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DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY
SCHOOL OF BIOLOGICAL SCIENCES
COLLEGE OF BASIC AND APPLIED SCIENCES
UNIVERSITY OF GHANA, LEGON
ANTI-INFLAMMATORY ACTIVITY AND THE MECHANISM OF ACTION OF
MORINDA LUCIDA BENTH.
BY
FREDERICK AYERTEY
(10507338)
THIS THESIS IS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY, CELL
AND MOLECULAR BIOLOGY, COLLEGE OF BASIC AND APPLIED SCIENCES,
UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILMENT OF THE
REQUIREMENT FOR THE AWARD OF MPHIL BIOCHEMISTRY DEGREE
DECEMBER, 2016
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DECLARATION
I, Frederick Ayertey, do hereby declare that except for references to other people’s work, for
which I have acknowledged, this report is the product of my own research carried out at the
Department of Biochemistry, Cell and Molecular Biology, Center for Plant Medicine Research
and Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, under the
supervision of Prof. Laud Kenneth Okine and Dr. (Mrs.) Regina Appiah-Opong.
FREDERICK AYERTEY (STUDENT)
SIGNATURE………………………….
DATE………………………………….
PROF. LAUD KENNETH OKINE (PRINCIPAL SUPERVISOR)
SIGNATURE………………………….
DATE………………………………….
DR. (MRS.) REGINA APPIAH-OPONG (CO-SUPERVISOR)
SIGNATURE………………………….
DATE………………………………….
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DEDICATION
I dedicate this thesis to the Almighty God for his protection and guidance, my parents, siblings
and all friends for their loyal support.
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ACKNOWLEDGMENTS
I acknowledge my supervisors Prof. Laud Kenneth Okine and Dr. (Mrs.) Regina Appiah-Opong
for their key guidance and support throughout the research period. Such a height would not have
been reached without the advice and encouragement of Dr. Alfred Ampomah Appiah also.
A special thank goes to Mr. Stephen Antwi, Mr. Bismark Asante (Pharmacology Department),
Mr. Henry Brew-Daniels, Mr. Benjamin Asante, Peter Bola (Phytochemistry Department), Mr.
Sylvester Kamintah (Microbiology), staff of the Animal house and all other staff of Centre for
Plant Medicine (CPMR), Mampong-Akwapim for the immense contributions to this research
work.
My sincere appreciation goes to Mr. Ebenezer Ofori-Attah (Clinical Pathology Department), Dr.
Mitsuko Ohashi and Mr. Michael Amoa-Bosompem (Parasitology Department), Dr. Kusi
Asamoah (Immunology Department) and all other staff of the Clinical Pathology, Parasitology
and Immunology Departments of Noguchi Memorial Institute for Medical Research (NMIR),
Legon-Accra, for their special assistance.
I am most grateful for the kind contributions of Prof. Takuhiro Uto of Nagasaki International
University, Japan, the West African Centre for Cell Biology of Infectious Pathogens
(WACCBIP), Department of Biochemistry, Cell and Molecular Biology (BCMB) and Japan
Agency for Medical Research and Development (AMED).
Finally, I would like to thank the staff and colleagues of the Department of Biochemistry,
Cell and Molecular Biology.
To everyone I say ‘Ayekoo’.
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TABLE OF CONTENTS
CONTENTS PAGE
DECLARATION............................................................................................................................ i
DEDICATION............................................................................................................................... ii
ACKNOWLEDGMENTS ........................................................................................................... iii
LIST OF TABLES ....................................................................................................................... ix
LIST OF FIGURES ...................................................................................................................... x
ABBREVIATIONS AND ACRONYMS.................................................................................... xi
ABSTRACT................................................................................................................................ xiv
CHAPTER ONE ........................................................................................................................... 1
1.0 INTRODUCTION.............................................................................................................. 1
1.1 BACKGROUND ..................................................................................................................... 1
1.2 PROBLEM STATEMENT AND JUSTIFICATION .......................................................... 3
1.3 HYPOTHESIS......................................................................................................................... 4
1.4 AIM/ SPECIFIC OBJECTIVES ........................................................................................... 4
1.4.1 Aim ........................................................................................................................................ 4
1.4.2 Specific Objectives ............................................................................................................... 4
CHAPTER TWO .......................................................................................................................... 5
2.0 LITERATURE REVIEW ...................................................................................................... 5
2.2 BOTANICAL DESCRIPTION OF M. LUCIDA. ................................................................ 6
2.3 GEOGRAPHICAL DISTRIBUTION OF M. LUCIDA ...................................................... 7
2.4 TRADITIONAL USES OF M. LUCIDA............................................................................... 7
2.5 SOME CHEMICAL CONSTITUENTS OF M. LUCIDA................................................... 9
2.6 PHARMACOLOGICAL PROPERTIES OF M. LUCIDA ............................................... 10
2.7 INFLAMMATION ............................................................................................................... 12
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2.7.1 Acute Inflammation ........................................................................................................... 13
2.7.2 Chronic Inflammation ....................................................................................................... 16
2.8 MEDIATORS OF INFLAMMATION ............................................................................... 18
2.8.1 Histamine ............................................................................................................................ 18
2.8.2 Serotonin ............................................................................................................................. 20
2.8.3 Bradykinin .......................................................................................................................... 20
2.8.4 Cytokines ............................................................................................................................ 21
2.8.5 Lipid Mediators (Eicosanoids).......................................................................................... 23
2.8.5.1 Prostaglandins and inflammation ................................................................................. 24
Regulation of COX-2 ................................................................................................................... 24
2.8.6 Free Radicals ...................................................................................................................... 26
2.8.6.1 Reactive oxygen species (ROS) and inflammation....................................................... 26
Nitric Oxide (NO) and inflammation .......................................................................................... 27
2.8.7.1 Regulation of iNOS synthesis ......................................................................................... 29
CHAPTER THREE .................................................................................................................... 32
3.0 MATERIALS AND METHODS .................................................................................... 32
3.1 MATERIALS ........................................................................................................................ 32
3.1.1 Animals ............................................................................................................................... 32
3.1.2 Plant Collection .................................................................................................................. 32
3.1.3 Chemicals and Reagents.................................................................................................... 32
3.2. METHODS ........................................................................................................................... 33
3.2.1 Extract Preparation ........................................................................................................... 33
3.2.2. Phytochemical Screening of HEML ................................................................................ 33
3.2.2.1. Qualitative phytochemical screening for hydrophilic constituents ........................... 34
Test for saponins .......................................................................................................................... 34
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Test for reducing sugars .............................................................................................................. 34
Phenolic compounds .................................................................................................................... 34
Test for cyanogenic glycosides .................................................................................................... 34
Test for alkaloids .......................................................................................................................... 35
Test for polyuronides ................................................................................................................... 35
3.2.2.2 Qualitative phytochemical screening for lipophilic constituents ................................ 35
Test for flavonoids........................................................................................................................ 35
Test for phytosterols and triterpenes ........................................................................................... 36
Test for anthraquinones............................................................................................................... 36
3.2.2.3 Thin layer chromatography profiling of HEML .......................................................... 36
3.2.3 Determination of Antioxidant Property of HEML ......................................................... 37
3.2.3.1 Quantification of total phenolic content ....................................................................... 37
3.2.3.2 Quantification of total flavonoids .................................................................................. 37
3.2.3.3 DPPH free-radical scavenging activity ......................................................................... 37
3.2.4 Determination of Median Lethal Dose (LD50) ................................................................. 38
3.2.5 Effect of HEML on Carrageenan-induced Paw Edema ................................................. 38
3.2.6. Anti-histaminic Activity of HEML .................................................................................. 39
3.2.8 Anti-pyretic Activity of HEML ......................................................................................... 40
3.2.9 Cell Culture ........................................................................................................................ 41
3.2.10 Cell Survival Assay .......................................................................................................... 41
3.2.11 Measurement of Cellular Nitric Oxide (NO) Levels ..................................................... 42
3.2.12 Determination of PGE2 in Cell Culture Supernatant ................................................... 42
3.2.13 Determination of Cytokine Levels in Culture Medium................................................ 43
3.2.14 Western Blot Analysis...................................................................................................... 43
3.2.15 Statistical Analysis ........................................................................................................... 44
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CHAPTER FOUR....................................................................................................................... 45
4.0 RESULTS ......................................................................................................................... 45
4.1. EXTRACT YIELD .............................................................................................................. 45
Table 4.1: Some groups of phytochemicals in HEML.......................................................... 45
RESULT................................................................................................................................... 45
4.3 TLC PROFILING OF HEML ............................................................................................. 46
4.4 TOTAL PHENOLIC AND FLAVONOID CONTENTS/ANTIOXIDANT ACTIVITY
OF HEML .................................................................................................................................... 47
4.4.1 Total Phenolic Content ...................................................................................................... 47
4.4.2 Total Flavonoid Content.................................................................................................... 47
4.4.3 DPPH Free Radical Scavenging Activity ......................................................................... 47
4.5 DETERMINATION OF MEDIAN LETHAL DOSE (LD50)............................................ 48
4.6 CARRAGEENAN-INDUCED PAW EDEMA IN RATS.................................................. 48
4.7 HISTAMINE-INDUCED PAW EDEMA IN RATS .......................................................... 50
4.8 SEROTONIN-INDUCED PAW EDEMA IN RATS ......................................................... 51
4.9 LPS-INDUCED FEVER IN RATS...................................................................................... 53
4.12 NITRIC OXIDE (NO) PRODUCTION AND iNOS EXPRESSION ............................. 56
4.13 LPS-INDUCED CYTOKINE EXPRESSION .................................................................. 58
CHAPTER FIVE ........................................................................................................................ 60
5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS .............................. 60
5.1 DISCUSSION ........................................................................................................................ 60
5.2 CONCLUSIONS .............................................................................................................. 65
5.3 RECOMMENDATIONS...................................................................................................... 65
REFERENCES............................................................................................................................ 67
APPENDICES ............................................................................................................................. 80
Appendix I: Standard Curve for Gallic Acid ........................................................................... 80
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Appendix II: Standard Curve for Quercetin............................................................................ 80
Appendix III: Standard Curve for Sodium Nitrite.................................................................. 81
Appendix V: Standard Curve for PGE2 ........................................................................................ 82
Appendix VI: Standard Curve for IL-1Β ...................................................................................... 82
Appendix VII: Standard Curve for TNF-α ................................................................................... 83
Appendix VIII: Standard Curve for IL-10 .................................................................................... 83
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LIST OF TABLES
Table 4.1 some phytochemical constituents screened for in HEML. ...................................................... 44
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LIST OF FIGURES
Fig.2.1: M. lucida plant;................................................................................................................................ 7
Fig.2.2: Chemical structures of tetracyclic irridroids isolated from M. lucida……………………………….11
Fig. 2.3: The metabolic pathway of arachidonic acid mediated by COX-1 and COX-2 ............................ 25
Fig 2.4: Generation of NO catalyzed by iNOS ........................................................................................... 28
Fig. 2.5: Co-activation of COX-2 by iNOS. . ............................................................................................. 30
Fig. 4.1: TLC profile of HEML; ................................................................................................................. 45
Fig. 4.2: Antioxidant activity expressed as DPPH free radical scavenging activity of HEML................... 46
Fig. 4.3: Effect of HEML and diclofenac on carrageenan-induced paw edema in rats; ............................. 48
Fig. 4.4: Effects of HEML or Chlorpheniramine on histamine-induced paw edema in rats. ...................... 49
Fig. 4.5: Effects of HEML or Granisetron (GRA) on serotonin-induced paw edema in rats...................... 50
Fig. 4.6: Effect of HEML on LPS-induced fever in rats;. ........................................................................... 52
Fig. 4.7: Effect of HEML or sulforaphane (SFN) on PGE2 release and COX-2 expression ...................... 53
Fig. 4.8: Effect of HEML or sulforaphane (SFN) on NO production and iNOS expression ...................... 54
Fig. 4.9: Effect of HEML on cytokine levels of LPS-activated RAW 264.7 cells...................................... 55
Fig. 4.10: Effect of HEML on cell viability of LPS-activated RAW 264 cell. …………………………...57
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ABBREVIATIONS AND ACRONYMS
AP: Activator protein
BHT: Butylated Hydroxytoluene
C/EBP: CCAAT/Enhancer-Binding Protein
CAM: Cellular Adhesion Molecules
cGMP: Cyclic Guanosine Monophosphate
CNS: Central Nervous System
COX: Cyclooxygenase
CPM: Chlorpheniramine
CPMR: Center for Plant Medicine Research
CRE: Cyclic AMP Response Element
DMEM: Dulbecco’s Modified Eagle’s Medium
DNA: Deoxyribonucleic Acid
DPPH: 2, 2-Diphenyl-1-picryl hydrazyl
EDRF: Endothelium-derived Relaxing Factor
ELISA: Enzyme-linked Immunosorbent Assay
eNOS: Endothelial Nitric Oxide Synthase
FBS: Fetal Bovine Serum
GAE: Galic Acid Equivalence
GIT: Gastrointestinal Tract
GPCR: G-protein Coupled Receptor
GRA: Granisetrone
HEML: Hydro-ethanolic leaf extract of Morinda lucida.
