 per individual stainless steel cage under
controlled room conditions: 23±2◦C RT, 50±10% humidity, and 12-hour light-dark cycle.
5.3.1.5 Study Design
The experimental protocol is summarized in Figure 5-2. Mice (n=99, 6 week old)
were divided into three treatment groups: 1) BD (n=35); 2) 2% asparagus (n=33); and 3)
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0.025% rutin (n=30), such that the mean BW of each group was not significantly different
from each other (p>0.05). Then each group of mice was fed their respective diet for 2
weeks, and during this period, food intake and BW of each group were recorded. After 2
weeks, the mice were given 2% (w/v) DSS (MP Biomedicals, USA, MW 36,000-50,000) in
the drinking water for 7 days to induce colitis. Half of the BD group received DSS-free
drinking water and served as healthy controls. During this time the mice continued on their
respective diet ad libitum. At the end of the 7 days, mice (BD+DSS and 0.025% rutin+DSS:
each n=11; and 2% asparagus+DSS: n=12) were sacrificed to evaluate the effects of
asparagus on symptoms and progression of acute colitis. The remaining animals were
switched back to normal drinking water in order to induce recovery.
During the DSS cycle, food and water intake were measured daily. Furthermore,
mice were monitored daily for DAI according to the procedures as described in section
4.2.3. Sacrifice methods as well as organ measurements were also the same as detailed in
section 4.2.3.
Figure 5-2: Experimental design

C57BL/6 mice were acclimatized on BD for 7 days and then switched over to experimental
diets for the remainder of the study. After 14 days of experimental diets, a 7-day DSS cycle
started and all mice received 2% DSS in their drinking water, except for half of the BD
group who received DSS-free water. At the end of the 7 days, a subset of mice was
sacrificed and the remaining mice started on a 5-day recovery cycle with normal drinking
water. Sacrifice occurred again at the end of the recovery cycle. During DSS-induced acute
colitis and recovery, DAI was monitored and diet and water intake were collected daily.
93
5.3.1.6 Colonic MPO Assay
Colonic MPO activity was measured by using the method as outlined in section 4.2.4.
5.3.1.7 Colon Histopathology
Colon cross sections were fixed, embedded, cut, and scored according to the
procedures described in section 4.2.5. The scoring system and representative sections for
colon erosion and goblet cell depletion during acute colitis are described in section 4.2.5
(Table 4-3 and Table 4-4, and Figure 4-3 and Figure 4-4). The scoring system and
representative histological sections for colon erosion and goblet cell depletion during
recovery are shown in Table 4-3 and Figure 5-3, and Table 4-4 and Figure 5-4, respectively.
Figure 5-3: Histological grading of colon erosion during recovery

Distal colon cross-sections were scored for erosion based on five scores: (1) normal with
intact crypts and surface epithelial cells; (2) loss of <1/3 of crypts, but surface epithelial
cells are still visible; (3) loss of 2/3 of crypts, but some crypt structures and most surface
epithelial cells are still visible; (4) loss of >2/3 of crypts, but surface epithelial cells are still
visible; and (5) loss of entire crypt and surface epithelial cells (20X magnification).
94
Figure 5-4: Histological grading of goblet cell depletion during recovery
Distal colon cross-sections were scored for goblet cell depletion based on five scores: (1)
normal with intact goblet cells; (2) loss of <1/3 of goblet cells; increase in number/size of
goblet cells; (3) loss of 2/3 of goblet cells; (4) loss of >2/3 of goblet cells; and (5) complete
loss of goblet cells (20X magnification).
5.3.1.8 Serum Cytokine Multiplex Analysis
The TH1 analytes that were measured included IL-10, IL-1β, IL-6, IL-17, TNF-α, and
IFN-ɣ. A TH17 magnetic bead-based multiplex sandwich immunoassay from the Bio-plex
Pro™ Assays (Bio-Rad Inc., CAN) was used. This assay contained dyed microspheres (i.e.
beads) conjugated with a monoclonal capture antibody that were specific for a target
protein. Serum samples were thawed and run in duplicates. According to the
manufacturer’s instructions, antibody-coupled beads were incubated with the serum
samples (1:3 dilution), and then incubated with biotinylated detection antibody to create a
sandwich complex. The final step was incubation with streptavidin-phycoerythrin (SA-PE)
conjugate to serve as a fluorescent reporter. The standards used consisted of 0.54 to
11,900 pg/mL. Bound molecules were read using a Bio-Plex 200 System (Bio-Rad Inc.,
95
CAN), which is based on Luminex fluorescent-bead-based technology with a flow-based
dual laser detector and high-speed digital processor.
5.3.1.9 Statistical Analyses
All values are expressed as means ± SEM. ORAC and FRAP antioxidant capacities
were analyzed by one-way ANOVA (SNK post-hoc test). Food and water intake were
analyzed by two-way ANOVA (SNK post-hoc test). DAI were analyzed by one-way ANOVA
on each day (SNK post-hoc test). For organ size parameters, one-way ANOVA (SNK post-
hoc test) was used for colon weight-to-length ratio and cecum weight, while Dunn’s post-
hoc test was used for spleen weight. MPO activity (with log10 transformed data), and
histopathology scores (colon erosion and goblet cell depletion) were analyzed by one-way
ANOVA (SNK post-hoc test). A difference with p<0.05 was considered significant. Serum
cytokines were analyzed by one-way ANOVA (Dunn’s post-hoc test for IL-1β, IL-6, and IL-
10, and SNK post-hoc for IL-17, IFN-γ, and TNF-α). Statistical analyses were performed
using Sigma Plot 12.0 (Systat Software Inc., USA).
Kaplan-Meier survival curves were generated using GraphPad Prism 5.03
(GraphPad Software Inc., USA) and analyzed by Log-Rank (Mantel Cox) χ2 test. Survival
curves of the DSS exposed treatment groups were considered significantly different from
healthy controls if the p value was less than the Bonferroni-corrected threshold which was
set at p<0.017.
96
5.3.2 Results
As indicated by the HPLC profile, rutin was not detected in BD, while 2% asparagus
and 0.025% rutin diets indeed contained equal detectable levels of the compound (Figure
5-5). Furthermore, there was no rutin breakdown during preparation or storage of the
rutin diets as quercetin was not detected. By using the standard curve, both 2% asparagus
and 0.025% rutin diets were validated to contain 0.025% rutin.
Figure 5-5: Determination of rutin in BD, 2% asparagus, and 0.025% rutin diets
Rutin was extracted from 0.5 g of BD, 2% asparagus, and 0.025% rutin diets through a
series of MeOH washes. A final 10 µL volume of each sample and standards (rutin and
quercetin) were used to detect rutin and quercetin by HPLC (360 nm). All peaks were
identified by comparison of retention times and UV absorbance spectra with the
commercial standards. Concentration of rutin was interpolated from the standard curve.
Each diet was run in triplicate.
5.3.2.2 Antioxidant Activity of Diets
The experimental diets, 2% asparagus and 0.025% rutin, were assessed for
antioxidant activity by two well-established assays, ORAC and FRAP. In both the assays, 2%
97
asparagus and 0.025% rutin had significantly higher antioxidant activities compared to BD,
yet 2% asparagus diet exhibited the most antioxidant activity (Figure 5-6).
A ORAC B FRAP
15 80
a
mol AAE/g DW
60
mol TE/g DW
a
10
b
40
5 b c
c 20
0 0
BD
BD
tin
tin
s
s
gu
gu
ru
ru
ra
ra
5%
5%
pa
pa
as
as
02
02
2%
2%
0.
0.
Figure 5-6: Antioxidant activity of BD, 2% asparagus, and 0.025% rutin diets
Antioxidant activities of BD, 2% asparagus, and 0.025% rutin diets were assessed in
triplicate by (A) ORAC and (B) FRAP assays. All data are expressed as the mean ± SEM.
Bars with different letters are significantly different from each other (p<0.05). Statistical
analysis was performed using one-way ANOVA (SNK post-hoc test).
5.3.2.3 Pre-DSS Food Intake and BW
Both 2% asparagus and 0.025% rutin diets were well tolerated by the mice as diet
intake and BW gain was comparable to mice in the BD group (Figure 5-7).
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A Diet Intake B Body Weight
4 20
3 15 BD
g/day/mouse
2% asparagus
% Change
2 10 0.025% rutin
1 5
0 0
4 6 8 10 12 14 0 2 4 6 8 10 12 14
Day Day
Figure 5-7: Pre-DSS food intake and BW

