ABSTRACT
The anti-inflammatory and tonic properties of methanol extract of Arachis hypogea leaves were studied in vitro and in vivo. Qualitative phytochemical screening revealed the presence of alkaloids, glycosides, fats and oils, phenols and lignins. Acute toxicity test showed that the extract was not toxic up to 500mg/kg body weight. There was a non-significant difference (p>0.05) in the rat paw oedema induced by egg-albumin for 1hr in group administered 10mg/kg b.w of aspirin (group 2) and those administered 200mg/kg and 400mg/kg of methanol extract of Arachis hypogea leaves (groups 3 and 4) respectively, when compared with the control (group 1) rats that were administered 5ml/kg b.w normal saline. However, there was a significant decrease (p<0.05) in the paw width of the rats after induction for 2,3,4,5 and 6hrs in groups 2, 3 and 4 when compared with group 1. In vitro study showed a significant (p<0.05) dose-dependent inhibition of platelet aggregation by the extract at the tested concentrations. Also, the extract significantly (p<0.05) decreased the hemoglobin concentrations in groups 3 and 4 when compared with groups 1 and 2. Tonicity studies using rabbit jejunum showed a significant (p<0.05) relaxation effect on that smooth muscle. At 14.28µg/ml, 29µg/ml and 57.14µg/ml, the extract inhibited in vitro acetylcholine-induced contraction of the rabbit jejunum by 84.21%, 86.84% and 89.47% respectively, which compared closely with the effect of atropine (90.21% at 0.28µg/ml). In conclusion, the results of this study strongly indicated that the methanol extract of Arachis hypogea leaves possesses anti-inflammatory and smooth muscle relaxant properties, suggesting that it contains substances with potent parasympatholytic and anti-inflammatory properties by relieving oedema and inhibiting platelet aggregation, prostaglandin synthase activity and reducing erythrocyte osmotic fragility.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Figures xiii
Abstract xiv
CHAPTER 1:
INTRODUCTION 1
1.1 Background of the Study 1
1.2 Aim of the Study 4
1.3 Specific
Objectives of the Study 4
1.4 Statement of Problem 5
1.5 Justification 5
CHAPTER 2:
LITERATURE REVIEW 6
2.2 Traditional and Pharmaceutical Uses 7
2.2.1 Pharmacological effects 9
2.2.1.1 Antioxidant
and hypolipidemic effects 10
2.2.1.2 Anti-inflammatory
effects 12
2.2.1.3 Opioid
receptor affinity 12
2.2.1.4 Sympathomimetic
effects 13
2.2.1.5 Immunomodulating
and anticancer effects 13
2.2.1.6 Endocrine
effects 14
2.2.1.7 Antimicrobial and anti-parasitic
effects 15
2.2.1.8 Sedative
effect 16
2.2.1.9 Hypotensive and haemostatic effects 17
2.2.2.1. Acetylcholine – a neurotransmitter 17
2.2.2.2 Targets of acetylcholine 18
2.2.2.3 Signal
transduction of muscarinic receptors 19
2.2.2.4 Contraindications and adverse effects 20
CHAPTER 3: MATERIALS AND
METHODS 21
3.1 Materials 21
3.1.1 Plant material 21
3.1.2 Animals 21
3.1.3 Instruments/equipment 21
3.1.4 Chemicals/reagents 22
3.1.4.1 Preparation of reagents
for phytochemical analysis 24
3.1.4.2 Anti-coagulant used for assay 25
3.1.4.3 Reagent preparation for
malondialdehyde (MDA) determination 25
3.1.4.4 Reagents used for prostaglandin synthase assay 25
3.1.4.5 Calculation of drug volumes and concentrations for tonicity studies 26
3.2 Procedures/protocol 26
3.2.1 Preparation of plant
material 26
3.2.2 Determination of
percentage yield of the ethanol extract of
Arachis hypogea leaves 27
3.2.3 Qualitative phytochemical
analyses of the extract 27
3.2.3.1 Test for alkaloids 27
3.2.3.2 Test for glycosides 27
3.2.3.3 Test for reducing sugars 28
