ABSTRACT
P. bifurcatum is a lower plant that has been used in ethnomedicine for the treatment of inflammation, cough and hypertension. The aim of the study was to evaluate the possible anti-inflammatory and hepatoprotective properties of methanol extract of P. bifurcatum. The acute toxicity test of the methanol extract showed no toxicity up to 5000 mg/kg body weight. The effect of the extract on inflammation was evaluated using fresh egg albumin-induced paw oedema in rats while its effect on liver damage was accessed using the carbon tetrachloride (CCl4)-induced hepatic damage and the following hematological, biochemical and antioxidant parameters measured, namely: packed cell volume(PCV), red blood cell(RBC), white blood cell(WBC), hemoglobin(Hb), platelet count(PLAT), bilirubin, alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), superoxide dismutase(SOD), catalase(CAT), reduced glutathione(GSH), malondialdehyde(MDA), c-reactive protein(CRP) as well as histopathologic evaluation. Administration of the methanol extract of P. bifurcatum (1000mg/kg) caused a progressive decrease in paw oedema(0.57±0.20, 0.47±0.02 and 0.25±0.02) at 1hr,2hr and 3hr, respectively, compared to the control(0.97±0.04,0.85±0.08 and 0.67±0.08) at the same period. The extract(1000mg/kg) also caused significant(p<0.05) increases in blood parameters (PCV-49.20±1.30, Hb-16.38±0.19, RBC-6.76±0.08, PLAT-146.20±0.66), GSH and CAT, no significant changes in SOD and organ to body weight ratio but significantly(p<0.05) reduced the WBC, ALT, AST, ALP, MDA and CRP compared to the untreated control. Histological sections of the liver in the two dose groups revealed severe coagulative necrosis of the hepatocytes in the centrilobular and midzonal areas of the hepatic lobules. The periportal hepatocytes were relatively normal compared to the untreated control. It is concluded from this study that methanol extract of P.bifurcatum possesses anti-inflammatory potential and can considerably ameliorate liver damage.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Table of Contents vi
List of Tables xii
List of Figures xiii
Abstract xvi
CHAPTER 1
INTRODUCTION
1.1
Background of the Study 1
1.2 Statement of the Problem 3
1.3 Aim 4
1.4
Objectives 4
1.5
Justification of Study 5
CHAPTER 2
LITERATURE REVIEW
2.1
Overview of P. bifurcatum 6
2.2
Taxonomy 6
2.3
Phytoconstituents of P. bifurcatum 7
2.4
Pharmacological properties of P.
bifurcatum 8
2.4.1
Antioxidant activity 8
2.4.2
Antimicrobial activity 8
2.5 Anti-Inflammatory
Properties of Some Medicinal Plants 9
2.5.1 Dichrostachys cinerea (Fabaceae) 9
2.5.2 Curcuma longa Linn (Zingiberacea) 9
2.5.3 Asparagus africanus (Liliaceae) 10
2.5.4 Zingiber officinale (Zingiberaceae) 11
2.5.5 Moringa oleifera (Moringaceae) 11
2.6 Hepatoprotective
Properties of Some Medicinal Plants 12
2.6.1 Aquilaria agallocha (Thymelaeaceae) 12
2.6.2 Phyllanthus
muellarianus (Euphorbiaceae) 12
2.6.3
Caesalpinia crista (Fabaceae) 13
2.6.4
Opuntia ficus-indica (Cactaceae) 13
2.6.5
Convolvulus arvensis (Convolvulaceae) 13
2.7
Inflammation 14
2.7.1
Mediators of inflammation 14
2.7.1.1
Vasoactive amines and peptides 14
2.7.1.2
Eicosanoids 15
2.7.1.3
Acute-phase protein 16
2.7.1.4
Inflammatory cytokines 16
2.8
Inflammation and Diseases 20
2.8.1
Inflammation and cardiovascular diseases 20
2.8.2
Inflammation and insulin resistance diabetes mellitus 21
2.8.3
Inflammation and cancer 21
2.8.4
Inflammation and osteoarthritis 23
2.9
Screening Models for Anti-Inflammatory Activity 24
2.9.1
Carrageenan-induced paw edema 24
2.9.2
Histamine/5-Hydroxytryptamine-induced paw edema 25
2.9.3