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HRP: Horse Radish Peroxidase
IFN-ɣ: Interferon-gamma
IL: Interleukin
iNOS: Inducible Nitric Oxide Synthase
LAM: Lipoarabinomannan
LD50: Lethal Dose at 50%
LFA: Lymphocyte Function Associated Antigen
LOX: Lipooxygenase
LPS: Lipopolysaccharide
MAC: Membrane Attack Complex
MAPK: Mitogen-Activated Protein Kinase
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
NF-κB: Nuclear Factor Kappa B
NKC: Natural Killer Cells
NMIMR: Noguchi Memorial Institute for Medical Research
nNOS: Neuronal Nitric Oxide Synthase
NO: Nitric Oxide
NSAIDs: Non-Steroidal Anti-inflammatory Drugs
PAF: Platelet-Activating Factors
PAMPs: Pathogen Associated Molecular Patterns
PGE2: Prostaglandin E2
PGI: Prostacyclin
PKC: Protein Kinase C
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PRR: Pathogen Recognition Receptors
PSGL: P-selectin Glycoprotein Ligand
QE: Quercetin Equivalence
ROS: Reactive Oxygen Species
SDR: Sprague-Dawley Rat
SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
SFN: Sulforaphane
TNF-α: Tumour Necrotic Factor- alpha
TXA: Thromboxane A
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ABSTRACT
A commonly used medicinal plant in African folk medicine for the treatment of various diseases
including inflammation is Morinda lucida Benth. However, scientific data supporting its anti-
inflammatory activity is scarce. In the current study, the anti-inflammatory activity of the
hydroethanolic leaf extract of Morinda lucida Benth (HEML) was assessed using carrageenan-
induced paw edema in female Sprague-Dawley rats (SDRs). The potential mode of action of
HEML was determined by assessing its effect on the levels of PGE2, NO, IL-1β, TNF-α and IL-
10, and also expressional levels of COX-2 and iNOS in RAW 264.7 cells in vitro. Its
phytochemical constituents were determined by standard methods, and its antioxidant activity
was investigated by DPPH free radical scavenging assay. The ability of HEML to significantly
reduce rat paw edema caused by carrageenan demonstrated its anti-inflammatory activity. HEML
also inhibited paw edema induced by histamine or serotonin, and further suppressed LPS-
induced fever in the SDRs, which indicates that both early and late phases of acute inflammation
were affected by the extract. Persistence of the late phase mediators may plunge the organism
into a chronic state of inflammation. Results obtained through the determination of levels of
PGE2, NO, IL-1β, TNF-α and IL-10 in culture supernatant of LPS-activated RAW 264.7 cells
showed that HEML reduced NO and PGE2 concentrations by downregulating iNOS expression
but not COX-2. It also suppressed the levels of pro-inflammatory cytokines IL-1β and TNF-α
possibly by boosting levels of the anti-inflammatory cytokine IL-10. HEML contained saponins,
reducing sugars, polyphenolics including flavonoids. It possesses antioxidant activity, which
may be due to its polyphenolic content, particularly flavonoids. The current findings provide
evidence-based scientific data to support the anecdotal use of M. lucida as treatment agent for
inflammation.
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CHAPTER ONE
1.0 INTRODUCTION
1.1 BACKGROUND
Inflammation is a complex mechanism and defensive process where biological tissues respond to
dangerous stimuli produced by pathogens, damaged cells or irritants (Ferrero-Miliani et al.,
2007). It provides the required physiology to get rid of infected or damaged cells, but the
aftermath can be undesirable. This is because, uncontrolled inflammation forms the basis of
some pathophysiological conditions such as gastritis, esophagitis, hepatitis, atherosclerosis, as
well as cancer (Schottenfeld and Beebe-Dimmer, 2006). Thus, what begins as a harmless
process to protect organisms, if not controlled can lead to deleterious consequences.
Evidence shows that prolonged inflammation is critical in the development of some
pathophysiological conditions like cancer (Khansari et al., 2009). Mast cells, basophils,
neutrophils, eosinophils, macrophages and monocytes are major cells which participate in the
process of inflammation.  These cells form the important source of a range of chemicals
including histamine, serotonin, bradykinin, cytokines, lipid mediators (eicosanoids), vascular
endothelial growth factors and reactive oxygen species (ROS) which mediate the inflammatory
processes (Boesiger et al., 1998; Grutzkau et al., 1998; Galli et al., 2005).
A major composition of bacterial (gram negative) outer membrane is made up of an endotoxin
lipopolysaccharide (LPS). The lipid part of LPS is found to activate many genes that express
inflammation including that of cyclooxygenases (COXs), inducible nitric oxide synthase (iNOS)
and cytokines among others (Kubes and McCafferty, 2000; Tanabe and Tohnai, 2002).
Prostanoids (prostaglandins) formation from arachidonic acid (obtained after the hydrolysis of
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membrane lipid by phospholipases) is catalyzed by COXs. Accumulated data indicate that there
are two isoforms of COXs, which are COX-1 and COX-2 expressed by different genes.
Constitutively, most tissues express COX-1 which synthesis basal levels of eicosanoids to ensure
homeostasis (Funk et al., 1991). Meanwhile, COX-2 expression is inducible and can be triggered
by inflammatory stimuli such as LPS (Kong et al., 2002 ). Increased expression of COX-2 is
attributed to most disorders associated with inflammation including lupus erythematosus,
multiple sclerosis, arthritis, Alzheimer’s disease and cancer (Kapoor et al., 2005; Wang et al.,
2007). Clinically, it has been shown that prostaglandins levels and COX-2 expression are higher
in transformed tissues than in normal tissues, indicating an important role of COX-2 in
tumourigenesis (Taketo, 1998; Buskens et al., 2002). Malignant cell proliferation can be
stimulated by enhanced levels of COX-2 (Subbaramaiah et al., 1998) which can also promote
angiogenesis (Lee et al., 2003), inhibit immune surveillance (Thomas et al., 2000) and suppress
apoptosis (D'Acquisto et al., 1997).
During arginine metabolism by nitric oxide synthases (NOSs), NO is generated endogenously to
play a significant role in killing of tumour cells, host intracellular defense against pathogens,
neurotransmission and prevention of platelet aggregation (Lorsbach et al., 1993; Kroncke et al.,
1998). In a biological system, NO is produced through a chemical reaction catalyzed by three
major isoforms of NOSs. These include neuronal nitric oxide synthase (nNOS) and endothelial
nitric oxide synthase (eNOS) which are expressed constitutively in neuronal and endothelium
cells, respectively. However, the expression of the third isoform, inducible nitric oxide synthase
(iNOS) is inflammation dependent and can be generated in stressed cells or cells (macrophages,
hepatocytes and endothelial cells) induced with LPS endotoxins, pro-inflammatory cytokines
(Nathan and Xie, 1994). The expression of iNOS leads to the production of large amount of NO.
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Excess NO production may play a significant role in the pathogenesis of cancer; thus can
damage DNA directly or indirectly by several mechanisms, interfere with DNA repairs and cause
post-translational modification by nitrosylation to initiate tumour formation and further drive the
process of tumourigenesis (Tamir et al., 1996; Wink et al., 1998). Thus NO production by iNOS
may reflect the degree of inflammation and cancer.
1.2 PROBLEM STATEMENT AND JUSTIFICATION
A common practice in the therapeutic approach to alleviate the symptoms associated with both
acute and chronic inflammatory diseases is the use of Non-Steroidal Anti-Inflammatory Drugs
(NSAIDs). Unfortunately, most NSAIDs have adverse effects on coagulation of blood,
gastrointestinal lining and the renal systems due in part to the inhibition of the housekeeping
enzyme COX-1 (Rainsford, 1999). Thus, most available therapeutic agents for the treatment of
inflammatory diseases lack specificity and are plagued with untoward side effects (Dhikav,
2002).
While current treatment modality targets inflammatory cells and mediators, there is evidence that
the extent of success with this approach could have been reached using plants (Dhikav, 2002).
Limitations of NSAIDs, including the usual adverse effects on the gastrointestinal tract and renal
system have sensitized the need for an alternative approach.  Moreover, some newer COX-2
specific inhibitors, claimed to be devoid of these adverse effects, have not lived up to these
claims (Celotti and Laufer, 2001), and hence the continuous efforts in search of more potent anti-
inflammatory drugs with minimal side-effects.
The increasing optimism in Ghana and other developing countries for the use of herbs and
safety-modified natural metabolites in the treatment of various diseases including inflammatory
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conditions has come of age. M. lucida Benth. (Rubiaceae) is one of the most popular medicinal
plants widely distributed in Africa. Although M. lucida has been used in Ghanaian folk medicine
in the treatment of inflammation, there is little scientific data to validate its use as such. This
work, therefore, seeks to evaluate the anti-inflammatory properties of hydroethanolic leaf extract
of M. lucida (HEML) and determine its possible mechanism. This would provide some scientific
evidence to support the use of the plant in the treatment of inflammation.
1.3 HYPOTHESIS
M. lucida has anti-inflammatory activity and this is expressed through the suppression of pro-
inflammatory mediators and/or elevation of anti-inflammatory mediators.
1.4 AIM/ SPECIFIC OBJECTIVES
1.4.1 Aim
To evaluate scientifically the anti-inflammatory effect of HEML and its possible mechanism of
action.
1.4.2 Specific Objectives
 To determine the phytochemical constituents and antioxidant activity of the HEML.
 To assess the anti-inflammatory activity of HEML using the Carrageenan-induced paw
oedema rat model.
 To determine the possible mechanisms of action of HEML as an anti-inflammatory agent
by examining its effect on selected inflammatory mediators.
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CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 MEDICINAL PLANT
It is believed that about 80% of the world’s population use medicinal plants for their primary
health care needs (Segar, 2012). Medicinal plants have long formed a key composition in
traditional medicine and a common element in Ayurvedic, homeopathic, naturopathic, traditional
oriental, Native American Indian medicine and African folklore. Records indicate that 74% of
plant-derived pharmaceutical medicines used in modern medicine directly correlates with their
traditional uses as plant medicines by native cultures. This has placed an urgent need on modern
pharmaceutical companies to engage in extensive research on the pharmacological values of
medicinal plants since these plants are utilized in herbal medicine.
The reason for high patronage of herbal medicines include its perceived lesser side effects,
affordability, accessibility and availability. They are also thought to be more effective especially
for chronic disease conditions that do not respond well to orthodox medicine (Grunert, 2011).