3.2.3.4 Test for flavonoids 28
3.2.3.5 Test for tannins 28
3.2.3.6 Test for saponins 28
3.2.3.7 Test for resins 29
3.2.3.8 Test for phenol 29
3.2.3.9 Test for carbohydrates 29
2.2.3.10 Test for oil 29
3.2.3.11 Test for proteins 29
3.2.5 Acute toxicity and lethal dose test (LD50) 29
3.2.6 Assays of biochemical
parameters 30
3.2.6.1 Assay of serum alanine aminotrainsferase (ALT) activity 30
3.2.6.2 Assay of serum aspartate aminotransferase (AST) activity 31
3.2.6.3 Assay of serum alkaline phosphatase (ALP) activity 31
3.2.6.4 Determination of serum
bilirubin concentration 32
3.2.6.5 Total serum
proteins 33
3.2.7 Determination of lipid
profile 34
3.2.7.1 Determination of serum total cholesterol concentration
(enzymatic end-point method) 34
3.2.7.2 Determination of
low-density lipoprotein (LDL)
cholesterol
Concentration 35
3.2.7.3 Determination of
high-density lipoprotein (HDL) cholesterol
Concentration 36
3.2.7.4 Determination of triacylglycerol (TAG)
concentration 37
3.2.7.5 Assay of prostaglandin
synthase activity 39
3.2.7.6 Isolation of the enzyme
containing fraction 39
3.2.7.7 Assay for the enzyme activity 40
3.2.7.8 Preparation of intestinal smooth tissue for in vitro isometric
contraction
effect of methanol extract of Arachis
hypogea leaves 42
3.2.8.0 Anti-inflammatory test 43
3.2.8.1 Anti-inflammatory test 43
3.2.11 Determination of lipid
peroxidation (malondialdehyde) 45
3.2.12 Assay of catalase activity 46
3.2.13 Assay of superoxide dismutase (SOD) activity 46
3.2.14 Determination of
vitamin E concentration 47
3.2.15 Determination of membrane stability (hypotonicitv-induced
haemolysis) 47
3.2.16
Determination
of anti-platelet aggregatory activity 48
3.2.17 Phospholipase
A2 activity test 49
3.3 Statistical Analysis 49
CHAPTER 4: RESULTS
AND DISCUSSION 50
4.1 Results 50
4.1.1 Phytochemical profile of
methanolic extract of Arachis hypogea leaves 50
4.1.2 Effect of
the methanol extract of Arachis hypogea leaves
on serum alanine aminotransferase activity of rats 51
4.1.3 Effect
of the methanol extract of Arachis
hypogea leaves on serum
aspartate
aminotransferase activity of rats 52
4.1.4 Effect
of methanol extract of Arachis hypogea leaves on serum alkaline
phosphatase activity 53
4.1.5 Effect
of methanol extract of Arachis hypogea leaves on serum
bilirubin, albumin and total protein concentration 54
4.1.6 Effect
of methanol extract of Arachis hypogea leaves on serum cholesterol concentration
of rats 55
4.1.7 Effect
of the methanol extract of Arachis
hypogea leaves on
serum low-density lipoprotein cholesterol
(LDL-cholesterol)
concentration of rats 56
4.1.8 Effect
of the methanol extract of Arachis
hypogea leaves on
serum high-density lipoprotein cholesterol
(HDL-cholesterol)
concentration of rats 57
4.1.9 Effect
of the methanol extract of Arachis
hypogea leaves on serum
triacylglycerol concentration of rats 58
4.1.10 Effect of the methanol extract of Arachis hypogea leaves on
prostaglandin synthase activity 59
4.2.1 In
vitro effect of acetylcholine on an isolated rabbit jejunum 60
4.2.2 In vitro effect of Arachis hypogea
on an isolated rabbit jejunum 61
4.2.3 In
vitro effect of atropin on
acetylcholine-induced contractions
on the isolated rabbit jejunum triacylglycerol
concentration of rats 62
4.2.4 In vitro effect of Arachis hypogea on
acetylcholine-induced contractions
on the isolated rabbit jejunum 63
4.2.5 Effect of methanol extract of Arachis
hypogea leaves on egg albumin-
induced hind paw edema of rats 64
4.2.6 Effect of methanol extract of Arachis hypogea leaves on
serum lipid peroxidation: malondialdehyde (MDA) of