Bradykinin-induced paw edema 26
2.9.4
Dextran induced paw edema 26
2.9.5
Lipopolysaccharide-induced paw edema 26
2.9.6
Granular pouch model 26
2.9.7
Complete Freund’s adjuvant-induced arthritis 27
2.10
Liver Toxicity 27
2.10.1
Stages of liver toxicity 28
2.10.2 Screening models for
hepatoprotective agents 29
2.10.3
Liver function tests 33
2.10.3.1
Biochemical indices 33
2.10.3.2
Histopathological assessment 35
2.10.3.3
Types of histological
preparations 35
CHAPTER 3
MATERIALS AND METHODS
3.1
Materials 37
3.1.1
Collection of plant materials 37
3.1.2 Instruments/ equipment 37
3.1.3 Chemicals/reagents 38
3.1.4 Drugs 39
3.2 Methods 40
3.2.1 Extract preparation 40
3.2.2 Determination of percentage yield of
methanol extract P. bifurcatum leaves 40
3.2.3 Phytochemical screening 40
3.2.3.1 Test
for alkaloids 40
3.2.3.2 Test
for steroids 40
3.2.3.3 Test
for flavonoids 41
3.2.3.4 Test
for tannins 41
3.2.3.5 Test
for saponins 41
3.2.3.6 Test
for terpenoids 41
3.2.3.7 Test
for phenolics 41
3.2.4
Experimental animals 42
3.2.5 Acute toxicity (LD50) 42
3.2.6.1
Anti-inflammatory test 43
3.2.6.2
Hepatoprotective activity 44
3.2.7
Assessment of liver function 45
3.2.7.1 Determination
of alkaline phosphatase (ALP) 45
3.2.7.2 Determination
of alanine aminotransferase (ALT) 46
3.2.7.3 Determination
of aspartate aminotransferase (AST) 47
3.2.7.4 Determination
of direct bilirubin (DB) and total bilirubin (TB) in
serum 48
3.2.8
Hematology parameters 48
3.2.8.1 Determination of erythrocyte count by hemocytometry 48
3.2.8.2 Determination of total leucocyte count by hemocytometry 49
3.2.8.3 Determination of
packed cell volume (PCV) 50
3.2.9 Antioxidant
estimation 51
3.2.9.1 Estimation of extent of lipid peroxidation 51
3.2.9.2
Assay of superoxide dismutase activity 52
3.2.9.3 Assay
for catalase activity 53
3.2.9.4
Reduced glutathione (GSH) estimation 53
3.2.9.5 Determination of total protein. 54
3.2.11 Organ weight to body weight ratio 57
3.2.12 Histopathological
examination 57
3.2.12.1 Tissue preparation 57
3.2.12.2 Slide
examination 58
3.3 Statistical analysis 58
CHAPTER
4
RESULTS
AND DISCUSSION
4.1 Results 59
4.1.1 Qualitative
phytochemical screening of methanol extract of
P.
bifurcatum leaves 59
4.1.2 Oral
Acute Toxicity Study of methanol extract of P. bifurcatum leaves 61
4.1.3
Effect of Methanol leaf extract of P. bifurcatum on egg albumin
induced paw edema 62
4.1.4 Effect
of methanol leaf extract of P. bifurcatum
on packed cell volume, hemoglobin concentration,
red blood cell and platelet counts of CCl4- intoxicated rats. 64
4.1.5 Effect
of methanol leaf extract of P. bifurcatum
on white blood cell count of CCl4- intoxicated rats 69
4.1.6 Effect
of Methanol leaf extract of P. bifurcatum
on serum alanine transaminase, aspartate transaminase,
alkaline phosphatase activity and bilirubin concentration of CCl4-intoxicated
rats. 71
4.1.7 Effect of Methanol
leaf extract of P. bifurcatum on
catalase, superoxide dismutase activity, reduced glutathione concentration and
malondialdehyde levels of CCl4-intoxicated rats. 77
4.1.8 Effect
of Methanol leaf extract of P. bifurcatum
on serum c- reactive protein activity of CCl4-intoxicated
rats. 82
4.1.9
Final body weight, organ weights, and
organ to body ratio 84
4.1.10 Histopathology
of the liver 86
4.2 Discussion
92
CHAPTER
5
CONCLUSION
AND RECOMMENDATIONS
5.1 Conclusion 102
5.2 Recommendations 102
REFERENCES
103
APPENDIX
LIST OF TABLES
4.1 Qualitative
phytochemical screening of methanol extract of P. bifurcatum leaves
60 4.2 Effect of methanol leaf
extract of P. bifurcatum on egg
albumin induced
paw edema 63
4.3
Final body weight, organ weights.