However, the most fascinating issue to discredit the world of herbal medicine is the inadequate
data and documentation to explain its anecdotal use. There is also the risk of self-dosing and
slow response to ailment as compared to modern medicine, which can treat sudden illnesses and
emergency conditions much more effective at a faster rate (Grunert, 2011), but often with
deleterious consequences. A typical example of such medicinal plant exploited for its medicinal
values and other economic gains in Africa is M. lucida.
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2.2 BOTANICAL DESCRIPTION OF M. LUCIDA.
M. lucida is a medicinal plant which belongs to the Rubiaceae family. It is known commonly as
Brimestone tree due to the yellow color of its wood. In Nigeria, especially among the Yoruba
natives of south-western Nigeria, it is referred to as ‘Oruwo’ (Dalziel, 1937), while in Ghana it is
popularly known as ‘Konkroma’ by the Akans and ‘Amake’ by the Ewes (Addo-Fordjour et al.,
2008). The medium sized tree or evergreen shrub is about 18-25 meters high, with the branches
often crooked or gnarled, projecting from a stem covered with both smooth and rough-forming
irregular grey-brown patches at the bark, often showing purple divisions (Abbiw, 1990). In
addition, the plant has slender branchlets and a dense crown. The leaves are broad, ovate and
tapering to the end, with sizes ranging normally from 7-15 cm long and 3.5-7.5 cm wide. The
display of the leaves around the branches is opposite and simple.
As a flowering plant, M. lucida produces white aromatic scented flowers from January to July
and September to October and also bears fruits from March to April (Irvine, 1961). The flowers
are bisexual and have narrow glabrous corolla tube of about 2.5 cm. The fruits produced are
classified as drupe; several are arranged together into an almost globose succulent syncarp 1-2.5
cm in diameter, which is soft and black when matured, pyrene compressed ovoid, up to 6.5 mm x
4 mm, dark red brown and one seeded.
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A B
Fig.2.1: M. lucida plant; (A) showing the whole plant and (B) showing the leaves.
2.3 GEOGRAPHICAL DISTRIBUTION OF M. LUCIDA
M. lucida thrives on grassland, exposed hillsides, thickets, forests, and often on termite mounds,
sometimes in areas which are regularly flooded, from sea-level up to 1300 m altitude. The plant
also grows in fringe forests and sometimes it takes over secondary clearings in rain forests.
2.4 TRADITIONAL USES OF M. LUCIDA
The indigenous style of treating or managing specific disease conditions from the native’s
perspective which may reflect the living conditions of people in a given environment is said to be
ethnomedicine. The concept of ethnomedicine is proven useful in studying indigenous
therapeutic agents particularly during drug discovery or development. This has enabled
researchers to understand fairly and suggest possible scientific explanations to the basis of most
native treatment modalities (Neuwinger, 2000).
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Various parts of M. lucida have been used extensively in traditional medicine in West Africa.
Nigerians use it as one of the top four medicinal plants in various preparations for the treatment
of fever. Decoctions and infusions made from the leaves, roots and bark may serve as remedy
against fever during childbirth and infectious diseases such as malaria, yellow fever,
trypanosomiasis among others (Adesida and Adesogan, 1972). Other disease conditions such as
diabetes, hypertension, cerebral congestion, dysentery, stomach-ache, ulcers, leprosy and
gonorrhea are also well managed or treated with M. lucida (Adesida and Adesogan, 1972).
As one of the ways of treating ringworm infections and itches in DR Congo, the leaves or stem
bark is used in combination with the root bark as dressings. Decoction of the leaves is also used
as remedy against jaundice in Côte d’Ivoire (Abbiw, 1990). Adewunmi et al. (1984) reported
that aqueous extract of the leaves is applied to the breast of women during weaning of their
infants not only due to its bitterness, but also to prevent infections. Traditionally, M. lucida is
reported to be used generally as laxative, analgesic and febrifuge while it is used locally in
Ghana and Nigeria for the treatment of irregular menstruation, jaundice, insomnia, wound
infections, abscesses and chancre (Makinde and Obih, 1985; Burkill et al., 1997; Raji et al.,
2005), however, the specific part was not stated. Some herbalists in Ghana often cold macerate
the fresh leaves in palm wine to form a bitter concoction. This is administered orally for few
days to patients with diabetes.
Apart from the medicinal values, the yellowish to red dye extracted from the wood of M. lucida
has significant benefits to the textile industries. For example, the root remains an important
source of yellow dye to feed the Kasai Province of Democratic Republic of Congo, where it may
be used with or without a mordant. In both Gabon and Nigerian, scarlet red of textiles is made
using the root bark as dye. In Ghana, the indigenes of the Ashanti Region during the grief period
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over the death of a chief require special clothing locally made and known as ‘Kobene’. ‘Kobene’
is cotton clothing dyed red using the root bark of M. lucida and represents the official dress for
the indigenes of the Ashanti kingdom for mourning a dead chief. By the process of fermentation
and reduction which are some steps in dyeing, the root of M. lucida is added to indigo vats to
generate a darker blue color in Cote d’ Ivoire. This process may not be complete without adding
leafy twigs of Saba comorensis. To obtain a pale green color of basket during basket weaving,
the young leaves of M. lucida is combined with the leaves of Philenoptera species.
In the food industry, the roots are used as chewing stick and flavoring agent in alcoholic
beverages. The hardy woody nature of M. lucida makes it very useful in energy generation hence
is an excellent source of charcoal (Burkill et al., 1997).
2.5 SOME CHEMICAL CONSTITUENTS OF M. LUCIDA
It has been documented that the major composition of M. lucida extracts are alkaloids,
anthraquinones and anthraquinols (Adewunmi et al., 1984). Among the 18 anthraquinones
known to have been isolated from the wood and bark of M. lucida are red dyes 1-methylether-
alizarin, rubiadin and their derivatives, lucidin, soranjidiol, damnacanthal, nordamnacanthal,
morindin, munjistin and purpuroxanthin. Other compounds such as Oruwalol and Oruwal have
been isolated and characterized from the stem (Adesogan, 1973). Recently in Ghana, a
tetracyclic iridoid named molucidin and its derivatives have been isolated and characterized from
the leaves of M. lucida through a bioassay guided fractionation process in the quest for drug
development against parasitic infections such as trypanosomiasis, malaria and leishmeniasis
(Suzuki et al., 2015; Kwofie et al., 2016). Kwofie et al. (2016) studied extensively the
mechanism of action of molucidins on trypanosome parasites. Other groups of phytochemicals
such as tannins, flavonoids and saponins have been detected and some isolated (Adewumi and
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Adesogan, 1984). Oruwacin has also been reported (Evans et al., 2002) to be isolated from the
roots of M. lucida and further confirmed the presence of anthraquinones also in the roots.
Furthermore, work done on the leaves showed the presence of ursolic and oleanolic acids which
are also classified as triterpenes (Evans et al., 2002).
2.6 PHARMACOLOGICAL PROPERTIES OF M. LUCIDA
Various in vitro models have been used to assess the biological and pharmacological effects of
extracts of M. lucida and further confirmed its activity by pre-clinical trials using animal models.
Most of these activities reflect and explain the traditional applications of extracts from the plant.
A typical example is as cited in a study, which demonstrated that extracts of the roots of M.
lucida possess analgesic and consequently a sedative effect in mice (Younos et al., 1990).
Zimudzi and Cardon (2005) also reported the antihypertensive activity of extracts of the leaf and
stem bark of M. lucida. Although the extracts showed a strong antihypertensive activity, it was
short acting, which may possibly be due to rapid metabolism and faster elimination of
metabolites from circulation but had no data to ascertain that. It was, therefore, recommended
that extracts of M. lucida has potentials as treatment agent for hypertension and cerebral
complications due to its strongly marked diuretic and tranquilizing ability. Most especially, M.
lucida extract did not only cause a remarkable reduction in contraction of the smooth muscles of
the uterus of both pregnant and non-pregnant women but also suppressed contractions induced
by both oxytocin and acetylcholine in the uterine walls (Elias et al., 2007).
The anthraquinone content has molluscidal activity against fasciola and schistosoma sp., which
has been attributed to the presence of oruwacin (Adewumi and Adesogan, 1984). The anti-
trypanosomal property of the hydroethanolic leaf extract has been demonstrated to be
pronounced in mice infected with Trypanosoma brucei and treated at a dose of 1000 mg/kg
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intraperitoneally (Asuzu and Chineme, 1990). In a bioassay-guided fractionation, Suzuki et al.
(2015) isolated a tetracyclic iridroid and two other derivatives from the chloroform fraction of 50
% (v/v) hydroethanolic leaf extract of M. lucida. These compounds have activity against
trypanosomes and were named as Molucidin, ML-2-3 and ML-F52. The side chain of the ML-2-
3 at the C-4 has carboxylic acid functional group. The other two derivatives (ML-F52 and
Molucidin) have functional groups which are methyl and ethyl esters of the main compound ML-
2-3, respectively. They also established possible mechanisms of action of ML-2-3 and ML-F52
on Trypanosoma brucei as induction of apoptosis and suppression of the expression of
paraflagellar rod protein 2 (PFR-2). Their research suggested that, the compounds isolated were
not only active against trypanosomes but other protozoans. They, however, failed to prove that
the isolate could be active against other protozoans since they had no data to support it.
COOH COOCH3 COOC2H5
ML-2-3 ML-2-2 (Mollucidin) ML-F 52
Fig.2.2: Chemical structures of tetracyclic irridroids isolated from M. lucida. (Suzuki et al., 2015).
During infection, there is inflammation triggered by the immune system just to notice and get rid
of the infection (Ferrero-Miliani et al., 2007). One key manifestation of inflammation during
infections is fever due to the release of prostaglandins E2 (PGE2). Although M. lucida has been
reported and evaluated for various pharmacological activities, there is limited research to show
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its anti-inflammatory, anti-fever and pain relieving effect, even though other species from the
Rubaecia family has been evaluated for their anti-inflammatory effect (Awe et al., 1998). The
leaf extracts of M. lucida has also been reported to have anti-malarial activity against
Plasmodium falciparum in vitro (Adeneye et al., 2008) and Plasmodium bergei in mice where
the leaf extract had significant inhibition on the schizontal stage of the parasite (Makinde and
Obih, 1985; Obih et al., 1985).
2.7 INFLAMMATION
As part of a complex defensive mechanism in an organism, the local build-up of catabolic
products leading to subsequent rise in tissue osmotic pressure and the concomitant attraction of
fluids accompanied with or without heat is said to be inflammation (Stankov, 2012). It can be
reduced to five macroscopic pathological phenomena, characterized by tumour or swellings of
the tissue resulting from fluid accumulation, elevated temperature and tissue redness due to
increased blood flow to the affected area, intense sensation due to noxious stimulus and finally
loss of function of organ(s) involved resulting from combination of factors. For example, in the
process of recovery from a toxic insult in the liver, there may be high levels of collagen
disposition into the extracellular space of the hepatocytes. This may harden the liver and impair
its function, leading to a disease condition referred to as liver cirrhosis.
The process of inflammation is key in the displacement of offending factors and total restoration
of tissue structure for proper functioning of the body. Quite often, the supposed beneficial
process has gone out of control plunging the organism involved into dreadful and irreversible
pathophysiological conditions. The inflammatory mechanism is, therefore, complex and may
lead to cell death or tissue damage. A number of factors including physical, biological and
chemical factors may trigger the whole cascade. Host cells, blood vessels and plasma proteins
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are some of the major players in the initiation and resolution stages of the mechanism.
Progression of inflammation is very intricate and can be divided into acute; where inflammation
lasts for only few days and chronic; where inflammation persist for longer duration.