The (A) average daily intake per mouse (n=8-10) and (B) % change in BW (n=30-35) was
measured every 3 days. Data points are expressed as the mean ± SEM.
5.3.3 Part 1: Acute Colitis Results
5.3.3.1 Food and Water Intake During DSS-induced Acute Colitis
During the 7 day DSS cycle, DSS caused a significant decrease in diet intake (Figure
5-8). By the end of the cycle, all DSS-treated groups were not significantly different from
each other in diet and water intake.
99
4 8
3 6
mL/day/mouse
BD
g/day/mouse
a a
BD+DSS
2 4 a a 2% asparagus+DSS
b b 0.025% rutin+DSS
1 2 b
b
0 0
2 4 6 8 2 4 6 8
Day Day
Figure 5-8: Food and water intake during DSS-induced acute colitis
(n=2-9) was measured over 7 days during 2% DSS-induced acute colitis. Data points are
expressed as the mean ± SEM. Within in each day, means with different letters are
two-way ANOVA (SNK post-hoc test). BD=basal diet, DSS=dextran sodium sulfate.
5.3.3.2 DAI in Acute Colitis
During acute colitis, DSS induced BW loss (Figure 5-9A), and an increase in stool
consistency (i.e. diarrhea) (Figure 5-9B) and stool blood (i.e. rectal bleeding) (Figure 5-9C),
which corresponds with previous studies [71, 78]. By the end of the DSS cycle, there were
no significant differences among DSS-treated groups with BW loss; however, 2%
asparagus+DSS significantly attenuated the onset of diarrhea and rectal bleeding. Through
taking the average of all three scores, the total DAI score was not significantly different
between DSS-treated groups (Figure 5-9D).
100
A Body Weight B Stool Consistency
6 3 a
ab
4 a a
a a b
a 2
% Change
2 ab a
Score
ab b
b b ab ab
0 a a
1 2 3 4 5 6 ab 7 1 a b
a
-2 b b
b b
b b c b c
-4 Day 0 BD
0 1 2 3 4 5 6 7
BD+DSS
2% asparagus+DSS
Day
0.025% rutin+DSS
C Stool Blood D DAI

2.5 2.5
2.0 a a 2.0
a a
ab b a
1.5 a b 1.5
a
Score
b
Score
a
1.0 a a b
1.0 a a
b 0.5 b
0.5 b b c b b
0.0
b c b c c c 1 2 3 4 5 6 7
0.0 -0.5
0 1 2 3 4 5 6 7
Day
Day
Figure 5-9: Effects of 2% asparagus and 0.025% rutin diets on DAI during DSS-
induced acute colitis
During the 7-day 2% DSS cycle, mice (n=6-27) were monitored daily for (A) % BW loss, (B)
stool consistency, and (C) stool blood. The average of all three scores was expressed as (D)
DAI. Means with different letters are significantly different from each other (p<0.05).
Within each day, some letters are representing more than one treatment group. Statistical
analyses were performed using one-way ANOVA (SNK post-hoc test) on each day. BD=basal
diet, DSS=dextran sodium sulfate.
5.3.3.3 Organ Size Parameters During Acute Colitis
During colitis, DSS increased colon weight to length ratio, and cecum and spleen
weights. There were no significant differences between DSS-treated groups with cecum
weight and colon weight to length ratio (Figure 5-10A and B); however, DSS-induced
spleen weight enlargement was significantly attenuated by 2% asparagus+DSS (Figure
5-10C).
101
A B 0.08
0.3 a
a a
a a a
0.06
b
Colon (g/cm)
Cecum (g)
0.2
b 0.04
0.1
0.02
0.0 0.00
SS
BD
SS
SS
S
BD
SS
SS
S
+D
+D
+D
+D
s+
s+
BD
in
BD
in
gu
gu
t
t
ru
ru
ra
ra
5%
pa
5%
pa
as
as
02
02
2%
2%
0.
0.
C 0.20
a
0.15
a
Spleen (g)
0.10 ab
b
0.05
0.00
SS
BD
SS
SS
+D
+D
s+
BD
in
gu
t
ru
ra
%
pa
25
as
0
2%
0.
Figure 5-10: Effects of 2% asparagus and 0.025% rutin diets on organ size
parameters during DSS-induced acute colitis
The effects of diets on (A) colon weight-to-length ratio (n=6-12) and extraintestinal organs:
(B) cecum (n=6-11) and (C) spleen weights (n=6-11) were measured during 7 days of 2%
DSS-induced acute colitis. All data are expressed as the mean ± SEM. Means with different
letters are significantly different from each other (p<0.05). Statistical analyses were
performed using (A and B) one-way ANOVA (SNK post-hoc test), and (C) one-way ANOVA
(Dunn’s post-hoc test). BD=basal diet, DSS=dextran sodium sulfate.
5.3.3.4 Colonic MPO Activity During Acute Colitis
In acute colitis, MPO activity was increased by DSS, but there was not a significant
difference amongst DSS-treated groups (Figure 5-11).
102
5 a
a a
4
Log10 MPO (u/g)