and organ to body ratio 85
LIST
OF FIGURES
4.1 Effect
of methanol leaf extract of P. bifurcatum
on Packed cell volume of CCl4- intoxicated
rats. 65
4.2 Effect
of methanol leaf extract of P. bifurcatum
on hemoglobin concentration of CCl4- intoxicated rats. 66
4.3 Effect
of methanol leaf extract of P. bifurcatum
on red blood cell count of CCl4-intoxicated rats. 67
4.4 Effect
of methanol leaf extract of P. bifurcatum
on Platelet count of CCl4-intoxicated rats. 68
4.5 Effect
of methanol leaf extract of P. bifurcatum
on white blood cell count of CCl4- intoxicated rats. 70
4.6 Effect
of methanol leaf extract of P. bifurcatum
on serum alanine transaminase activity of CCl4-intoxicated
rats. 72
4.7 Effect
of methanol leaf extract of P. bifurcatum
on serum aspartate transaminase activity of
CCl4-intoxicated rats. 73
4.8
Effect of methanol leaf extract of P.
bifurcatum on serum alkaline phosphatase activity of CCl4-intoxicated
rats 74
4.9
Effect of methanol leaf extract of P.
bifurcatum on serum total bilirubin concentration of CCl4-intoxicated rats. 75
4.10
Effect of methanol leaf extract of P.
bifurcatum on serum direct bilirubin concentration of CCl4-intoxicated
rats. 76
4.11 Effect
of methanol leaf extract of P. bifurcatum
on superoxide dismutase activity of CCl4-intoxicated
rats. 78
4.12 Effect
of methanol leaf extract of P. bifurcatum
on catalase activity of CCl4-intoxicated rats. 79
4.13
Effect of methanol leaf extract of P.
bifurcatum on Reduced glutathione concentration of CCl4-intoxicated rats. 80
4.14
Effect of methanol leaf extract of P.
bifurcatum on Malondialdehyde concentration of CCl4-intoxicated rats. 81
4.15
Effect of methanol leaf extract of P. bifurcatum on serum c-reactive
protein activity of CCl4-intoxicated
rats. 83
4.16 Histological
sections of the liver of rats given distilled water (normal rats) 87
4.17 Histological
sections of the liver of rats given Carbon tetrachloride (2 mg/kg)
88
4.18 Histological sections of
the liver of rats given Silymarin (50 mg/kg) and Carbon tetrachloride (2 mg/kg) 89
4.19 Histological
sections of the liver of rats given P.
bifurcatum (500 mg/kg) and Carbon tetrachloride
(2 mg/kg) 90
4.20 Histological
sections of the liver of rats given P.
bifurcatum (1000 mg/kg) and Carbon tetrachloride
(2 mg/kg) 91
CHAPTER 1
INTRODUCTION
1.1
BACKGROUND
OF STUDY
The body’s complex biological response to damaging
stimuli such as infections, damaged cells or allergies is known as Inflammation
(Ferrero-Miliani et al., 2007). Immune cells,
blood vessels, and chemical mediators are all part of this protective response.
Inflammation aims at clearing out damaged cells and tissues that have been
destroyed by the initial injury and the inflammatory process thus initiating
tissue regeneration (Abul and Andrew, 2009). Chronic inflammation produces a
constant influx of reactive oxygen species, which overwhelm the antioxidant
defenses, disrupt DNA, and induce ageing and diseases.
Microorganisms,
physical agents, chemicals, erroneous immune responses, and tissue death
can cause inflammation. Anti-inflammation is achieved by reducing
inflammatory reactions with herbal remedies or non-steroidal anti-inflammatory
drugs (NSAIDs). Over time, scientists have discovered that plant materials can
be used to create new anti-inflammatory drugs.
The
Liver is the organ responsible for drug metabolism and elimination (Singh et
al., 2012). Hepatotoxicity is defined as liver impairment or damage caused
by an excessive amount of drugs or xenobiotics such as acetaminophen, cadmium
chloride, ethanol, carbon tetrachloride (CCl4), and galactosamine
(Novarro and Senior, 2006).
Hepato-toxicants are substances that cause damage to the Liver. They result from
overdosing on certain medications, industrial chemicals, and natural chemicals
such as microcystins, medicinal herbs and dietary supplements (Papay et al.,
2009).