2.7.1 Acute Inflammation
The initial stage of a typical inflammatory process is marked by an acute phase, where the
cascade begins with primary response of the immune and vascular system right after infection or
damage to sterile tissues. It represents the first line of defense against injury, nonspecific but
immediately lethal which is characterized by various changes in microcirculation, thus plasma
exudation and emigration of leukocytes from blood vessels to the site of injury (Lawrence et al.,
2002). The response is rapid and persists for a short while, normally before the immune response
becomes established. This is because, the primary role of acute inflammation is targeted at
eliminating the injurious agent as soon as possible. Acute inflammation also acts as a
homeostatic mechanism to the benefit of the host in a reparative process where affected tissues
are restored to normal (Gabay and Kushner, 1999). A hallmark of acute inflammation is the
infiltration of polymorphonuclear leukocytes, thus leukocytes with looped nucleus (Maton et al.,
1997). Leukocytes are group of white blood cells (WBCs) which make up the cells of the
immune system. They consist of the broadest category of cells including neutrophils,
eosinophils, basophils, lymphocytes and monocytes. They are derived from hematopoietic stem
cells from the bone marrow and aid in protection against invasion. They are present throughout
the body in circulation, thus the blood and lymphatic systems (Maton et al., 1997).
Sentinel cells are basic guard cells which include mast cells, dendritic cells and resident
macrophages. Their major role is to protect the body against invasion and this makes them
critical players during inflammation. Pathogens possess pathogen associated molecular patterns
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(PAMPs) which recognize and bind to pathogen recognition receptors (PRRs) present on the
surface of the sentinel cells (Janeway and Medzhitov, 2002; Kawai and Akira, 2010). This cause
their activation and consequent release of acute phase mediators such as histamine, bradykinin
and serotonin from mast cell especially, interleukin-1 (IL-1) and tumour necrosis factor alpha
(TNF- α) by resident macrophages and dendritic cells. Unlike macrophage and dendritic cells,
mast cell activation is more rapid because mediators involved are already synthesized and stored
in the vesicles.  Macrophage and dendritic cell activation is followed by the release of mediators
such as IL-1, IL-6, TNF-α, prostaglandins (PGs) and NO which are synthesized in delayed
process.
Histamine released from degranulation of mast cells act on endothelial cells lining the walls of
terminal arterioles and capillaries and post-capillary venules to release nitric oxide (NO) and
prostaglandins (e.g. prostacyclin-2 (PGI2) and prostaglandin E2 (PGE2)). The NO and
prostanoids generated cause the relaxation of vascular smooth muscle cells leading to
vasodilatation of the blood vessels. This enables more blood to rush through causing redness of
the inflamed area. Vasodilation of blood vessels means the pulling apart of actin filaments at
adherence junction linking endothelial cells from each other, creating gaps to allow plasma
exudates into the interstitial spaces and hence tumour or edema formation. Plasma exudate
contains proteins like that of the complement system, kallikrein-kinin system and coagulation
factors.
The C3b complement protein found among plasma exudate coats the pathogen membrane
surface for easy opsonization and phagocytosis by phagocytes (Martin and Blom, 2016). Other
complement proteins such as C3a and C5a are regarded as anaphylatoxin and are essential for the
formation of membrane attack complex (MAC) in the membrane of the pathogen to cause
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osmotic lysis. C3a and C5a also have their receptors located on mast cells. They bind to them
and cause positive feedback loop by activating mast cells to release more histamine. Kallikrein-
kinin system also consists of proteins such as bradykinin and kallidin. Bradykinin and kallidin in
plasma exudate act on sensory neurons and cause pain and loss of function. They also act on B2
receptors on endothelial cells to cause positive feedback loop activation.
Coagulation factors have no specific role to play in circulation but once they are leaked into the
interstitial spaces and come into contact with collagen and tissue factors, the intrinsic or extrinsic
coagulation cascade is set-up. This results in the transformation of fibrinogen (factor-1) into
fibrin (factor 1A). Fibrin then polymerizes into fibrin strands which are joined together to form a
very dense mesh network known as fibrin mesh work (Doolittle, 1973). Fibrin mesh work
restricts the spread of pathogens and also prevents external pathogen from entry.
To recruit phagocytes (e.g. neutrophils and macrophages) to the site of injury, endothelial cells
release already synthesized and sequestered receptors stored in weibel-palade bodies. These
receptors are known as P-selectin. Selectins are cellular adhesion molecules (CAMs) with single-
chain transmembrane glycoproteins, which facilitate attachment of cells and are calcium-
dependent in binding (Cotran and Mayadas-Norton, 1998). During the process, the weibel-palade
bodies fuse with the apical membrane of the endothelial cells and release the P-selectin onto its
surface. Neutrophils for example have on their membrane surface P-selectin glycoprotein ligand-
1 (PSGL-1) which binds to P-selectin on the surface of the endothelial cells to slow down the
rate of movement. The process of rolling of neutrophils begins by breaking and re-bonding with
other P-selectin secreted on the surface of endothelial cell, until a weak interaction is formed
between lymphocyte function associated antigen-1 (LFA-1) on the neutrophil membrane surface
and intracellular adherence molecule-1 (ICAM-1). The weak interaction is due to the non-
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activation of LFA-1. Platelet activating factors (PAF) released by the endothelial cells activate
LFA-1 by binding to a platelet activating factor receptor (PAFR) present on the neutrophil. This
finally halts the rolling neutrophil to enable it to push through the space created in between the
endothelial cells and gets into the interstitial space in a process known as diapedesis. C3b
receptors on the neutrophil membrane bind to C3b protein coatings on pathogen membrane and
invaginate to form a phagosome. Lysozymes stored in vesicles in neutrophils are released to fuse
with phagosome and digest the pathogen. Persistence of acute phase mediators due to the
injurious agent may set the system into the chronic stage of inflammation.
2.7.2 Chronic Inflammation
Chronic inflammation is a prolonged (takes weeks, months or years) and an active inflammation
in which tissue injury and healing proceed simultaneously (Lazzarino et al.,2016). It is
characterized by infiltration of mononuclear cells such as macrophages, lymphocytes and plasma
cells. It is caused by prolonged infection and can be better explained by the pathogenesis of
tuberculosis.
Microorganisms can escape clearance by the immune system leading to prolonged infection and
activation of several immune cells resulting in chronic inflammation. For instance, during
Mycobacterium tuberculosis infection in the lungs, resident alveolar macrophages and tissue
dendritic cells engulf the mycobacterium to form a phagosome and tend to eliminate it by fusing
the lysosome containing hydrolytic enzymes with the phagosome (Steinberg and Grinstein,
2009). Mycobacterium cell wall component; lipoarabinomannan (LAM), prevents the fusion
process by suppressing phagosomal maturation by the alveoli macrophages. This affects and
interferes with the associated cell signaling cascade to switch cytokines response from pro-
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inflammatory to anti-inflammatory (Nigou et al., 2001; Briken et al., 2004; Pathak et al., 2005;
Vergne et al., 2005) to enable the microorganism to survive.
The pro-inflammatory cytokines released at the initial phase of the infection leads to the
recruitment of more inflammatory cells from the blood stream (Briken et al., 2004). Infected
cells become activated and release IL-12 and IL-18 at a local lymph node where they activate T-
lymphocytes. The activated T-lymphocyte release interferon gamma (IFN-γ) which induces
natural killer (NK) cell activity and enhance the fusion of lysosome with the phagosome, just to
eliminate the microorganism. The IFN-γ released also acts on endothelial cells lining blood
vessels to attract monocytes from tissues to differentiate into macrophages where they are
recruited to the site of infection. The consequent activation of the macrophages is associated with
the release of pro-inflammatory cytokines such as TNF-α and IL-1 and results in high
inflammation (North and Jung, 2004; Korbel et al., 2008). The activated macrophages crowd and
fuse to form giant cells. The site of infection becomes necrotic and surrounded by activated T-
lymphocytes causing reactions leading to granuloma formation and granuloma inflammation.
Chronically activated macrophages release cytokines which stimulate fibroblast proliferation and
collagen production in a healing process. This produces a scar (fibrosis) at the inflammatory site
and impair the normal tissue function of the specific organ involved (Welin et al., 2011).
Apart from persistent infections, other factors such as autoimmunity and prolonged exposure to
potentially toxic substances are of importance in chronic inflammation. These factors delay
hyperactivity and prevent the system from attaining the optimum resolution during acute
inflammation (O'Byrne and Dalgleish, 2001; Dalgleish and O'Byrne, 2002). Chronic
inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and chronic allergic
diseases (e.g. bronchial asthma) might have occurred through the activation of immune cells by
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auto-antigens and common environmental agents like smoke, dust and hay. In most autoimmune
diseases, there is a ‘self-antigen’ which continually activates T cells and results in significant
tissue damage. Chronic inflammation has been attributed to the development of degenerative
diseases such as atherosclerosis, arthritis, inflammatory bowel disease, aging and other
neurodegenerative central nervous system depression (O'Byrne and Dalgleish, 2001; Dalgleish
and O'Byrne, 2002).
2.8 MEDIATORS OF INFLAMMATION
Immune reaction elicited by the body to tissue damage involves a myriad of antibodies, cells,
proteins, antimicrobial peptides and connective tissue features which interact to prevent the
damage thereby restoring normal tissue integrity (Davidson, 1992). Vasodilation and consequent
increase in vascular endothelial permeability at the initial phase of local inflammation, as a result
of infection or tissue injury is associated with the recruitment of large number of leukocytes from
peripheral blood to the site inflamed (Butcher et al., 1999). The leukocytes involved in the
inflammatory process include basophils, neutrophils, eosinophils and monocyte which
differentiate into macrophages at the inflammatory site. Other cells which mediate inflammation
are mast cells and dendritic cells. Upon activation, these cells release chemical agents which do
not only propagate the whole inflammatory process but also act as crucial players for resolving
it. Some of these chemical mediators are histamine, serotonin, bradykinin, cytokines,
chemokines, lipid mediators (eicosanoids) and reactive oxygen species (ROS) (Boesiger et al.,
1998; Grutzkau et al., 1998; Galli et al., 2005).
2.8.1 Histamine
Histamine [2-(4imidazolyl)-ethylamine] is a vasoactive and endogenous short acting biogenic
amine which is obtained from the catalysis of histidine by the enzyme histidine decarboxylase. It
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is synthesized and stored in granules of mast cells, basophils and platelets and known to regulate
Th-1 and Th-2 antigen specific immune responses (Packard and Khan, 2003). It was first
described as an anaphylactogen due to its high tendency of mimicking anaphylaxis and since
then, research has clearly elucidated its role as far as inflammatory response is concerned. For
example, during allergic reaction, histamine acts as a chemical neurotransmitter to cause skin,
nose, throat and lung irritation (Zampeli and Tiligada, 2009). The ability of histamine to bind its
specific receptors is key to affecting cellular activities and this may represent a basic regulatory
mechanism to control the undesirable effects elicited.  There are four major G-protein coupled
receptors (GPCR) subunits designated as H1, H2, H3 and H4 known to be acted upon by
histamine (Dy and Schneider, 2004; Akdis and Simons, 2006). These receptors are widely
distributed and differentially expressed in various cell types such as endothelial cells,
macrophages, dendritic cells, T- and B-lymphocytes (Zampeli and Tiligada, 2009).
Activation of H1 receptor results in histamine-induced vascular permeability (Zampeli and
Tiligada, 2009). Just like H1 receptor, H2 receptor type in addition to mediating vascular
permeability also serve as a more important secretagogin to stimulate gastric acid secretion. H3
receptors are known to be localized in the CNS but very little is known about their role in
humans (Parsons and Ganellin, 2006). H4 receptors are found on cells with hematopoietic origin
and hence are regarded as major players during inflammation (Parsons and Ganellin, 2006). Due
to this, H4 and H2 receptor antagonists may serve as promising drug candidates for treating
inflammatory conditions and lower the risk of gastrointestinal tract disorders (e.g. peptic ulcer)
like those associated with treatment of inflammation by non-steroidal anti-inflammatory drugs
(NSAIDs).