b
3
SS
SS
BD
S
DS
+D
+D
s+
tin
BD
u
ag
ru
ar
5%
p
as
02
2%
0.
Figure 5-11: Effects of 2% asparagus and 0.025% rutin diets on myeloperoxidase
(MPO) activity during acute colitis
The effects of diets on distal colon MPO (n=5-12, in duplicate) after 7 days of 2% DSS-
induced colitis. All data are expressed as the mean ± SEM. Means with different letters are
significantly different from each other (p<0.05). Statistical analyses were performed using
one-way ANOVA (SNK post-hoc test) on log10 transformed data. BD=basal diet,
DSS=dextran sodium sulfate.
5.3.3.5 Acute Colitis Colon Histopathology: Colon Erosion and Goblet Cell
Some of the earliest histological signs of DSS-induced colitis are: increases in crypt
distortion; epithelial cell loss; mucin-producing goblet cell loss; presence of ulcers;
cryptitis; and crypt abscesses [71, 78, 83, 85]. The epithelium, containing a dense network
of innate and adaptive immune cells, is central in monitoring the integrity and health of the
colonic mucosa by eliciting appropriate responses to invading bacteria and antigens [99]. It
has been found that the removal of DSS results in recovery, including a restitution of
normal colonic architecture and an increase in crypt cell proliferation as an integral part of
the repair process [81, 86].
103
In this study, there were no effects observed between DSS-treated groups during
colitis with regards to colon erosion (Figure 5-12A) and goblet cell depletion (Figure
5-12B).
A Colon Erosion B Goblet Cell Depletion

5 5
a
4 4 a a
3 3
Score
Score
2 2
b
1 1
0 0
SS
S
S
SS
S
BD
DS
DS
DS
DS
+D
+D
+
s+
s+
T+
tin
BD
BD
gu
u
RU
ag
ru
ra
ar
5%
pa
p
25
as
as
02
0
2%
2%
0.
0.
Figure 5-12: Histopathology of distal colon erosion and goblet cell depletion during
acute colitis
The effects of 2% asparagus and 0.025% rutin diets on (A) colon erosion (n=11-12) and
(B) goblet cell depletion (n=6-12) during 7 days of 2% DSS-induced acute colitis. Data are
expressed as the mean ± SEM. Means with different letters are significantly different from
each other (p<0.05). Statistical analyses were performed using one-way ANOVA (SNK post-
hoc test). BD=basal diet, DSS=dextran sodium sulfate.
5.3.4 Part 2: Colitis Recovery Results
5.3.4.1 Food and Water Intake During Colitis Recovery
Once DSS was removed and replaced with normal drinking water again during the 5
day recovery cycle, the DSS-treated mice began to increase their diet and water intake by
day 3 (Figure 5-13). By the end of the 5 day recovery cycle, all DSS-treated groups were
normally consuming diet and water comparable to the BD group.
104
5 6
mL/day/mouse
a a
g/day/mouse
a a a BD
a 4
3 ab DSS
ab ab ab ab 2% asparagus
2 ab bc ab
2 0.025% rutin
b
b c b b b
1
0 0
1 2 3 4 5 1 2 3 4 5
Day Day
Figure 5-13: Food and water intake during colitis recovery

(n=2-5) was measured over 5 days of colitis recovery with water. Data points are expressed
as the mean ± SEM. Within in each day, means with different letters are significantly
different from each other (p<0.05). Within each day, some letters are representing more
than one treatment group. Statistical analyses were performed using two-way ANOVA (SNK
post-hoc test). BD=basal diet, DSS=dextran sodium sulfate.
5.3.4.2 Kaplan-Meier Survival Curve in Colitis Recovery
During the recovery period, the survival rate was notably different between groups
(Figure 5-14). As mentioned in the methods section 5.3.1.5, mice were removed from the
study and terminated if their BW loss exceeded 20% of their initial body weight and if they
experienced excessive diarrhea and stool blood loss. Based on these parameters, 50% (6 of
12) of the mice in the BD+DSS group remained in the study throughout the 5 day recovery
period, while 67% (10 of 15) and 85% (11 of 13), remained in the study from the 2%
asparagus+DSS and 0.025% rutin+DSS groups, respectively. This resulted in a significant
difference in survival between the DSS group (p=0.0057) and the healthy controls (BD),
while the survival of the asparagus (p=0.031) and rutin (p=0.17) groups did not differ from
healthy controls. These results indicate that asparagus and rutin supplemented diets
improved colitis recovery, with rutin being more effective than asparagus.
105
Figure 5-14: Impact of 2% asparagus and 0.025% rutin diets on survival during
colitis recovery in mice
Kaplan-Meier survival curve demonstrating the % of mice (n=12-15) which survived
within different treatment groups during the 5 day recovery period with water following a
7-day 2% DSS-induced acute colitis. Curve was analyzed by Log-Rank (Mantel Cox) χ2 test
and treatment groups were considered significantly different from healthy controls if the p
value was less than the Bonferroni-corrected threshold which was set at p<0.017. BD=basal
diet, DSS=dextran sodium sulfate.
5.3.4.3 DAI in Colitis Recovery
At the start of the recovery cycle, BD-fed mice who previously received DSS
continued to lose BW until day 4, while mice fed 0.025% rutin+DSS significantly started to
gain BW compared to those fed BD+DSS and 2% asparagus+DSS (Figure 5-15A). At day 4,
0.025% rutin+DSS significantly improved colitis recovery in terms of BW gain compared to
the BD+DSS group, followed by the 2% asparagus group, though not significantly different
from mice in the BD+DSS. With stool consistency and stool blood, there were no significant
differences amongst DSS-treated groups by the end of the DSS cycle (Figure 5-15B). There
was a decrease in stool blood by all DSS-treated groups throughout the recovery period,
which was diminished to healthy control levels by day 5 (Figure 5-15C). Lastly, the total
DAI score decreased in all treatments groups by day 7, but 0.025% rutin+DSS group
106
significantly decreased this score, followed by mice in the 2% asparagus+DSS group
(Figure 5-15D).
A Body Weight B Stool Consistency

