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Title page i
Certification ii
Dedication iii
Acknowledgement iv
Table of Content v
List of Figures vii
List of Tables vii
Abstract viii
1.0 Introduction 1
1.1 Justification 4
1.2 Objectives 4
2.0 Literature review 5
2.1 Diabetes Mellitus 5
2.1.1 Pathophysiology of Diabetes Mellitus 5
2.1.2 Classification of Diabetes Mellitus 7 Type 1 diabetes 7 Type two diabetes 8
2.1.3 Prevalence and incidence of type 2 diabetes 10
2.1.4 Key enzymes linked to type-2 diabetes mellitus 12
2.1.5 Alpha glucosidase inhibitors 13
2.1.6 Alpha amylase inhibitors 13
2.2 Free radicals and oxidative stress 14
2.3 Hyperglycemia and oxidative stress 15
2.4 Lipid peroxidation 17
2.5 Antioxidants 20
2.5.1 Endogenous antioxidants 21
2.5.2 Dietary antioxidants 23
2.6 Polyphenols 25
2.7 Gallic acid 27
2.8 Acarbose 28
3.0 Materials Methods 29
3.1 Materials 29
3.1.1 Sample collection 29
3.1.2 Aqueous preparation 29
3.1.3 Chemicals and reagents 29
3.2 Methods 30
3.2.1 Alpha glucosidase inhibition assay 30
3.2.2 Alpha amylase inhibition assay 30
3.2.3 Fe2+ chelation assay 31
3.2.4 Inhibition of lipid peroxidation and thiobarbituric acid reactions 31
3.2.5 Determination of ferric reducing antioxidant property 32
3.2.6 2, 2′-azino-bis(-3-ethylbenzthiazoline-6-sulphonate scavenging ability 32
3.2.7 1, 1-diphenyl–2-picrylhydrazyl (DPPH*) free radical scavenging ability 33
3.3 Data Analysis 33
4.0 Results 34
5.0 Discussion 44
6.0 Conclusion and recommendation 49
6.1 Conclusion 49
6.2 Recommendation 49
Figure 2.1 Diagram showing pathogenesis of type 2 diabetes 10
Figure 2.2 Map showing the prevalence of diabetes mellitus 12
Figure 2.4 The antioxidant pathway 25
Figure 2.5 Molecular structure of Gallic acid 28
Figure 2.6 Molecular structure of Acarbose 29
Figure 4.1 Effect of Gallic acid on α- Glucosidase inhibitory ability
of Acarbose in vitro 35
Figure 4.2 Effect of Gallic acid on α- amylase inhibitory ability
of Acarbose in vitro 36
Figure 4.3 Effect of Gallic acid on Fe2+ chelating ability of Acarbose in vitro 38
Figure 4.4 Effect of Gallic acid on Inhibition of Fe2+ induced lipid by
acarbose peroxidation in Rat’s pancreas in vitro. 39
Figure 4.5 Effect of Gallic acid on Ferric reducing antioxidant properties
of acarbose in vitro 41
Figure 4.6 Effect of Gallic acid on DPPH radical scavenging ability
of acarbose in vitro 42
Figure 4.7 Effect of Gallic acid onABTS* scavenging ability
of acarbose in vitro 43
Type 2 diabetes mellitus (T2DM) is a chronic progressive disease that has continued to be a
global heath and economic burden. Acarbose is an antidiabetic drug, which acts by inhibiting
alpha amylase and alpha glucosidase; while gallic acid is a simple phenolic acid that is
widespread in plant foods and beverages such as tea and wine.This study therefore, sought to
investigate the influence of gallic acid on α-amylase and α-glucosidase inhibitory and antioxidant
properties of acarbose (in vitro). Aqueous solution of acarbose and gallic acid were prepared to a
final concentration of 25µM each. Thereafter, mixtures of the samples (50% acarbose + 50%
gallic acid; 75% acarbose + 25% gallic acid; 25% acarbose + 75% gallic acid) were prepared.
The results showed that the combination of 50% acarbose and 50% gallic acid showed the
highest α-glucosidase inhibitory effect, while 75% acarbose + 25% gallic acid showed highest α- amylase inhibitory effect. Furthermore, all the samples caused the inhibition of Fe2+
lipid peroxidation (in vitro) in rat pancreatic tissue homogenate, with the combination of 50%
acarbose and 50% gallic acid causing the highest reduction in the malondialdehyde content. In
addition, all the samples showed antioxidant properties (ferric reducing property, 2, 2′-azino-bis
(-3-ethylbenzthiazoline-6-sulphonate (ABTS*) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
radicals scavenging abilities, and Fe2+ chelating ability). Therefore, the combinations of gallic
acid with acarbose could be employed in the management of T2DM with the comparative
advantage of possible reduction of the side effects of acarbose; nevertheless the combination of
50% acarbose and 50% gallic acid seems the best combinatory therapy for the management of
type 2 diabetes mellitus.
Diabetes Mellitus (DM) commonly referred to as diabetes is a group of metabolic
diseases characterized by hyperglycemia (High blood sugar levels over a prolonged period), either because the pancreas does not produce enough insulin, or because the cells do not respond
to the insulin that is produced (David and Gardner, 2011). The main symptoms of high blood
sugar include polyuria (frequent urination), polydipsia (increased thirst) and polyphagia
(increased hunger). If left untreated, this chronic disease can cause many complications (Cooke
and Plotnick, 2008).