rats 65
4.2.7 Effect of the methanol extract of Arachi hypogea leaves on
serum
catalase activity of rats 66
4.2.8 Effect
of the methanol extract of Arachi hypogea
leaves on
serum
superoxide dismutase activity of rats 67
4.2.9 Effect
of the methanol extract of Arachi hypogea
leaves on
serum vitamin E concentration of rats 68
4.3.1 Effect of methanol extract of Arachis
hypogea leaves on
membrane stability (osmotic fragility) 69
4.3.2 In
vitro effect of methanol extract of Arachis
hypogea leaves
on platelet aggregation 70
4.3.3 In
vitro effect of methanol extract of Arachis
hypogea leaves on
phospholipase A2 activity 72
4.3.4 Effect of methanol extract of Arachis
hypogea leaves on in vivo
platelet indices in rats 73
4.4 Discussion 74
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 80
5.1 Conclusion 80
5.2 Recommendations 80
References 81
Appendix
LIST OF FIGURES
|
FIGURE
|
|
PAGE
|
|
2.1
|
Schematic
representation of acetylcholine synthesis, release, action and breakdown in
neuronal cells at a cholinergic nerve terminal.
|
20
|
|
4.1
|
Effect of the methanol
extract of Arachis hypogea leaves
on serum alanine aminotransferase
|
52
|
|
4.2
|
Effect of the methanol extract of Arachis hypogea leaves on serum aspartate
aminotransferase activity of rats
|
53
|
|
4.3
|
Effect of methanol extract of Arachis hypogea leaves
on serum alkaline phosphatase activity of rats.
|
54
|
|
4.4
|
Effect of
methanol extract of Arachis hypogea leaves on serum Bilirubin, albumin
and total protein concentration of rats.
|
55
|
|
4.5
|
Effect of the methanol extract of Arachis hypogea leaves on serum
cholesterol concentration of rats
|
56
|
|
4.6
|
Effect of the methanol extract of Arachis hypogea leaves on serum
low-density lipoprotein cholesterol (LDL-cholesterol) concentration of rats
|
57
|
|
4.7
|
Effect of the methanol extract of Arachis hypogea leaves on serum
high-density lipoprotein cholesterol (HDL-cholesterol) concentration of rats
|
58
|
|
4.8
|
Effects
of the methanol extract of Arachis
hypogea leaves on serum triacylglycerol concentration of rats
|
59
|
|
4.9
|
In
vitro effect
of acetylcholine on an isolated rabbit jejunum
|
61
|
|
4.10
|
In
vitro effect of Arachis hypogea methanol leaves extract of Arachis hypogea
|
62
|
|
4.11
|
Effects of
atropine on acetylcholine induced smooth muscle contractions
|
63
|
|
4.12
|
Effects of Arachis hypogea on acetylcholine
induced smooth muscle contractions
|
64
|
|
4.13
|
Effect of methanol extract of Arachis hypogea leaves
on egg albumin- induced hind paw edema of rats
|
65
|
|
4.14
|
Effect of
methanol extract of Arachis hypogea
leaves on serum lipid peroxidation: malondialdehyde (MDA) of rats
|
66
|
|
4.15
|
Effect of the methanol extract of Arachis hypogea leaves on serum
catalase activity of rats
|
67
|
|
4.16
|
Effect of the methanol extract of Arachis hypogea leaves on serum
superoxide dismutase activity of rats
|
68
|
|
4.17
|
Effect of the methanol
extract of Arachis hypogea leaves
on serum vitamin E concentration of rats
|
69
|
|
4.18
|
Effect of
methanol extract of Arachis hypogea leaves on membrane stability
(osmotic fragility)
|
70
|
|
4.19
|
Effect
of methanol extract of Arachis hypogea leaves on in vivo platelet
indices in rats
|
74
|
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Inflammation is a complex pathophysiological
response of vascularized tissue to injury arising from various stimuli,
including thermal, chemical or physical damage, ischemia, infectious agents,
antigen-antibody interactions and other biologic processes (Clark, 2002).