Hepatotoxicity
is caused by direct toxicity of the main compound or a reactive metabolite from
an immune response affecting liver cells, biliary epithelial cells, and liver
vasculature (Deng et al., 2009). Liver disease is one of the world's
most serious health issues. Using several of these hepatotoxins,
hepatoprotective drugs have been researched extensively. However, studying
promising hepatoprotective medicinal plants using carbon tetrachloride-induced
liver injury has been effective. Its toxicity is primarily due to formation of
reactive oxygen species (ROS). Reactive oxygen species bind to polyunsaturated
fatty acids (PUFAs) forming hydroxyl or peroxyl radicals, damaging the cell
membrane and altering enzyme activity (Khan et al., 2016). Centrilobular
steatosis, inflammation, apoptosis, and necrosis are all effects of CCl4
exposure (Manibusan et al., 2007).
Medicinal
plants are currently in high demand and, their popularity is growing. A
medicinal plant is any plant that has medicinal properties. These plants
include a diversity of compounds employed in drug discovery (Rasool
Hassan, 2012). Different types of seeds,
roots, leaves, fruit, skin, flowers and even plants may be utilized as
medicinal plant components. Most medicinal plants have active compounds with
direct or indirect therapeutic effects and are employed as medicinal agents.
Phytochemicals
are referred to as biologically active naturally occurring compounds that are
useful to humans (Hasler, 1999). They lack nutritional value, unlike
vitamins and minerals. They do, however, have an impact on several bodily
functions. They function with minerals and dietary fibre to protect the body
from disease, slow the ageing process, and lower the risk of various ailments,
including cancer, heart disease, stroke, and high blood pressure.
The
"staghorn fern," Platycerium bifurcatum (Cav) C. Chr is an
epiphyte that grows naturally on tree branches and trunks in tropical,
subtropical, and rain forest environments (Bode and Oyedapo, 2011). Because it
lacks roots and reproduces through spores rather than flowers, P. bifurcatum
is considered a lower plant. It is propagated via spores and belongs to the
Polypodiaceae family. It has been utilized in ethnomedicine in addition to
ornamental purposes. Young leaves of P. bifurcatum have been reported to
have various uses in Nigeria which includes preventing miscarriages in women
two months after conception (Flora and Ubah, 2006). The plant can also be
employed for treating oedema, coughs, and hypertension (Mensah et al.,
2006).
1.2 STATEMENT
OF PROBLEM
One
of the most critical public health issues is liver disease. Despite the use of
hepatoprotective drugs, the number of liver disorders has continued to
rise. Chronic hepatic injury, primarily caused by alcohol abuse, xenobiotic
toxicity, chronic infections, such as those caused by the hepatitis C virus,
bile duct damage, and non-alcoholic liver disease, are the most common causes
of these disorders (Younissi et al., 2018). It is the main cause of
death in most developing nations, particularly among people with metabolic
syndrome, obesity, and type 2 diabetes (Loomba and Sanyal, 2013).
Nonsteroidal
anti-inflammatory drugs (NSAIDS) affect the gastric mucosa, kidney,
cardiovascular system, liver, and hematopoietic system. The adverse effects are
most likely related to Cyclooxygenase inhibition, which prevents the generation
of prostaglandins (Sostres et al., 2010).
Several
plant components have been used for treatment of various ailments with few or
no side effects worldwide, and some of these plant materials, which contain a
large number of beneficial phytoconstituents, are poorly utilized and may
contain numerous pharmacological properties.
1.3 AIM
This
study aims to investigate the potential anti-inflammatory and hepatoprotective
effects of methanol extract of P. bifurcatum leaves on carbon
tetrachloride (CCl4) induced liver damage in Sprague Dawley rats.
1.4 OBJECTIVES
The objectives of the study were as follows:
1. Determine
the phytochemical properties of methanol extract of P. bifurcatum.
2. Determine
the oral acute toxicity of methanol extract of P. bifurcatum
3. Evaluate
the effect of methanol extract of P. bifurcatum on hind paw edema in
rats.
4. Determine
the effect of methanol extract of P. bifurcatum on biochemical,
hematological and pathophysiologic parameters of rat liver damaged with carbon
tetrachloride.
1.5 JUSTIFICATION OF
STUDY
Medicinal
plants are said to offer a variety of pharmacological properties. These plants
have been utilized, but many others, notably lower plants, have remained
unexplored. Researchers have continued to investigate the systemic effects of
these plant preparations in the hopes of discovering new drugs or improving the
potency of those that already exist.
P.
bifurcatum is one of these lower plants that has
been used in traditional medicine to treat inflammation, hypertension, cough,
and other diseases. The purpose of this study is to confirm the ethnomedical
use of the P. bifurcatum leaf using methanol as the extraction solvent.
This will also serve as a criterion for recommending the plant's continued
usage in ethnopharmacology.
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