4
10
a a a
a a 3 a a
a a
ab
a a
0 b
Score
% Change
b b
1 2 3b 4 5b 2 a
bc a
c b b
b
-10 c b 1
b b
c b b b
b b
0
-20 Day 0 1 2 3 4 5 BD
DSS
Day 2% asparagus
C Stool Blood D DAI 0.025% rutin
2.5 2.5
a a a
2.0 a 2.0 a a a
a a b a
1.5 1.5 a
a a b b
Score
Score
a ab
1.0 1.0
b
0.5 ab 0.5
bc b c c
b b b b c
0.0 c 0.0
1 2 3 4 5 1 2 3 4 5
-0.5 -0.5
Day Day
Figure 5-15: Effects of diets on DAI during colitis recovery

During the 5-day recovery cycle, mice (n=6-15) were monitored daily for (A) % BW loss,
(B) stool consistency, and (C) stool blood. The average of all three scores was expressed as
(D) DAI. Data are expressed as the mean ± SEM. Means with different letters are
sulfate.
107
5.3.4.4 Organ Size Parameters During Colitis Recovery
During recovery, colon weight-to-length ratio was also not significantly different
between DSS-treated groups (Figure 5-16A), and there was no DSS effect observed with
cecum weight (Figure 5-16B).
A 0.4
B 0.08
a a a
0.3 0.06
Colon (g/cm)
Cecum (g)
0.2 0.04
b
0.1 0.02
0.0 0.00
BD
BD
tin
tin
SS
SS
us
us
+D
+D
ag
ag
ru
ru
r
r
BD
BD
5%
5%
pa
pa
as
as
02
02
2%
2%
0.
0.
Figure 5-16: Effects of 2% asparagus and 0.025% rutin diets on organ size
parameters during colitis recovery
The effects of asparagus and rutin diets on (A) colon weight-to-length ratio and (B) cecum
weight were measured during colitis recovery. All data are expressed as the mean ± SEM.
Means with different letters are significantly different from each other (p<0.05). Statistical
analyses were performed using one-way ANOVA (SNK post-hoc test). BD=basal diet,
5.3.4.5 Colonic MPO Activity During Colitis Recovery
Similar to what was observed in acute colitis, at the end of the 5 day recovery cycle,
MPO activity was increased by DSS, but there was not a significant difference amongst DSS-
treated groups (Figure 5-17).
108
6
a a
a
Log10 MPO (u/g)

4
b
tin
BD
su
DS
ag
ru
ar
5%
p
as
02
2%
0.
Figure 5-17: Effects of 2% asparagus and 0.025% rutin diets on myeloperoxidase
(MPO) activity during colitis recovery
The effects of diets on distal colon MPO (n=5-9, in duplicate) after 5 days of colitis recovery
with water. All data are expressed as the mean ± SEM. Means with different letters are
one-way ANOVA (SNK post-hoc test) on log10 transformed data. BD=basal diet,
5.3.4.6 Colitis Recovery Colon Histopathology: Colon Erosion and Goblet Cell
Depletion
Unlike what was observed in acute colitis, during recovery, significant effects were
observed between treatment groups for colon erosion (Figure 5-18) and goblet cell
depletion (Figure 5-19). The BD+DSS group was the slowest in improving colon erosion,
while 2% asparagus and 0.025% rutin significantly regenerated crypts and surface
epithelial cells compared to BD+DSS, with 0.025% rutin having the greatest impact on
colon erosion recovery (Figure 5-18). These patterns were also seen with goblet cell
depletion. The 0.025% rutin-fed mice had the greatest impact in increasing the
regeneration of mucin-producing goblet cells, followed by 2% asparagus (Figure 5-19).
109
Thus, the decreased survival rate in mice fed BD+DSS during colitis recovery may
potentially be attributed to slow improvements in colon architecture and goblet cell
regeneration.
A Colon Erosion
6
a
b
4 c
Score
0
SS
tin
s u
+D
ag
ru
ar
BD
5%
p
as
02
2%
0.
Figure 5-18: Histopathology of distal colon erosion during colitis recovery

The effects of 2% asparagus and 0.025% rutin diets on (A) colon erosion (n=9-13) during 5
days of colitis recovery with water. (B) Representative sections are shown (10X
magnification). Data are expressed as the mean ± SEM. Means with different letters are
one-way ANOVA (SNK post-hoc test). BD=basal diet, DSS=dextran sodium sulfate.
110
A Goblet Cell Depletion
6
a
b
4
c
Score
2
d
tin
SS
s
BD
u
+D
ag
ru
ar
BD
5%
p
as
02
2%
0.
Figure 5-19: Histopathology of distal colon goblet cell depletion during colitis
recovery
The effects of 2% asparagus and 0.025% rutin diets on (A) goblet cell depletion (n=6-13)
during 5 days of recovery with water. (B) Representative sections from recovery are
shown (10X magnification). All data are expressed as the mean ± SEM. Means with different
letters are significantly different from each other (p<0.05). Statistical analyses were
performed using one-way ANOVA (SNK post-hoc test). BD=basal diet, DSS=dextran sodium
sulfate.
111
5.3.4.7 Serum Cytokine Multiplex Analysis in Colitis Recovery
Mouse serum from the recovery phase showed that DSS significantly increased IL-
1β and IL-10, but mice fed 0.025% rutin attenuated these increases (Figure 5-20A and C).
DSS also was shown to increase IL-6, IL-17A, and IFN-γ, and TNF-α during recovery (Figure
5-20B, D-F), but there were no significant differences amongst DSS-treated groups, which
may be due to the large variances.
A B C
500 200 400
a
400 a
a
150 300
IL-1 (pg/mL)
IL-10 (pg/mL)
IL-6 (pg/mL)
a a
300 ab ab
100 200
200 b a
100 50 100
b
b
0 0 0
tin
BD
s
SS
SS
in
SS
in
BD
BD
s
s
gu
gu
gu
t
t
+D
+D
+D
ru
ru
ru
ra
ra
ra
BD
BD
BD
5%
pa
5%
5%
pa
pa
as
as
as
02
02
02
2%
2%
2%
0.
0.
0.
D E F
1500 500 1500
a a a
400
IL-17A (pg/mL)
TNF- (pg/mL)
a
IFN- (pg/mL)
1000 a a 1000 a
a a
300
500 200
500
b 100 b b
0
0 0
tin
BD
SS
us
tin
BD
s
SS
SS
BD
tin
us
+D
ag
gu
ru
+D
+D
ru
r
ru
BD
ra
ra
5%
pa
BD
BD
5%
pa
5%
pa
as
02
as
as
2%
02
02
0.
2%
2%
0.
0.
Figure 5-20: Serum TH1 cytokine multiplex analysis in colitis recovery