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2.8.2 Serotonin
Serotonin, also known as 5-hydroxytryptamine (5-HT) formed from tryptophan. It is a
monoamine molecule found in mast cells and platelets distributed in the gastrointestinal tract
(GIT) and the central nervous system (CNS). It serves as a neurotransmitter and so modulates
behaviors such as cognition, mood, aggression, mating, feeding, and sleep in the brain (Nichols
and Nichols, 2008). It also acts as vasoactive amine, which increases vascular permeability and
act as vasodilator of capillaries to bring about contraction of nonvascular smooth muscle
(Borissova et al., 1994). Thus, during acute inflammation, serotonin plays a major role by
increasing vascular permeability through vasodilation of blood vessels. To facilitate and
accelerate the rate of leukocytes emigration from microcirculation to the site of inflammation,
serotonin increases the levels of intracellular cyclic guanylate monophosphate (cGMP) to induce
a chemotactic effect so that polymorphonuclear granulocytes migrate to the inflamed site
(Paegelow et al., 1985; Kelm and Schrader, 1990). McCorkle et al. (1990) reported that the
presence of 5-HT antagonist could suppress leukocyte emigration by blocking serotonin effect.
2.8.3 Bradykinin
Bradykinin is a peptide which is endogenously produced through the kallikrein-kinin system
(Raidoo and Bhoola, 1998). Its effect is mediated in the body through B1 and B2 receptors (Chao
et al., 1987; Hall, 1997). These receptors are transmembrane and members of G-protein coupled
receptors (GPCR). The B2 receptor is expressed constitutively on various cell types such as
endothelial cells, nerve fibers, leukocytes, and mast cells (Bhoola et al., 1992; Calixto et al.,
2000) while the B1 receptor is known to be inducible and expressed in most stressed tissues. The
expression of the latter may be enhanced by cytokines production during shocks and
inflammation (Marceau et al., 1998; McLean et al., 2000).
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Bradykinin is an acute phase inflammatory mediator. During inflammation, bradykinin released
by endothelial cells binds to the B2 receptor and activates it by exchanging a guanosine
diphosphate (GDP) bound to the receptor for a guanosine triphosphate (GTP). The alpha subunit
(αq) of the heterotrimeric protein coupled to the receptor dissociates and binds to activate
membrane phospholipase-Cβ (PL-Cβ). Activated PL-Cβ act on and hydrolyze membrane lipid
phosphatidylinositol-4,5-bisphosphate (PIP2) to produce inositol triphosphate (IP3) and
diacylglycerol (DAG) (Hilgemann, 2007). Both IP3 and DAG serve as secondary messengers and
important signaling molecules which control diverse cellular events especially by stimulating the
release of intracellular calcium from the endoplasmic reticulum of endothelial cells. Influx of
calcium in the cytosol leads to activation of protein kinase C (PKC), which may control signaling
activity of the cell. DAG may also serve as a precursor to arachidonic acid generation and hence
prostaglandins formation. Thus, there is enough evidence to suggest that bradykinin has a direct
effect on cellular levels of arachidonic acid (Burch and Kniss, 1988; Kaeser et al., 1988) and
induction of calcium dependent release of glutamate and cytokine expression (Parpura et al.,
1994; Schwaninger et al., 1999). Bradykinin also induces the expression of adhesion molecules,
vasodilation of arteries, facilitation of leukocyte infiltration and protein extravasation in post
capillary venules (Calixto et al., 2000). B2 receptor activation can lead to the co-expression of B1
receptors (Phagoo et al., 1999). This, however, suggests that the induced expression and
activation of B1 receptors can take part in the cascade resulting in tissue injury and systemic
inflammation.
2.8.4 Cytokines
Cytokines can be defined as small proteins which are specific in their actions and are secreted by
cells to mediate cellular communications. Cytokines released by lymphocytes are known as
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lymphokines, those released by monocytes are termed as monokines, those released by
leukocytes or act on leukocytes are known as interleukins, while chemokines are cytokines with
chemotactic activity. As far as inflammation is concerned, cytokines can be classified as pro-
inflammatory or anti-inflammatory.
Pro-inflammatory cytokines are those which promote inflammation whereas those cytokines
which reduce or prevent inflammation by suppressing the activities of pro-inflammatory
cytokines are called anti-inflammatory cytokines (Charles and Dinarello, 2000). This concept is
fundamental to cytokine biology and may be based on the fact that, during inflammation, certain
encoding genes are upregulated. For example, genes responsible for the synthesis of pro-
inflammatory mediators such as phospholipase A2 (PL-A2), cyclooxygenases-2 (COX-2) and
inducible nitric oxide synthase (iNOS) are increased in expression during inflammation. This
increases the expression of enzymes and proteins responsible for the synthesis of pro-
inflammatory mediators.
Interleukine-1 (IL-1), intereukine-6 and tumour necrotic factor alpha (TNF-α) or cachectin are
some few examples of pro-inflammatory cytokines which may exert their effects through the
induction of endothelial adhesion molecules for leukocyte interaction. Cytokine expression can
be induced by endotoxins such as LPS, but other cytokines can be formed through stimulation by
IL-1α, IL-1β and TNF-α and in some cases, interferon-ɣ (IFN-ɣ) after their expression. During
fever, TNF-α, IL-1α and IL-1β enhances the production of PGE2 through the vascular
endothelium of the hypothalamus (Warren, 1990) and stimulate T-cell proliferation.  TNF-α also
serve as a trimer (Smith and Baglioni, 1987) to regulate apoptotic pathways, nuclear factor-kB
(NF-kB) activation of inflammation and active stress-activated protein kinases. It is one of the
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products of activated macrophages/monocytes, fibroblasts, mast cells, and some T and natural
killer (NK) cells (Aggarwal, 1992).
Anti-inflammatory cytokines are regarded as immunoregulatory peptides which control the
response of pro-inflammatory cytokines by acting in concert with specific cytokine inhibitors
and soluble cytokine receptors (Zhang and An, 2007). Some anti-inflammatory cytokines include
IL-4, IL-10, IL-11, IL-13 and transforming growth factor-beta (TGF-β) which suppress the
expression of pro-inflammatory cytokines and vascular adhesion molecules. This implies that, a
ratio closer to 1.0 may ensure a proper balance between the pro- and anti-inflammatory
cytokines, which is critical in determining the outcome of a disease (Charles and Dinarello,
2000). IL-10 happens to be one of the potent anti-inflammatory cytokines which represses the
expression of TNF-α, IL-6 and IL-1 by activated macrophages. It can counter-regulate both the
synthesis and function of pro-inflammatory cytokines in multiple states by increasing the
expression of endogenous anti-cytokines and down-regulating the receptors for pro-
inflammatory cytokines.  It has been realized that low levels of IL-10 and other anti-
inflammatory cytokines such as IL-4 in the blood could be key to chronic pain (Uceyler et al.,
2006) while acute administration of IL-10 protein suppressed the development of pain mediated
by the spine in different animal models (Wieseler-Frank et al., 2004).
2.8.5 Lipid Mediators (Eicosanoids)
The lipid mediators are generated through the catabolism of arachidonic acid, where the
availability of arachidonic acid is a rate-limiting factor to the pathway. Arachidonic acid is
produced in all cells from the hydrolysis of membrane phospholipids. It acts as the single but
essential substrate to the production of active mediators of inflammation known as eicosanoids.
Eicosanoids are made up of products of cyclooxygenases; prostaglandins (PG) and thromboxane
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(TXA), 5-lipoxygenase (5-LOX); leukotrienes and 5- hydroxyeicosatetraenoic acid and 12-
lipoxygenases(12-LOX); 12-hydroxyeicosateteraenoic acid (Borgeat and Samuelsson, 1979).
2.8.5.1 Prostaglandins and inflammation
Prostaglandins are twenty-carbon fatty acid derivative formed from the enzymatic metabolism of
arachidonic acid through the enzyme cyclooxygenases (COXs). They act generally in both
autocrine and paracrine manner and mediate various physiological processes including mucosal
defense. Prostaglandins released in the stomach stimulate mucus and bicarbonate secretion,
increase mucosal blood flow and resistance of cytotoxin-induced injury to epithelial cells. As a
result, the inhibition of prostaglandins especially in the stomach can be very detrimental and a
critical event to consider in the development of pathophysiological conditions related to the
stomach as those elicited by the NSAIDs administration.
There are two isoforms of the COXs namely COX-1 and COX-2 which are encoded by different
genes. COX-1 is constitutively expressed and is known to generate basal levels of prostanoids in
order to maintain homeostasis (Funk et al.,1991). On the contrary, COX-2 is only found in many
cells associated with inflammation such as macrophages, endothelial cells and fibroblast and is
expressed upon stimulation (Hla et al., 1993). Tumour and transformed cells also express high
level of COX-2 and so have increased levels of prostaglandins (Lupulescu, 1996).  COX-2 for
this reason has important role to play in inflammation and carcinogenesis, hence its regulation
may be relevant and very promising to avoiding diseases associated to inflammation.
Regulation of COX-2
COX-2 can be regulated at the transcriptional level. The cis-acting elements present in the
promoter region of the COX-2 gene include NF-kB site, CCAAT/enhancer-binding protein
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(C/EBP) site and cyclic AMP-response element (CRE) site which are critical in the regulatory
process. The NF-kB pathway leads to the activation of transcriptional factor NF-kB upon LPS-
stimulation and mediates the activation of many cytokines and other products of inflammation. It
has been reported that, the inhibition of NF-kB impairs transcription of COX-2 mRNA
(D'Acquisto et al., 1997; Inoue and Tanabe, 1998). The C/EBP is a family of proteins
(transcriptional factors) which interacts with the CCAAT (cytosine-cytosine-adenosine-
adenosine-thymidine) box motif present in the promoter region of the COX-2 gene (Sorli et al.,
1998). CRE is also essential for both basal and induced COX-2 transcriptional levels in most
cells by binding to cyclic AMP response element binding proteins (CREB) and activator protein-
1 (AP-1) (Wadleigh et al., 2000).
Arachidonic Acid
COX-1 COX-2
(Constitutive (Inducible)
)
Physiological stimuli Inflammatory stimuli
Prostaglandin G eg. LPS, cytokines,2
growth factors
PGD Prostaglandin H2 TXA
2 2
PGE PGF PGI22α
2
Fig. 2.3: The metabolic pathway of arachidonic acid mediated by COX-1 and COX-2. (Uto et al., 2012).
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2.8.6 Free Radicals
Molecular species which are independent in their existence because they contain in their atomic
orbital unpaired electrons are classified as free radicals. Cellular activity requires oxygen to
generate energy in the form of adenosine triphosphate (ATP) by the mitochondrial complexes
through the electron transport chain (ETC) system. Consequently, in the process, free radicals are
generated as by products which include reactive oxygen species (ROS). Although these species
can be both beneficial and detrimental to the organism, a balance between the two effects may be
crucial to the life of an organism. Thus, ROS may be beneficial at lower or moderate levels as far
as cellular activity is concerned.  However, they exert a deleterious effect at increased levels by
causing oxidative stress (Halliwell and Gutteridge, 2007).
2.8.6.1 Reactive oxygen species (ROS) and inflammation
It is estimated that at least 5% of the oxygen inhaled into the human system is channeled into
ROS generation (Trenam et al., 1992). This means that cells under aerobic condition are likely to
experience the toxic insults of ROS. Superoxide anion (O2.), oxygen singlet (
1O2), hypochlorite
(HOCl), hydroxyl radical (OH.), nitric oxide (NO.), peroxy-nitrite radical (ONOO-), hydrogen
peroxide (H2O), among others are some potent examples of ROS. The level of stability of these
species determines the degree of reactivity (Aitken and Fisher, 1994).