Inflammation, a fundamental protective response, can be harmful in conditions
such as life-threatening hypersensitivity reactions to insect bite, drugs,
toxins, and in chronic diseases, such as rheumatic arthritis, lung fibrosis and
cancer (Simon et al., 2000).
Inflammatory response is brought about or mediated by inflammatory mediators
such as chemokines, cytokines, cell adhesion molecules and extracellular matrix
proteins (Simon et al., 2000), which when in excess, are deleterious (Liu and Hong,
2002). Phytochemicals are divided according to their functions in plant
metabolism into two groups: which are primary and secondary constituents;
Primary constituents comprise common sugars, amino acids, proteins and
chlorophyll, while secondary constituents consists of alkaloids, terpenoids and
phenolic compounds (Krishnaiah et al.,
2009) as well as flavonoids, tannins, saponins, essential oils and many more (Edeoga
et al., 2005). The medicinal values
of plants lie in these bioactive phytochemical constituents that produce
definite physiological actions in the human body (Akinmoladun et al., 2007).
Phytochemicals are
natural bioactive compounds found in plants, such as vegetables, fruits, flowers,
leaves and roots that work with nutrients and fibers to act as a defense system
against diseases or more accurately, to protect against disease. Unlike vitamins and minerals, they have no
nutritional value. They can, however, influence various body processes. They
work together with nutrients and dietary fibers to protect the body against
diseases, slow the aging process and reduce the risk of many diseases such as
cancer, heart disease, stroke and high blood pressure (Igwenyi et al., 2011).
Peanut, also known as groundnut (Arachis hypogaea),
is a crop of global importance. It is widely grown in both the tropics
and subtropics, being important to both smallholder and large commercial
producers. It is classified as both a grain legume,
and because of its high oil content, an oil crop (EBTP, 2015).World annual production is about 46
million tonnes.
Very unusually among crop plants, peanut pods develop under the ground.
Cultivated
peanut (A. hypogaea) has two sets of chromosomes from two different
species, thought to be A. duranensis
and A. ipaensisarise (Seijo et al 2007;Kochert et al 1996;
Moretzsohn
et al, 2013). The two species' chromosomes combine by
hybridization and doubling, to form what is termed an amphidiploid
or allotetraploid. Genetic analysis
suggests this hybridization event probably occurred only once and gave rise to A. monticola, a wild form of
peanut that occurs in a few restricted locations in northwestern Argentina, and, by artificial selection to A.
hypogaea. (Seijo
et al 2007; Kochert et al., 1996; Husted, 1936; Halward et al., 1992).
The process of domestication through artificial selection made A. hypogaea
dramatically different from its wild relatives. The domesticated plants are
more bushy and compact, and have a different pod structure and larger seeds.
The initial domestication may have taken place in northwestern Argentina, or in
southeastern Bolivia, where the peanut landraces with the most wild-like features are
grown today (Krapovickas
and Gregory; Krapovickas Gregory, 2007).
From this primary center of
origin, cultivation spread and formed secondary and
tertiary centers of diversity in Peru,
Equador, Brazil,
Paraguay and Uruguay.
Subspecies-fastigiata types- are more upright in their growth habit and
have a shorter crop cycle. Subspecies hypogaea types spread more on the
ground and have longer crop cycles (Krapovickas and Gregory; Krapovickas Gregory, 2007).