Serum from mice fed 2% asparagus and 0.025% rutin diets during the 5 day recovery were
analyzed for TH1 cytokines by multiplex analysis: (A) IL-1β (n=8), (B) IL-6 (n=8), (C) IL-10
(n=8-9), (D) IL-17A (n=7-8), (E) IFN-γ (n=8-9), and (F) TNF-α (n=8-9). All data are
expressed as the mean ± SEM). Means with different letters are significantly different from
each other (p<0.05). Statistical analyses were performed using (A-C) one-way ANOVA
(Dunn’s post-hoc test), and (D-F) one-way ANOVA (SNK post-hoc test). BD=basal diet,
112
5.3.5 Discussion
The objectives of this study were to determine the effects of whole cooked
asparagus and 0.025% rutin-supplemented diets during DSS-induced acute colitis and
recovery in C57BL/6 mice. After feeding the mice 2% asparagus or 0.025% rutin in their
diet for 2 weeks prior to and during DSS colitis induction, the 2% asparagus diet reduced
some clinical symptoms of colitis (e.g. stool consistency, stool blood, and spleen
enlargement). Though, these improvements were not reflected in histological scores with
asparagus diet. During recovery, both 2% asparagus and 0.025% fed mice had significantly
greater survival than the DSS group as they did not differ from healthy controls.
Furthermore, 0.025% rutin was more effective than 2% asparagus in recovery in terms of
increased diet and water intake, BW gain, total DAI score, improvement in histological
scores, and reductions in IL-1β and IL-10 levels in serum. Overall, this was the first study to
show the effects of rutin in recovery and whole asparagus during acute colitis and
recovery.
In Chapter 4, rutin was observed in DSS-induced colitis, and it was found that mice fed
0.025% rutin significantly reduced spleen enlargement. When cross-comparing between
Chapter 4 and this study, 0.025% rutin was analogous between studies, producing similar
clinical, molecular, and histological data in colitis. During colitis, mice fed 2% asparagus
significantly improved some clinical symptoms compared to those fed 0.025% rutin fed (e.g.
stool consistency and stool blood). It is likely that the matrix of the asparagus is contributing
anti-inflammatory and/or anti-oxidative effects. Prior to starting the intervention, an analysis
of the antioxidant capacities of all diets was completed by FRAP and ORAC assays. It was
found that the 2% asparagus diet had significantly higher antioxidant capacity compared to
113
the 0.025% rutin diet. This indicates that 2% asparagus contains additional anti-oxidative
compounds, including phenolics other than rutin. The attenuation in clinical colitis symptoms
could be attributed to the anti-oxidative nature of rutin and quercetin [212, 213, 262, 263], as
well as other bioactives (e.g. vitamins, minerals) and unidentified phenolic compounds within
asparagus [238, 269, 270]. It is probable that prior to colitis, the bioactives in asparagus
matrix prepared the colon and/or increased the endogenous antioxidant defense system for
potential insults. Therefore, after colitis is initiated, asparagus bioactives could then directly
eliminate ROS and/or maintain a balance in the antioxidant defense system [212, 213, 238,
262, 263, 269]. It is not likely that the anti-inflammatory effects of asparagus during active
colitis could be from mainly dietary fibre as there were no significant differences seen with
the cecum weight (usually known to be an indicator of dietary fibre fermentation [241]). It is
very probable that there was not enough dietary fibre in the 2% asparagus diet. Although
asparagus itself is a good source of dietary fibre, a level of 2% asparagus in diet is minimal.
Vitamin K, which is well known for its role in blood clotting, is found abundantly in
asparagus. This vitamin could have aided in reducing the onset of rectal bleeding. Vitamin K
injections have been used to treat infants who have had potentially fatal rectal bleeding in the
first month of life [271].
The most prominent effects were observed in the recovery phase when DSS was
removed and C57BL/6 mice were given normal drinking water for 5 days. Mice fed 0.025%
rutin significantly consumed more diet and water starting on days 1 and 2, respectively,
compared to 2% asparagus and DSS groups, which is an indication of increased recovery.
This may explain why 0.025% rutin-fed mice recovered quicker than BD+DSS and 2%
asparagus groups in terms of BW gain and total DAI score, thereby improving in other
114
parameters (e.g. histopathology scores). Mice fed 2% asparagus also significantly improved
in these measures, but not as much as 0.025% rutin-fed mice. One reason could be because
there was overall reduced diet intake (including asparagus diet intake) as a result of DSS-
induced acute colitis, thus there would be a reduction in obtaining anti-oxidative and anti-
inflammatory rutin and other bioactives in asparagus matrix during active colitis. Another
reason could be due to a bioavailability issue; there could be delays in releasing rutin from
certain components of the asparagus matrix, such as soluble fibre [272], whereas rutin would
be readily free in the 0.025% rutin diet to exert effects during recovery.
Moreover, there were significant differences seen with serum cytokine levels during
recovery. The expression of IL-1β, TNF-α, IL-6, and IL-10 has been observed to increase as
early as 1 day post-DSS treatment [88]. After 5 days of DSS followed by 7 days of water
treatment in C57BL/6J mice, the expressions of TNF-α, IL-1β, IL-6, IL-10, and IFN-γ [88] were
increased, and by 28 days of water, there were high levels of IL-1β, IL-6, and IL-17 [83]. In
this study, high levels of TNF-α, IL-1β, IL-6, IL-10, IL-17, and IFN-γ induced by DSS were also
observed. However, mice fed 0.025% rutin diets had mildly reduced IL-1β and IL-10 levels in
their serum compared to BD+DSS and 2% asparagus groups. These results coincide with
previous studies regarding rutin and quercetin with IL-1β. Quercetin, released from rutin, has
been found to significantly inhibit IL-1β levels in BMDM [172]. Furthermore, a recent study
discovered that the dose-dependent inhibition of IL-1β by quercetin leads to an inhibition of
LPS-induced DC activation, which is the most potent APC [273]. There has not been in vitro
studies looking at rutin in regards to IL-1β, but one study has observed that rutin can reduce
colitis severity in vivo by inhibiting IL-1β mRNA and protein expression [176]. There is also a
lack of studies observing quercetin and rutin with regards to IL-10 levels, but it is likely that
115
these two compounds are either directly or indirectly reducing levels of neutrophils, T cells, B
cells, and NK cells that are producing IL-10 [104, 108, 109]. In this study 0.025% rutin was
more effective in reducing IL-1β and IL-10 levels during recovery than 2% asparagus. This
may be because 0.025% rutin-fed mice consumed more diet compared to 2% asparagus-fed
mice during the 5-day recovery, thereby obtaining more rutin and its respective aglycone,
quercetin. There could also be a bioavailability issue occurring, resulting in a delay of rutin
release from specific components of the asparagus food matrix, such as soluble fibre [272], to
exert effects.
There may be two reasons why rutin was not found to be beneficial in colitis, but was
during recovery, and these include: 1) the dose of DSS and 2) distinct mediators and
mechanisms involved in colitis and recovery. The dose of DSS to initiate colitis is very vital,
especially with the specific mouse strain used as evidence has suggested that there are
differences in susceptibility to DSS in various mouse strains [81]. For instance, BALB/c mice,
which are often used in DSS-induced colitis studies, are less susceptible to DSS compared to
IQI/Jic [82] or C57BL/6 mice [83]. Although the DSS dose used (2%) in C57BL/6 mice is
comparable with other studies using C57BL/6 [274-276], it is still possible that this DSS dose
was too strong, thereby masking any protective effects of rutin during colitis. Secondly, a
relapsing and a recovering state are two distinct phases, producing dissimilar mediators and
mechanisms of inflammation and repair. This can be supported by the evidence that there are
different levels of cytokines secreted and detected during colitis and recovery. Levels of IL-