Even though endogenous systems may be available to cater for ROS without the cell being
affected, any imbalance between ROS generated and the available endogenous antioxidant
system may put the cell under stress resulting in oxidative damage as observed in most diseases
associated with inflammation. Mononuclear cells such as macrophages and lymphocytes as well
as polymorphonuclear leukocytes like neutrophils and eosinophils, due to their phagocytic action
tend to generate excessive quantity of ROS, which is essential to combat any microbial attack.
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The excessive ROS generated in the process affect cellular function and results in cell or tissue
damage by elevating the level of inflammation (Trenam et al., 1992) leading to pathological
states like cancer (Wiseman and Halliwell, 1996), atherosclerosis (Witztum, 1994),
neurodegenerative disorders (Lebovitz et al., 1996), Parkinson disease (Jenner, 2003),
rheumatoid arthritis (Halliwell, 1995) and premature aging (Orr and Sohal, 1994).  ROS act as
activators of immune cells monocytes/macrophages specially to synthesize TNF-α, IL-1β and IL-
6 to promote inflammation (Beckman and Koppenol, 1996), which initiate the inflammatory
cascade.
Nitric Oxide (NO) and inflammation
It was reported that intact endothelium is required for the relaxation of blood vessels in response
to acetylcholine (Furchgott and Zawadzki, 1980) and the endothelium-derived relaxing factor
(EDRF) responsible was identified as NO or its derivatives (Palmer et al., 1987). NO is a
signaling molecule which plays a significant role in regulation of physiologic processes
including vasodilation, neurotransmission and inflammation (Prast and Philippu, 2001; Ignarro,
2002).
Some biological importance of NO such as killing of tumour cells, defense of host system
against intracellular pathogens, inhibition of platelet aggregation and acting as a neurotransmitter
(Moncada et al., 1991; Alderton et al., 2001) cannot be overemphasized. However, excess
production of NO in a biological system has long been identified as a potent mediator of
inflammation (Korhonen et al., 2005). Excessive NO levels can also affect the DNA of an
organism by direct or indirect damage through several mechanisms (Tamir et al.,1996),
interfering with their repair (Jaiswal et al., 2000) and also causing nitrosylation of DNA, which
is a good post-translational modification to potentially initiate and drive tumourigenesis (Wink et
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al., 1998), and hence cancer. Under chronic conditions sustained and high levels of NO could
confer multiple damages and lead to accumulation of mutated genes such as that of the tumour
suppressor gene p53 (Forrester et al., 1996; Chazotte-Aubert et al., 2000) which contributes to
malignant transformation. As a result, NO has been described as endogenous mutagen (Wink et
al., 1998), inhibitor of apoptosis and oncogene enhancer (Li et al., 1997; Ambs et al., 1998).
2.8.7 Enzymes Involved in NO Synthesis
Endogenous synthesis of NO in living systems is mediated by the different isoforms of nitric
oxide synthases (NOSs) through the oxidative deamination of L-arginine (Sharma et al., 2007) to
citrulline and then the consequent release of NO. The three isoforms of NOS include endothelial
nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide
synthase (iNOS). Both eNOS and nNOS are constitutively expressed in endothelial and neuronal
tissues, whilst iNOS is inducible mostly in macrophages, hepatocytes (Kupffer cells) and
endothelial cells during inflammation (Nathan and Xie, 1994; Alderton et al., 2001). Excess
generation of NO has been attributed to the activation of iNOS, which is believed to play a key
role in inflammation (Maeda and Akaike, 1998). Unlike eNOS and nNOS, iNOS is calcium-
independent and can easily be activated by the release of proinflammatory cytokines such as IL-
1,TNF-α, IFN-ɣ and also other endotoxins such as lipopolysaccharides (LPS) (Nakayama et al.,
1992).
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L-Arginine
Inflammatory stimuli e.g.
iNOS LPS, cytokines.
NO
Inflammation and its
associated
Pathophysiological
conditions such
Fig 2.4: Generation of NO catalyzed by iNOS. (Uto et al., 2012)
2.8.7.1 Regulation of iNOS synthesis
The active form of iNOS with catalytic activity is a homodimeric protein of approximate size of
260 kD (Mollace et al., 2005). The regulation of NO production by iNOS is mainly at the
transcriptional level and its mRNA stability (Xie et al., 1993). This is because the mechanism of
iNOS activation involves de novo transcription and biosynthesis of new protein.  Thus,
transcription may occur through binding of transcriptional factors, including activator protein-1
(AP-1) and nuclear factor kappa-B (NF-kB) to the cis-acting elements of the promotor region
(Marks-Konczalik et al., 1998). AP-1 is a transcriptional factor, minimally activated under
normal physiologic conditions but dramatically activated under inflammatory stimuli including
that of bacterial LPS (Guha et al., 2001). NF-kB represents another transcription factor to
regulate the iNOS gene and is activated by inflammatory responses elicited during viral and
bacterial infections (Marks-Konczalik et al., 1998).
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There is about 66% homology between murine and human iNOS at the 5’ flanking region, which
has sequences conserved for binding transcriptional factors like that of NF-kB (Chartrain et al.,
1994). In a review by Mollace et al. (2005), it was reported that further release of NO during
inflammation can activate the NF-kB pathway and lead to the co-activation of COX-2 (Fig 2.4).
Similar immunological stimuli which activate iNOS are also found to induce guanosine
triphosphate cyclohydrolase. Guanosine triphosphate cyclohydrolase is an enzyme which
synthesizes and supply iNOS with the cofactor tetrahydrobiopterin (BH4) (Salvemini and
Masferrer, 1996). However, many antioxidants (e.g. pyrrolidine-dithiocarbamate and
diethydithiocarbamate), glucocorticoids such as dexamethasone, thrombin, macrophage
deactivation factor, TNF-α, platelet-derived growth factor, IL-4, IL-8 and IL-10, all inhibit iNOS
induction (Di Rosa et al., 1990; Mollace et al., 2005).
A
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Fig. 2.5 Co-activation of COX-2 by iNOS. Increased NO levels by iNOS during inflammation co-
activates COX-2 (A) through the NF-κB pathway (B). (Mollace et al., 2005).
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CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 MATERIALS
3.1.1 Animals
Female Sprague-Dawley Rats (SDRs) of weight 180-200 g were acquired from the Animal
Experimentation Session of the Centre for Plant Medicine Research (CPMR), Mampong-
Akwapem, Ghana. Animals were fed ad libitum with feed obtained from Ghana Agro Food
Company, Tema, Ghana and housed in metallic cages lined with soft wood shavings as beddings.
The animals were kept under standard laboratory conditions (temperature 28 ºC ± 2 ºC, relative
humidity 60-70 % and normal 12 h cycle of light and dark) and allowed access to sterilized
drinking water ad libitum. This animal study was approved by the Ethics Committee of the
CPMR, Mampong-Akwapem, Ghana. The animals were handled in accordance with
internationally accepted principles of laboratory animal use and care (EEC Directive of 1986:
86/609 EEC).
3.1.2 Plant Collection
Dried leaves of Morinda lucida Benth were obtained from the Plant Development Department
(PDD) of the CPMR, Mampong-Akwapem, Ghana. It was authenticated by a Senior Botanist in
the department and a voucher specimen number CSRPM/8510 was assigned and kept at the
herbarium of the PDD.
3.1.3 Chemicals and Reagents
Antibodies against COX-2 and iNOS were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Fetal bovine serum (FBS) came from Equitech-Bio (Kerrville, TX). LPS
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(Escherichia coli) was purchased from Sigma-Aldrich (St. Louis, Island, NY). Carrageenan,
diclofenac, histamine and serotonin were obtained from Sigma-Aldrich (St. Louis, USA).
Absolute ethanol and methanol were purchased from Hayman Limited (Loughborough, England,
UK). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from
Fluka BioChemika (Buchs, Switzerland). TLC plates (Kieselgel 60 F254) were purchased from
Merck, Damstadt, Germany.
3.2. METHODS
3.2.1 Extract Preparation
The air-dried pulverized leaves of M. lucida Benth (500 g) were cold-macerated in 5 L of 70 %
v/v ethanol-water for 72 h with periodic stirring. It was then filtered and the crude extract
obtained was concentrated under low temperature (40 ºC ± 2 ºC) and reduced pressure using the
rotary evaporator (EYELER) to remove ethanol. The residue was re-extracted with the 70%
ethanol three times to ensure exhaustive extraction. The aqueous-based concentrated crude
extract was freeze-dried and the powdered extract was labelled as hydroethanolic extract of M.
lucida (HEML) and kept until used in an air-tight desiccator, with silica gel as desiccant, in a
cool dry place. This was to prevent possible absorption of moisture, which may facilitate the
growth of molds and compromise the general stability of the extract.
3.2.2. Phytochemical Screening of HEML
Some phytochemical constituents present in HEML were ascertained with reference to methods
described previously by Trease and Evans (1989), in which 10 cluster of compounds screened
for were grouped as hydrophilic or lipophilic.
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3.2.2.1. Qualitative phytochemical screening for hydrophilic constituents
The HEML (2 g) was reconstituted in 20 ml of distilled water, sonicated to dissolve and
aliquoted for testing for the presence of hydrophilic phyto-constituents qualitatively.
Test for saponins
An aliquot (2 ml) of the reconstituted extract in water was shaken for 30 seconds to 1 minute.
The formation of a froth which persisted for about 10 minutes indicated a positive test for
saponins.
Test for reducing sugars
Equal volumes of Fehling’s solutions A and B (1 ml each) were mixed freshly and added to 2 ml
aliquot of HEML. The mixture was heated for 10 minutes in a water bath and the formation of a
brick-red precipitate was indicative of a positive test for reducing sugars.
Phenolic compounds
To 2 ml of reconstituted extract was added 2-3 drops of ferric chloride (FeCl3). A blue-black
coloration indicated the presence of phenolic compounds.
Test for cyanogenic glycosides
To an aliquot of the reconstituted extract (2 ml) was added 4 ml of chloroform. A strip of filter
paper stained in picric acid was placed at the neck of the test tube containing the mixture making
sure that it did not touch the contents of tube. The tube was plugged loosely with a clean cotton
wool to keep the picric paper firmly in place and to prevent any possible extrusion of mixture
during heating. The preparation was heated on a water bath for about an hour.  A change in color
of picric paper from yellow to brownish-red was indicative of a positive test for cyanogenic
glycosides.
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Test for alkaloids
An aliquot (5 ml) of the reconstituted extract was basified with 1 ml of 25% ammonia (NH3)
solution in a separating funnel. An equal volume of chloroform was added, shaken and left to
settle. The chloroform layer was collected into a test tube and evaporated off on water bath. The
residue was reconstituted in 5 ml of 2 M HCl and filtered. Mayer’s reagent (1 ml) was added and
the appearance of a creamy precipitate was indicative of a positive test for alkaloids.
Test for polyuronides
The presence of violet or blue precipitate upon addition of about 2-3 drops of acetone to 2 ml of
reconstituted HEML was indicative of a positive test.
3.2.2.2 Qualitative phytochemical screening for lipophilic constituents
Dry extract of HEML (10 g) was suspended in 100 ml of distilled water and sonicated for 5
minutes to dissolve. The reconstituted extract was refluxed on water bath for 2 h after adding 10
ml of 2 M HCl to extract lipids. The refluxed solution of HEML (5 ml) was shaken with 20 ml of
diethyl ether in a separating funnel and left to settle. The inorganic layer was carefully siphoned
off, leaving the organic layer (diethyl ether) which was screened for lipophilic phytochemicals.