Peanuts grow
well in Northern Nigeria, where they are used to make all sorts of local snacks
like Kuli-kuli, Tamfili and other. In the Igbo land, virtually every
traditional occasion is graced with groundnut paste locally called Okwa-ose. In
West African countries like Ivory Coast,
Burkina Faso, Ghana,
Nigeria, and Senegal,
they are also used for both culinary and agricultural purposes. Malian meat stew called maafe
is made from groundnut paste. In Ghana,
peanut butter is used for peanut butter
soup known as nkate nkwan. Crushed peanuts may
also be used for peanut candies called nkate cake and kuli-kuli respectively, as well as other local foods
such as oto
(Ghanaian cuisine). Peanut butter is also an
ingredient in Nigeria's "African salad". Its powder is an important
ingredient in the spicy coating for kebabs in Nigeria
and Ghana.
1.2 AIM OF THE STUDY
The
aim of this study was to investigate the anti-inflammatory and tonic properties
of methanol extracts of Arachis hypogea leaves on some biochemical indices.
1.3 SPECIFIC OBJECTIVES OF THE STUDY
·
Qualitative determination
of the phytochemical composition of methanol extracts of Arachis hypogea leaves.
·
Determination of acute toxicity LD50 of methanol
extract of A. hypogeal on albino rats
·
Determination of the
effect of methanol extract of Arachis
hypogea on hind paw edema of rats.
·
Determination of the
effect of methanol extract of Arachis
hypogea on prostaglandin synthase activity
·
Determination of the
effect of methanol extract of Arachis hypogea
on phospholipase A2 activity
·
Determination of the
effects of methanol extracts of Arachis
hypogea on platelet aggregatory activity.
·
Determination of the
effect of methanol extract of Arachis
hypogea on smooth muscle contraction/relaxation
·
Determination of the
effects of methanol extracts of Arachis
hypogea on membrane fragility.
·
Determination for the
effect of methanol extract of Arachis
hypogea leaves on activities of some liver enzymes (ALT, AST and ALP) and albumin,
total bilirubin and total protein.
·
Determination of the
effect of methanol extract of Arachis
hypogea on the concentrations of selected lipids: low-density
lipoprotein-cholesterol (LDL), high-density lipoprotein-cholesterol (HDL), triacylglycerol
(TAG) and total cholesterol.
·
Determination of the
effect of methanol extract of Arachis
hypogea on lipid peroxidation
·
Determination of the
effect of methanol extracts of Arachis
hypogea on two antioxidant enzymes (catalase and superoxide dismutase).
1.4 STATEMENT
OF PROBLEM
Peanut, also known as groundnut (Arachis hypogaea) is
a crop of economical value globally, grown for culinary, medicinal and commercial
purposes. It is classified as both a grain legume
and an oil crop because of its high oil content. A
great number of anti-inflammatory drugs (both steroids and non-steroidal
anti-inflammatory drugs) and parasympathomimetics are extensively used for the
treatment of acute and chronic inflammatory and muscle conditions (Rang et al., 2003). Among these drugs, none
has proved to be curative. They suppress rather than abolish the inflammatory
and muscle disorders thereby providing
symptomatic relief and are usually accompanied by severe adverse effects such
as gastrointestinal irritations, ulcers, bone marrow depression, hypertension,
myocardial infarction and muscular degenerations among others (Klein et al.,2012; Liew et al., 2013). There is therefore, the need to search for more
potent and less toxic anti-inflammatory and parasympathomimetics drugs from
medicinal plants as there is the worldwide green revolution which is reflected
in the belief that herbal remedies are safer and less damaging to the human and
animal systems than synthetic drugs (Williamson et al., 1996).
1.5 JUSTIFICATION
There
is an increasing divergence towards the use of plant based drugs in
therapeutics, worldwide and specifically the developing countries. This may be
because the nation is host to hundreds of thousands of plants species, many of
which have medicinal values (Ojieh et al.,
2013). Many of these plants have been exploited while a host of others remain
uninvestigated. Researchers have continued to explore the systemic effects of
these plant preparations with the intention to discover new drugs and or increase
the potency of existing ones. Arachis hypogea is one of such medicinal plants that are being
used to treat various ailments.
The
widespread ethnomedicinal use of the leaf extract of the plant for the
management of diseases including inflammation and epilepsy spurred this study aimed
to evaluate the anti-inflammatory and tonic properties of methanol extract of Arachis
hypogea leaves.
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