1β, TNF-α, IL-6, and IL-10 are usually seen 1 day post-DSS colitis induction [88]. And after 5
days of DSS treatment followed by 7 days of water, the same levels of cytokines are present
but there is an addition of IFN-γ [88], and by 28 days of water, elevated cytokine levels are
116
reduced to only IL-1β, IL-6, and IL-17 [83]. Therefore, it is possible that rutin and asparagus
may be interacting differently with certain mediators of inflammation during colitis and
recovery.
Overall, this study demonstrated for the first time that during acute colitis, C57BL/6
mice fed 2% asparagus had the greatest reductions in colitis severity as indicated by the
decrease in DSS-induced diarrhea onset and rectal bleeding, and attenuation in DSS-
induced spleen hypertrophy. Yet, during recovery, 0.025% rutin-fed mice recovered the
quickest in terms of BW gain and total DAI, reductions in IL-1β and IL-10, and crypt,
epithelial and goblet cell regeneration. Mice fed 2% asparagus also recovered quicker than
the control group. It is likely that 0.025% rutin-fed mice recovered quicker than the 2%
asparagus group because they consumed more diet than mice fed 2% asparagus, thereby
obtaining an increased level of rutin. However, there has been research suggesting that
food matrices, including dietary fibre, can interfere or delay the release of antioxidants
[272]. Therefore, to further understand the role of cooked asparagus in gut health
(diseased and healthy states), future bioavailability and bioaccessibility studies are
warranted.
117
Chapter 6 : Thesis Overview, Future Directions, and
Implications
118
6.1 Thesis Overview
This thesis was comprised of three studies using C57BL/6 mice: 1) chemical and
proximate characterization of asparagus flour and an assessment of the suitability of
supplementing asparagus in C57BL/6 mice diets; 2) an investigation of the effects of rutin
on symptoms and disease progression in DSS-induced acute colitis in C57BL/6 mice; and 3)
an assessment of whole cooked asparagus and purified rutin in DSS-induced acute colitis
and recovery. Asparagus is known to be a good source of micro- and macronutrients,
dietary fibre, and phenolic compounds, yet, for this thesis, the phenolic compounds of
asparagus, particularly rutin and quercetin were emphasized.
In Chapter 3, the objectives were to first characterize the macronutrients and rutin
concentration in cooked asparagus, and then the second objective was to test if asparagus
would be palatable to C57BL/6 mice. Cooked asparagus was found to contain high mounts
of rutin, protein, and fibre, particularly soluble fibre, low in dietary fat and available
carbohydrates, and rich in minerals. For the second objective, 12% asparagus was
supplemented in mouse diet, because it was equivalent to 0.1% rutin, which was the level
that has seen to be the most effective in reducing colitis in ICR mice by Kwon et al. (2005)
[176]. One week into the feeding, it was discovered that the mice were not tolerating the
diet well compared to the BD group as indicated by the decrease in diet intake and BW
gain. Thus, the 12% asparagus was modified with BD to create two more doses, 3 and 6%
asparagus (0.025 and 0.05% rutin, respectively) in powder form. It was concluded that
12% asparagus (0.1% rutin) was not tolerable with C57BL/6 mice, yet, 3% asparagus
(0.025% rutin) was.
120
In Chapter 4, the effects of rutin (0.025, 0.05, and 0.1%) on DSS-induced acute colitis
in C57BL/6 mice were examined. As seen with ICR mice [176], we wanted to verify if 0.1%
rutin would also have the greatest reductions in colitis severity in C57BL/6 mice. Yet, after
giving the respective diets for 3 weeks, we did not see significant differences amongst rutin
doses for most of the measured parameters, except that rutin was able to attenuate stool
consistency changes leading to diarrhea compared to BD+DSS mice. Also, the most
significant difference observed was an attenuation of DSS-induced spleen hypertrophy by
0.025% rutin. Therefore, based on these results, rutin can alleviate DSS-induced acute
colitis in C57BL/6 mice, but not dose-dependently.
In Chapter 5, I investigated the effects of consuming whole cooked asparagus and
rutin in C57BL/6 mice during acute colitis and recovery, mimicking the relapse and
recovery states of UC. Based on Chapters 3 and 4, we used an asparagus dose of 2%,
containing 0.025% rutin. Before the intervention began, an analysis of the diets for
antioxidant activity by FRAP and ORAC assays was completed. The 2% asparagus diet had
significantly higher antioxidant activity compared to 0.025% rutin, which denotes that 2%
asparagus must contain other anti-oxidative bioactives and phenolic compounds. Yet, it
must be kept in mind that human digestive systems may be more efficient in releasing
phenolics from food matrices than lab chemical extraction methods, so the antioxidant
activity of 2% asparagus diet may be greater than what was reflected by FRAP and ORAC
assays. One of the limitations of current chemical extraction methods of phenolic
compounds is that they cannot fully represent human digestive system. During acute
colitis, mice fed 2% asparagus significantly attenuated DSS-induced colitis symptoms.
Though in recovery, 2% asparagus fed mice had significant improvements in colitis
121
symptoms, mice fed 0.025% rutin had greater improvements in disease symptoms
compared to the 2% asparagus group. It is unknown at this point whether these effects are
because 0.025% rutin-fed mice ate more at the beginning, or if it is a bioavailability issue. If
it is the latter, then there could have been a delay in releasing rutin from certain
components of the asparagus food matrix, such as soluble fibre [272], whereas rutin is
readily free in the 0.025% rutin diet. However, from past research, these beneficial effects
can be attributed to the anti-oxidative and anti-inflammatory properties of rutin and
quercetin [172, 176, 210-213, 216, 248, 261, 262, 268, 269, 277].
Overall, this thesis has determined: the amounts of macronutrient, fibre, and rutin,
and the suitability of using cooked asparagus in animal diet (Chapter 3); the effect of rutin
in alleviating DSS-induced acute colitis symptoms and disease progression (Chapter 4); and
the effects of whole cooked asparagus and purified rutin in DSS-induced acute colitis and
recovery (Chapter 5), all with C57BL/6 mice This is the first study to demonstrate that
whole cooked asparagus is beneficial to colon health during relapse (inflammatory) and
recovery states of experimental UC.
6.2 Future Directions
6.2.1 Asparagus Characterization
Currently, a full characterization of asparagus bioactives has not been completed,
including phenolics. From the FRAP and ORAC analyses, it appears that the asparagus diet
contains other phenolic compounds aside from rutin. A full characterization of all the
bioactives and phenolic compounds in asparagus would be helpful in understanding how
asparagus can affect colon health in terms of the individual bioactive it contains, as well as
122
determine if there are any interactions (synergism or antagonism) between bioactives.
Mass-spectrometry combined with liquid chromatography is an efficient and highly
accurate method that can be used to identify unknown bioactives in asparagus, including
determining the structure of those compounds [278-280].
6.2.2 Bioavailability and Bioaccessibility
Bioaccessibility studies are needed to understand how accessible rutin or other
bioactives from asparagus is to the colon in order to then elucidate the mechanisms
involved with asparagus bioactives in colon health. Direct interactions between phenolics
and components of food, such as dietary fibre, proteins, and polysaccharides, can occur,
which can affect absorption [281]. For instance, with regard to flavonols, much higher
plasma concentrations were obtained when quercetin glucosides were given to fasted
volunteers in the form of a water-alcohol solution [282] than when an equivalent quantity
was ingested in foods like onions, apples, or a complex meal [283, 284]. A recent review has
also established that dietary fibre can limit the bioavailability of phenolic compounds in
fruits and vegetables. The authors concluded that the main reasons were because: 1) the