Test for flavonoids
About 4 ml of the diethyl ether layer were evaporated on a water bath to dryness. The residue
was dehydrated using anhydrous Na2SO4, reconstituted in 8 ml methanol and divided into two
portions of 4 ml each.  Magnesium (Mg) ribbon and concentrated HCl were added to one
portion.  A reddish or pinkish coloration indicated the presence of flavonoids.
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Test for phytosterols and triterpenes
About 4 ml of the diethyl ether layer was aliquoted into a test tube and placed on a water bath to
evaporate completely the diethyl ether. Anhydrous Na2SO4 was added to absorb any moisture
present. The residue was dissolved in equal volumes of chloroform and acetic anhydride (1:1).
Drops (2-3) of concentrated H2SO4 were added to the mixture.  A green coloration indicated the
presence of phytosterols but a wine coloration indicated the presence of triterpenes.  A brownish
layer dividing the content of the test tube indicated the presence of both phytosterols and
triterpenes.
Test for anthraquinones
To 2 ml aliquot of the diethyl ether layer was added 1-2 drops of 25% NH3 solution (v/v). A red
coloration was a positive test.
3.2.2.3 Thin layer chromatography profiling of HEML
The hydroethanolic extract obtained was spotted both on a normal and reverse phase silica gel
coated on aluminum sheet and dried with a hand drier (Revlon Handheld drier, RVDR5034,
USA). The normal phase (NP) TLC plate was run with chloroform/methanol (7:1 v/v) and the
reverse phase (RP) TLC plate was run with methanol/water (2:1 v/v). Both plates were
developed with 10% (v/v) H2SO4 and heated at 110 ºC for visualization and the retention factor
(Rf) was calculated as:
Rf = Distance travelled by spot
Distance travelled by solvent (solvent front)
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3.2.3 Determination of Antioxidant Property of HEML
3.2.3.1 Quantification of total phenolic content
Total phenolic compounds or polyphenols present in HEML was measured using the Folin-
Ciocalteau (FC) colorimetric method (Pattanayak et al., 2012).  Gallic acid was used to plot a
standard curve (Appendix I) from which the total phenolic compounds present in the extract was
estimated under the same conditions. In brief, 10 µl of 5 mg/ml of HEML dissolved in distilled
water was mixed with 0.79 µl of distilled water and 50 µl of FC reagent in a 96 well plate. After
8 minutes of incubation, 150 µl of 0.09 M Na2CO3 was added and incubated for further 2 h at
room temperature. The absorbance of the mixture was read at 750 nm with a microplate reader
(Infinite m200pro TECAN, Grodig, Austria) and in triplicate. Results were expressed as
microgram of Gallic acid equivalent (GAE) per milligram of dry sample.
3.2.3.2 Quantification of total flavonoids
The total flavonoid content of the HEML was estimated by colorimetry (Pattanayak et al., 2012).
Quercetin was used to plot a standard curve (Appendix II) from which the total flavonoid content
present of the extract was estimated under the same conditions. About 100 µl HEML (5 mg/ml in
methanol) were added to 100 µl of 2% AlCl3 (w/v) in methanol and incubated for 20 minutes at
room temperature. The absorbance of the mixture was read at 415 nm using a microplate reader
and in triplicate. The total flavonoid content was expressed as microgram of quercetin present
per milligram of dry sample.
3.2.3.3 DPPH free-radical scavenging activity
The free radical scavenging activity of HEML was determined according to the method described
previously by Appiah-Opong et al. (2015). About 100 µl of 2, 2-diphenyl-1-picryl hydrazyl
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(DPPH) in methanol (0.5 mM) was added to 100 µl each of varying concentrations of HEML and
allowed to stand for 20 minutes at room temperature (25 ºC) in the dark. Absorbance was
measured at 517 nm. Butylated hydroxytoluene (BHT) (0.97-500 µg/ml) was used, under
identical conditions, as a positive control. Percentage antioxidant was determined by the
equation:
% Antioxidant = (A - B) × 100%
A
Where A optical density of blank, and B is the optical density of sample.
The concentration of HEML or positive control (BHT) at 50% antioxidant activity was
determined; effective concentration at 50% value (EC50). The experiment was carried out in
triplicate.
3.2.4 Determination of Median Lethal Dose (LD50)
The acute toxicity of HEML was explored using a standard scale described by Hodge and
Sterner, (2005). A single dose of 5000 mg/kg HEML was administered orally to female SDRs of
body weight 180-200 g (n=6). The rats were observed over 48 h period for number of deaths and
general behavior. Surviving animals were observed for additional 12 days for signs of toxicity
such as abnormalities in feeding, breathing, locomotion, lachrymation, and pilo-erection.
3.2.5 Effect of HEML on Carrageenan-induced Paw Edema
The effect of HEML was evaluated using the method described previously by Winter et al.
(1962) with slight modifications. Female SDRs of body weight 180-200 g were grouped into 5
different metallic cages with each cage containing 6 animals (n=6). The initial paw volume of the
right hind limbs of the animals was taken at baseline using the plethysmometer (7140, UGO
Basil Ltd, Camerio VA, ITALY). Sterile carrageenan, 0.1 ml 1% (w/v) in normal saline, was
injected (s.c) into the sub-planter tissue of the right hind paw of each rat. The paw volume of the
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right hind paw of the rats was taken after 30 minutes to ascertain some level of edema after
which drug/extract was administered. Paw edema was monitored 1 h post drug/extract
administration at 1 h interval for 4 h. Treatment groups received either diclofenac (100 mg/kg) as
positive control or HEML (100, 300 and 600 mg/kg) while control rats received sterilized water.
Percentage change in paw edema was calculated in both controls and treatment groups as
follows:
% Change in paw edema = (Vt- Vo) × 100
Vo
Where Vt is the paw volume at time t, V0 is the paw volume at baseline.
3.2.6. Anti-histaminic Activity of HEML
The anti-histaminic effect of HEML was assessed in a histamine-induced paw edema assay using
histamine as a phlogistic agent in a method described earlier (Singh and Pandey, 1996). Freshly
prepared histamine (1% w/v dry histamine in saline, 0.1 ml) was injected (s.c) into the sub-
planter tissue of the right hind paw of the SDRs (180-200 g, n=6) to cause edema formation.
Edema was monitored after the first 30 min and then 1 h interval for 4 h using a plethysmometer
(7140, UGO Basil ltd, Camerio VA, ITALY) after edema induction. Control animals received
distilled water (1 ml) while treated groups received chlorpheniramine (CPM, 4 mg/kg) or HEML
(100, 300 and 600 mg/kg) orally 1 h prior to induction of edema. Paw edema was calculated as
described in section 3.2.5.
3.2.7 Anti-serotogenic Activity of HEML
The effect of HEML was assessed in serotonin-induced paw edema assay, where serotonin was
used as a phlogistic agent in a method described earlier (Singh and Pandey, 1996). Freshly
prepared serotonin (1% w/v serotonin in saline, 0.1 ml) was injected (s.c) into the sub-planter
tissue of the right hind paw of each SDR (180-200 g, n=6) to induce edema. Paw edema was
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monitored post induction at first 30 min and then at 30 min intervals for 4 h using a
plethysmometer (7140, UGO Basil Ltd, Camerio VA, ITALY). Control animals received
distilled water (1 ml) while treated groups received serotonin antagonist Granisetron (0.1 mg/kg)
as positive control or HEML (100, 300 and 600 mg/kg) orally 1 h prior to inducing edema. Paw
edema was calculated as described in section 3.2.5.
3.2.8 Anti-pyretic Activity of HEML
The disruption of E. coli cell wall to expose lipopolysaccharide (LPS) was done according to
methods described previously (Rezania et al., 2011) with slight modifications. E. coli
(ATCC25922) suspension containing 1.29 x 109 cells was obtained from the Microbiology
Department of CPMR. It was centrifuged at 10,000×g for 5 minutes and the pellet was washed
twice and re-suspended in 5 ml of 0.5 M PBS (pH=7.2) containing 0.15 mM CaCl2 and 0.5 mM
MgCl2. The mixture was sonicated on ice for 15 minutes.  The anti-pyretic effect of HEML was
assessed using method described earlier (Santos and Rao, 1998) with slight modifications.
Briefly, female SDRs were fasted overnight. The rectal temperature of the animals was taken in a
temperature controlled room at 25± 2ºC prior to injection of LPS exposed from E. coli cell wall
in PBS (15 mg wet weight E. coli per ml of PBS; i.p) with a digital thermometer. The rectal
temperature of the test animals was taken after 30 min of LPS injection and animals with
differences in temperature were randomly selected into five different groups (n=6). The groups
were treated orally with or without HEML at 100, 300 and 600 mg/kg or acetaminophen at 150
mg/kg suspended in distilled water. The control group received only distilled water. The rectal
temperatures of the rats were monitored at 1 h intervals for 4 h after HEML or acetaminophen
treatment. The percentage change in temperature was calculated as:
% Change in Temperature = (T1- T0) × 100
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T0
Where T0 is the basal temperature reading and T1 is the temperature reading at each hour.
3.2.9 Cell Culture
Murine macrophage-like RAW 264 cells were obtained from the Clinical Pathology Department,
Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana, Legon. The
cells were cultured in a humidified incubator at 37°C in 5% CO2 using Dulbecco’s modified
Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-
streptomycin solution. To eliminate LPS and growth factors interferences on the true features of
macrophages, all experiments were carried out under serum-free conditions.
3.2.10 Cell Survival Assay
Cell viability was determined by MTT assay (Uto et al., 2012).  RAW 264.7 cells (2 x 104 cells/
well) were seeded in 96 well micro-titer plates and incubated for 24 h, after which cells were
starved by culturing in serum-free medium for another 2.5 h to eliminate the influence of FBS.
They were then treated with or without 50, 16.67, 5.56, 1.85 and 0.62 mg/ml of HEML or
sulforuphane (SFN) and incubated for 30 min. The cells were stimulated with or without 50
ng/ml LPS and incubated for 12 h. To each well, 20 µl of MTT solution in PBS was added and
further incubated for 4 h to generate formazan product. The formazan product was made soluble
by adding 150 µl of acidified isopropanol (0.04 N HCL-isopropanol) in 1 % v/v Triton-X and
incubated overnight in the dark. The levels of formazan product generated was determined by
measuring absorbance at 570 nm using a microplate reader (Infinite m200pro TECAN, Grodig,
Austria). The data generated was presented as a ratio of the optical density of treatment group to
that of the control group. Percent cell survival was evaluated using the formula:
% Cell survival = (A1- Ao) × 100
A2
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Where A1 = the absorbance of the test experiment (cells pretreated with HEML or SFN), AO =
the absorbance of the test control (reaction mixture with test sample but without cells) and A2 =
the absorbance of the negative control (untreated cells).
3.2.11 Measurement of Cellular Nitric Oxide (NO) Levels
The concentration of nitric oxide in culture supernatant was determined by measuring the
concentration of nitrite produced in an assay using the Griess reaction (Archer, 1993). RAW
264.7 cells (1 x 106 cells/well) were seeded in a 48-well plate and left overnight for attachment.
The cells were then starved for 2.5 h by aspirating the culture medium and replacing with serum-
free medium just to eliminate FBS influence. The cells were treated with or without HEML (50,
16.67, 5.56, 1.85 and 0.62 mg/ml) for 30 min and stimulated with 50 ng/ml of LPS and incubated
for further 12 h. The cell culture supernatant was transferred into microtubes. Equal volumes of
cell culture supernatant (100 µl) were mixed with the Griess reagent (1% w/v sulfanilamide in
5% v/v phosphoric acid and 0.1% w/v naphthylethylenediamine dihydrochloride in water) in a
96 well plate and incubated for 10 min. The absorbance was read at a wavelength of 550 nm
using (Infinite m200pro TECAN, Grodig, Austria). Concentration of nitrite generated in cell
culture supernatant was estimated by extrapolation from a standard curve (Appendix III) plotted
with the optical density of sodium nitrite at concentrations (0, 1.25, 2.5, 5, 10, 20, 40 µM).