Type 2 diabetes is the most common form of diabetes (Shi and Hu 2014); it is
characterized by insulin resistance, which may be combined with relatively reduced insulin
secretion (David and Gardener, 2011), leading to hyperglycemia and ultimately malfunctioning
of the pancreatic β-cells. Prolonged hyperglycemia results in increased generation of reactive
oxygen species (ROS) and alteration of endogenous antioxidants (Ohkuwa et al., 1995).
Oxidative stress resulting from the hyperglycemic condition in Type 2 diabetes has been
implicated in the impairment of the pancreatic β-cells and diabetes complications such as
diabetes nephropathy (damage to the kidney) (Shukla et al., 2003), diabetes retinopathy (damage
to the eye) (Bearse et al., 2004; Hove et., 2004) and diabetes neuropathy (damage to the nerves
of the body) (Seki et al., 2004).
A practical approach to reducing the postprandial hyperglycemia is to retard the
absorption of carbohydrates after food intake (Oboh and Ademiluyi, 2013). This could be
achieved through the inhibition of α-amylase and α-glucosidase present in the gastrointestinal
tract (shim et al., 2003). Inhibitors of these enzymes slow down carbohydrate digestion time,
causing a reduction in the rate of glucose absorption and consequently blunting the postprandial
plasma glucose rise (Rhabasa- Lhoret and Chiasson, 2004). The dietary saccharides are first
broken down to monosaccharides by certain gastrointestinal enzymes, since only
monosaccharides can be absorbed from the intestinal lumen. Polysaccharides are hydrolyzed to
oligosaccharides and disaccharides by α-amylase and intestinal α- glucosidase further hydrolyzes
it to glucose before being absorbed into the intestinal epithelium entering the blood circulation
(Oboh et al., 2011).
Several reports have been published on established enzyme (α-glucosidase and α- amylase) inhibitors such as Acarbose, Miglitol, voglibose, nojirimycin and 1- deoxynojirimycin
and their favorable effects on blood glucose levels after food uptake (Kim et al., 2005). Enzyme
inhibitors may also act as effective anti-obesity agents (Kotowaroo, et al., 2006). This could be
due to inhibition of saccharide assimilation, by inhibiting starch breakdown (Oboh et al., 2010).
The reduced amount of amylase available for the breakdown enables complex saccharides to
have a better chance for travelling through the gastrointestinal tract (GIT) without being
assimilated, which are eventually excreted from the body instead of being converted into storage
fat (Oboh et al., 2010).
Acarbose is an oral alpha-glucosidase and alpha amylase inhibitor for use in the
management of Type 2 diabetes mellitus (Wang et al., 2014). It is chemically known as O-4,6- dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]amino]-α-D- glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucose (Bayer Healthcare
Pharmaceuticals, 2011). The antihyperglcemic action of acarbose results from a competitive,
reversible inhibition of pancreatic alpha amylase and a membrane bound intestinal alpha
glucoside hydrolase enzyme. Acarbose is shown to reduce and slow down the intestinal
absorption of glucose, which subsequently minimize the postprandial rise of blood glucose and
insulin concentration (Wang et al., 2014). It was first extracted from the culture broths of
actinomycetes by Puls and his colleagues in the 1970s, and was applied in clinical studies for
more than 10 years (Coniff and Krol, 1997; Scheen, 1998; Junger et al., 2000). It reversibly
inhibits alpha-glycosidases that exist in the brush-border of the small intestinal mucosa (Clissold
and Edwards, 1988). Acarbose does not cause hypoglycemia and its minor gastrointestinal side
effects can be prevented by gradual dosage increments (Wang et al., 2014).
In recent years, there has been an increased interest in the application of antioxidants to
medical treatment, as information is available linking the development of human diseases to
oxidative stress (Giustarini et al., 2009). Natural foods are known to contain natural antioxidants
that can scavenge free radicals. Small molecule dietary antioxidants, such as vitamin C, vitamin
E and carotenoids have procreated particular interest as defenses against degenerative diseases
(Kohlmier and Hastings, 1995; Stampfer and Rimm, 1998). However, some studies have
indicated that phenolic acids are considerably more potent antioxidants than vitamin C and
vitamin E (Vinson et al., 1995; Cao et al., 1997). Phenolic compounds form a substantial part of
plant foods, most of them have shown antioxidant properties both in in vitro and in vivo studies
(Rice- Evans et al., 1996).
Gallic acid is a ubiquitous natural product with various industrial applications including
ink dyes, tanning products, and paper (Eslami et al., 2010). Recent studies have documented that
gallic acid and its esters [e.g., (-)-epi-gallocatechin-3-gallate] exert antioxidant, anticancer,
antiviral, and many other biological effects (Sohi et al., 2003; Tachibana et al., 2004;
Sameermahmood et al., 2010).
Acarbose is an established antidiabetic drug that inhibits α- glucosidase and α- amylase
(key enzymes relevant to type 2 diabetes) activities, but with well reported deleterious side
effects. Gallic acid is a phenolic acid which is ubiquitous in many food/ natural sources; it can be
classified as one of the dietary antioxidants. Consequently, this aims to investigate the effect of
gallic acid on the enzyme (α- glucosidase and α- amylase) inhibitory and antioxidant properties
of acarbose in vitro.
The specific objectives of this project are to:
 Evaluate the effect of gallic acid on the in vitro inhibitory effect of acarbose on α- glucosidase and α- amylase
 Evaluate the effect of gallic acid on antioxidant properties of acarbose.


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