phenolic compounds are not well released from fruit and vegetable matrices; 2) dietary
fibre entraps the phenolic compounds during digestion in the upper intestine, and 3) some
antioxidants may be bound to polysaccharides and thus require enzymatic hydrolysis to be
absorbed, but this is restricted by dietary fibre matrices formed in the chyme [272].
Therefore, to assess the bioavailability of rutin from asparagus, a feeding study with
cooked asparagus or purified rutin in healthy subjects is needed. The amount of rutin and
123
quercetin metabolites can be measured in either blood serum or plasma, fecal, or urine
samples [285].
6.2.3 Mechanisms of Action of Asparagus on Colon Health
6.2.3.1 Gut Barrier Integrity
The preservation of gut barrier integrity, including: mucosal barrier; luminal
epithelial cell and tight junction interactions; and a balance between elimination of
damaged cells and generation of new cells in intestinal crypts; is very important in
preventing antigen penetration, injury, and stress of the colon [286]. There has been
evidence to suggest that certain food components can increase intestinal gut barrier
integrity. For instance, substantial amounts of dietary fibre, both soluble and insoluble, in
the diet can increase colonic mucosal production in rats [287]. Fermentable fibre can also
provide SCFAs that are an important energy source for colonocytes [288]. Feeding
asparagus to healthy subjects and assessing their colons could indicate whether or not
asparagus can increase gut barrier integrity and prepare the colon for potential impending
insults from toxins and pathogens, for instance. Assessments could be through histological
analyses (Hematoxylin & Eosin for colon morphology and Alcian Blue for mucus-producing
goblet cells) and gene expressions of MUC2 [289, 290]. MUC2 is a gel-forming mucin that
makes up the mucosal barrier and is known to be the first line of defense in protecting
against damaging agents within the lumen [90]. It is possible that quercetin may have
effects on MUC2 and other MUC genes within the colon. It has been found that quercetin
can inhibit human neutrophil elastase-induced MUC5AC expression in human airway
epithelial cells [291], so it may be possible that phenolics can affect MUC genes involved in
124
the colon. This could further elucidate the mechanisms of action of asparagus with regards
to colon health.
6.2.3.2 Antioxidant Status
Aside from potentially enhancing gut barrier integrity, asparagus may also be able to
increase the endogenous antioxidant defense system in a healthy state prior to colitis or in
remission prior to relapse. There has been research demonstrating that the antioxidant
capacities of serum or plasma from healthy subjects are increased after the ingestion of
antioxidant-rich foods [292-294]. From FRAP and ORAC analyses, it was determined that
asparagus diet contained the highest antioxidant capacities compared to BD and rutin diets.
However, it is unknown whether asparagus diet increased antioxidant levels systemically
in the blood and/or locally at the colon tissue. Therefore, analyses of the antioxidant
capacity in serum and/or colon tissue are warranted through FRAP, ORAC, or GSH assays
[120, 295].
6.2.3.3 Direct Effects on NF-κB Inflammatory Pathway
Though serum cytokines were measured after the 5 day recovery cycle, an analysis
of serum cytokines during acute colitis still needs to be completed. Since it is vital to
understand which cytokines are elevated and/or attenuated during active colitis by the
treatments, I will be completing this analysis sometime in the near future using the same
multiplex kit as described in section 5.3.1.8. It is also essential to complete to be able to
compare with the recovery serum cytokine data.
One main pathway that may be playing a major role in the inflammatory process of
DSS-induced colitis is the NF-κB pathway. NF-κB exists in the cytoplasm of cells in an
125
inactive form associated with inhibitory proteins [296]. When NF-κB is exposed to
activation signals such as TNF-α or ROS binding to cell surface receptors, the inhibitory
proteins associated with NF-κB are broken down. An increased activation of NF-κB leads to
increased expression of proinflammatory cytokines and immunoregulatory mediators
[296]. Certain components of the diet (e.g. butyrate, polyphenols, omega-3 PUFAs) have
been demonstrated to be effective inhibitors of NF-κB activation [297-299]. Hence,
asparagus, which is rich in polyphenols and other anti-inflammatory bioactives, may be
interacting with the NF-κB pathway. In order to confirm this, NF-κB assessments from
colon tissue using an ELISA kit should be completed [300].
6.2.3.4 Colonic Microflora Profile
The human intestinal tract consists of a complex community of microbiota (1011 to
1012 cells/gram) that is critical in influencing bodily health [301]. The intestinal microflora
varies between individuals and these variations are dependent on non-dietary (e.g.
geographic location, occupation) and dietary factors [301]. A diet consisting of prebiotics
(e.g. fructo-oligosaccharides, inulin) can significantly increase fecal concentrations of
bifidobacteria, which are considered key players in health and well-being, including
reducing disease risk in humans [288, 302]. Thus, asparagus matrix may also have the
ability to enhance health-promoting bacteria prior to colitis.
During colitis, however, evidence has suggested that there is a shift in bacteria
populations in UC patients [303] and in rodents of DSS-induced colitis to pro-inflammatory
Gram-negative intestinal bacteria [304]. Most phenolics, such as rutin, in food are in the
form of esters, glycosides, or polymers that cannot be absorbed in their native form. They
must be hydrolyzed or cleaved by intestinal enzymes (e.g. β-glucosidase) or by colonic
126
microflora, respectively, before they can be absorbed. Therefore, it is vital to verify if
microflora (e.g. Bacteroides population producing β-glucosidase) is decreased during colitis
in C57BL/6 mice, and whether or not this affected rutin metabolism. To assess microflora
populations prior to and during colitis, rutin metabolites can be identified in fecal or colon
tissue samples via culturing techniques and/or polymerase chain reaction-denaturing
gradient gel electrophoresis [301, 305].
6.3 Implications
Overall, the findings of this thesis are influential to managing symptoms of UC, a
prevalent condition of remitting and relapsing inflammation of the large intestine [1]. As
previously discussed, current conventional therapies for UC have several limitations,
including severe side effects, drug dependence, and antibiotic resistance [142-146]. Thus,
these limitations encourage the use of natural dietary interventions among UC patients,
such as PUFAs, prebiotics, and phenolic compounds. Currently, the link between diet,
etiology, and signs and symptoms of UC is unclear. Yet, it has been noted by UC patients
that they often experience food sensitivities, particularly with cereals, milk, eggs,
vegetables, and citrus fruits [306]. These food sensitivities could be dependent on the time
they are eaten (i.e. during relapse or remissive states) [307]. Certain dietary foods, such as
high meat or alcoholic beverage intake, have also been identified to increase the likelihood
of relapse in UC patients [308].
In the end, the aim for IBD therapy is to downregulate inflammation, reduce the
incidence of relapse, increase the healing time, and/or maintain remission without the
reliance of pharmacotherapy and surgical intervention. Nutrition currently offers a
127
prospective role in meeting these aims, but without additional research, it remains
uncertain whether the modification of diets would have a role in IBD management. As
demonstrated in this thesis, cooked whole Guelph Millennium asparagus can be beneficial
during relapse and recovery of experimental UC. This advocates for increased public
awareness of the health benefits of asparagus. An increased rate of asparagus consumption
could also be economically profitable for farmers and for Ontario as Southern Ontario
grows most of the Guelph Millennium asparagus.
128
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The Effects of Cooked Whole Asparagus (Asparagus Officinalis L.) and Its Purified Bioactive, Rutin (PDFDrive)

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The Effects of Cooked Whole Asparagus (Asparagus officinalis L.

In partial fulfillment of requirements

Guelph, Ontario, Canada

Jenifer Thi Lu Advisor: Dr. Lindsay Robinson

rutin, on colitis symptoms and disease progression in mice using a chemically-induced

recovery, asparagus-fed mice were improving in terms of regenerating crypts, surface

recovery phases of ulcerative colitis.

Alison Duncan who accepted to chair my defense.

Inflammatory bowel disease (IBD) is a spontaneous chronic, relapsing, and

dramatically distinct with regards to disease location, morphology, and histopathology. In

share many inflammatory similarities such as epithelial barrier dysfunction, but

histologically, UC usually consists of more neutrophil infiltration, goblet cell mucin

stabilize in those high-incidence populations, while continuing to increase in low-incidence

therapeutic and preventative treatments.

1.2 Risk Factors of IBD

As a multifactorial disorder, the development of UC is influenced by several factors

environmental factors [2, 19-21].

Population-based studies suggest that genetic susceptibility plays a role in the

were 15% and 4%, respectively [28].

susceptibility genes involved in IBD using genome-wide association scanning in large

cell response, to just name a few [40, 41].

1.2.2 Immune System and Microflora

it initiates an amplified response to bacteria, causing inflammation [44]. A recent study

IL-1β processing, suggesting initiation and/or promotion of mucosal inflammation [45].

infections and an activated mucosal immune system.

A continuous stimulation of the mucosal immune system can lead to uncontrolled

inflammation and subsequent defects to the intestinal epithelial barrier function,

1.2.3 Environmental Factors

Emigration [52-54], industrialization, and urbanization may explain the increase in UC

sedentary indoor workers have an increased risk [55].

progression of UC is not conclusive. The most consistent association noted in dietary

Interestingly, it has been widely publicized that there is an inverse relationship

rising incidence worldwide.

1.3 Animal Models of IBD

colitis can be characterized as genetic [gene knockout (KO), transgenic], immunological

(adoptive cell transfer), or chemical-based [67].

Gene KO models involve ‘knocking-out’ or making a specific gene or DNA region

1.3.2 Dextran Sodium Sulfate Model of Colitis

1.3.2.1 DSS Overview

DSS is a polyanionic derivative of dextran, a complex polymer of straight and

branched chains of glucose. Dextran is synthesized from sucrose by specific lactic-acid

IBD is known to be a complex multifactorial disease, which is not reproducible in

perceived as a representative model of experimental colitis as it mimics symptoms of UC,

duration of exposure of DSS [78].

Mice can show different susceptibilities and responsiveness to DSS-induced colitis,

and/or resisting inflammatory damage.

through the mucosal membrane to exert its effects [84].

1.3.2.2 Clinical, Histological, and Molecular Features of DSS-induced Colitis

permeability; immune cell infiltration (e.g. neutrophils, macrophages, and lymphocytes)

on mouse strain [83].

during inflammatory processes. Thus, MPO activity is an indicator of the degree of

Furthermore, the expression of proinflammatory cytokines (e.g. IL-10, IL-12, interferon

and therapeutic options for colitis.

1.3.2.3 Pathogenesis of DSS-induced Colitis

sulfation of mucins, have increased susceptibility to colitis [93]. So it is probable that

inflammation [85, 94].

DSS in Sprague-Dawley rats increased epithelial apoptotic index by 20-fold compared to

balance between apoptosis and proliferation of colonic epithelial cells.

experimental model of human UC, clinically, histologically, molecularly, and pathogenically.

experimental models that are currently available, as previously discussed, it is possible to

mediators and oxidative stress in IBD pathogenesis will only be discussed.

1.4.1 Inflammatory Mediators in Intestinal Immunity

Cytokines can be categorized into either pro- or anti-inflammatory