3.2.12 Determination of PGE2 in Cell Culture Supernatant
The levels of PGE2 in cell culture supernatant was determined using PGE2 enzyme-linked
immunoassay (ELISA) kit (R&D systems, USA) and according to the manual provided by the
manufacturer. In brief, RAW 264.7 cells (1 x106 cells) were seeded in 6 cm3 petri dish overnight
and incubated for 24 h. Cells were then starved by replacing spent medium (DMEM) with a
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serum-free medium and incubated for 2.5 h, just to avoid the interferences FBS. Cell treatment
was done with or without HEML at 16.67, 5.56, 1.85 µg/ml or SFN at 40 µM (positive control)
for 30 min before stimulation with 50 ng/ml LPS for 12 h. Culture supernatant was removed and
the quantity of PGE2 released was determined by reading absorbance at 450 nm with a
microplate reader (Infinite m200pro TECAN, Grodig, Austria). The concentration of PGE2 was
extrapolated from a PGE2 standard curve (Appendix V).
3.2.13 Determination of Cytokine Levels in Culture Medium
The concentrations of pro-inflammatory (IL-1β and TNF-α) and anti-inflammatory (IL-10)
cytokines released into the cell culture medium were determined using sandwich enzyme-linked
immunoassay (ELISA) kit (R&D systems, USA) and according to the manual provided by the
manufacturer. In brief, RAW 264.7 cells (1 x106 cells) were seeded in 6 cm3 petri dish overnight
and incubated for 24 h. Cells were then starved by replacing spent medium (DMEM) with a
serum-free medium for 2.5 h, just to avoid the influence of FBS. Cells were then treated with or
without HEML at 16.67, 5.56, 1.85 µg/ml or SFN at 40 µM for 30 min before stimulated with 50
ng/ml LPS for 12 h. Culture supernatant was removed to determine the amounts of IL-1β, TNF-α
and IL-10 by reading absorbance at 450 nm with an Ultra Microplate Reader ELx 808IUI (Bio-
Tek Instruments, Inc, USA) with their concentrations estimated from their respective standard
curves (Appendices VI, VII and VIII).
3.2.14 Western Blot Analysis
Western blot analysis was performed according to methods described previously by Hou et al.
(2004). Cultured cells (RAW 264.7, 1 x106 cells) starved in serum-free medium were incubated
with or without HEML at 16.67, 5.56, 1.85 µg/ml or SFN at 40 µM for 30 min before exposure
to 50 ng/ml of LPS for 12 hrs. The cells were removed from medium, washed with phosphate
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saline buffer (PBS), harvested and lysed using a RIPA buffer. Cell lysate was centrifuged at
10,000 g for 10 min at 4 ºC and the supernatant was collected. Protein concentration in the lysate
(supernatant) was determined from a BSA standard curve (Appendix IV) and according to
method described by (Bradford, 1976). Cell lysates (supernatant) were denatured at 70 ºC for 10
minutes in SDS sample buffer and equal protein (40 µg/ml) was loaded and run using Invitrogen
NuPAGE 12 % Bis-Trisgel (SDS-PAGE). The proteins were electrophoretically transferred to a
polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, USA) to blot. The
membrane after blotting was incubated overnight at 4°C with specific primary antibody; anti-
goat iNOS or COX-2 at 1:200 dilutions. It was further incubated for an hour using a secondary
antibody anti-goat horseradish peroxidase (HRP) antibodies at 1:2,000 dilution. Substrate
solution (Immobilon Western; Millipore, USA) was added to the membrane. A camera system
EzCapture II coupled with ATTO cooled charge device (CCD) (ATTO Corporation, Japan) was
used for the detection of bound antibodies. The intensities of the bands were quantified using
Image J software. The band intensities of the individual samples were normalized to that of the
internal control (Actin bands). The relative density of the bands (cells treated HEML/SFN and
exposed to LPS) were determined with respect to the negative control (cells exposed to only
LPS).
3.2.15 Statistical Analysis
Significant variations between all treatment and untreated groups were performed using one-way
analysis of variance (ANOVA). In order to identify where significance lies, Newman-Keuls post-
test was performed just after the ANOVA. P values of ≤ 0.05 were considered significant. All
graphs and analysis were done using GraphPad prism software version 5.01.
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CHAPTER FOUR
4.0 RESULTS
4.1. EXTRACT YIELD
The aqueous-based concentrated crude extract prepared from 500 g of air-dried pulverized M.
lucida leaves yielded 46.04 g of powdered extract. This represents 9.21 % (w/w) yield.
4.2. PHYTOCHEMICAL SCREENING
Phytochemical screening of HEML showed the presence of saponins, reducing sugars,
polyphenols and flavonoids but the absence of polyuronides, cyanogenic glycosides, alkaloids,
antraquinones, triterpenes and phytosterols (Table 4.1).
Table 4.1: Some groups of phytochemicals in HEML
GROUP OF PHYTOCHEMICALS RESULT
Saponins Present
Polyuronides Absent
Cyanogenic Glycosides Absent
Reducing Sugars Present
Alkaloids Absent
Polyphenols Present
Antraquinones Absent
Flavonoids Present
Triterpenes Absent
Phytosterols Absent
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4.3 TLC PROFILING OF HEML
The TLC profile of HEML was displayed on both normal (Plate A) and reversed (Plate B) phase
silica gels coated on aluminum sheet to reveal the possible non-polar and polar constituents of
the extract (Fig. 4.1). There were 8 spots with the following Rf: 0.92, 0.87, 0.77, 0.75, 0.74, 0.66,
0.40 and 0.26 when separated on the normal phase and 7 spots with the following Rf: 0.87, 0.78,
0.71, 0.62, 0.20, 0.15 and 0.09 when separated on the reversed phase silica gels.
A B
Fig. 4.1: TLC profile of HEML: The extract was spotted on both normal phase (NP) plate A and
reversed phase (RP) plate B. The NP plate was run with chloroform/methanol (7:1) and developed with
10% v/v H2SO4 and heated at 110 ºC to display the components (8 spots). The RP plate was run with
methanol/water (2:1) and developed with 10% v/v H2SO4 and heated at 110 ºC to show the constituents (7
spots).
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4.4 TOTAL PHENOLIC AND FLAVONOID CONTENTS/ANTIOXIDANT ACTIVITY
OF HEML
4.4.1 Total Phenolic Content
The total phenolic present was estimated to be 2.23 mg GAE/100 mg of HEML from a standard
curve plotted with 0.39- 250 µg/ml of Gallic acid (Appendix I).
4.4.2 Total Flavonoid Content
The total flavonoid content was estimated to be 0.15 mg QE/100 mg of HEML from a standard
curve plotted with 0.78-50 µg/ml of Quercetin (Appendix II).
4.4.3 DPPH Free Radical Scavenging Activity
The effective concentration at 50% inhibition (EC50) of HEML to that of the standard butylated
hydroxytoluene (BHT) estimated from Figs. 4.2 A & B were 0.017 ± 0.004 and 0.962 ± 0.024
mg/ml, respectively.
EC50= 0.017± 0.004 mg/ml
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EC50= 0.962± 0.024 mg/ml
Fig. 4.2: Antioxidant activity expressed as DPPH free radical scavenging activity of (A) HEML and
(B) BHT. Results are means of triplicate determinations. The EC50 value was estimated to be 0.017±
0.004 mg/ml for BHT (A) and 0.962± 0.024 mg/ml for HEML (B).
4.5 DETERMINATION OF MEDIAN LETHAL DOSE (LD50)
A single oral dose of 5000 mg/kg HEML administered to the Sprague- Dawley rats did not cause
any mortality after 48 h and 12 days thereafter. There were no observable signs of toxicity since
experimental animals showed normal locomotion, breathing, gaiting, lachrymation and no signs
of pilo-erection. The median lethal dose of HEML was, therefore, estimated to be greater than
5000 mg/kg
4.6 CARRAGEENAN-INDUCED PAW EDEMA IN RATS
There was a 100% increase in paw volume in untreated control animals within 2 hours with a
10% dip by the 4th hour.  There was a decrease in paw volume with the different doses of HEML
in 2 hours (30-50%) with relatively no further change in paw volume in the 300 and 600 mg/kg
HEML doses by the 4th hour but a steady decline in respect of the 100 mg/kg dose at this hour
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(20%). The positive control, diclofenac, showed a trend similar to the HEML dose at 100 mg/kg
but with slightly higher reductions in paw volume (Fig. 4.3 A).  These results are depicted in the
areas under the curve (AUC) over the 4-hour period (Fig. 4.3 B), which showed diclofenac with
a 50% significant reduction (p<0.001) in paw volume followed by HEML at 100 mg/kg (40%;
p<0.01) and 300 mg/kg (35%; p<0.05). The reduction in paw volume seen with HEML at 600
mg/kg (20%) was not statistically significant (p>0.05) from the untreated control.
Fig. 4.3: Effect of HEML and diclofenac on carrageenan-induced paw edema in rats; (A) time-
course curves and (B) areas under the curve (AUC). Data are presented as means ± S.E.M. (n=6). Value
statistically significant compared with untreated control; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
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4.7 HISTAMINE-INDUCED PAW EDEMA IN RATS
Pretreatment of animals with or without HEML or Chloramphineramine (CPM) as positive
control followed by histamine challenge 1 hour later, showed an increase (62%) in paw volume
of untreated histamine-challenged control animals relative to normal controls within 30 minutes
followed by reduction in paw volume to 50% by 4 hours post-histamine challenge.  All the other
animal treatment groups showed a similar trend.  The changes seen with the HEML-treated
groups at 300 and 600 mg/kg (60% at 30 minutes to 30-35% at 4 hours) were not significantly
different from the untreated control.  However, the increase in paw volume seen with HEML at
100 mg/kg at 30 minutes after histamine challenge was similar to CPM at 4 mg/kg (42%) which
decreased to about 5% by the 4th hour, albeit to a slightly higher degree with CPM within the
period (Fig. 4.4A). These changes were reflected in the AUC in which there were significant
reductions in paw volume as seen with HEML at 100 mg/kg (65%; p<0.01) and CPM (75%;
p<0.001) relative to the untreated control.  No significant reductions in paw volume were seen
with HEML pretreatment at 300 and 600 mg/kg over the period after histamine challenge (Fig.
4.4B).
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*
Fig. 4.4: Effects of pretreatment with HEML/Chlorpheniramine (CPM) on suppression of
histamine-induced paw edema in rats with time post-histamine challenge; (A) time-course curves and
(B) areas under the curve (AUC). Data are presented as means ± S.E.M. (n=6). Value statistically
significant compared with untreated control; ** p ≤ 0.01, *** p ≤ 0.001.
4.8 SEROTONIN-INDUCED PAW EDEMA IN RATS
Pretreatment of animals with or without HEML or Granisetron (GRA) as positive control
followed by serotonin challenge 1 hour later, showed an increase (25%) in paw volume of
untreated serotonin-challenged control animals relative to normal controls within 30 minutes
followed by a gradual increase in paw volume to 55% by 4 hours post-serotonin challenge.  All
the other animal treatment groups showed a first rise in paw volume (22-35%) 30 minutes post-
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serotonin challenge, with the HEML at 600 mg/kg being 10% higher than the control, followed
by a second rise 1 or 1½ hours later.  There were gradual declines in these animal treatment
groups thereafter to 5-25% change in paw volume (Fig. 4.5A).  These results are depicted in the
AUC in which there were significant reductions in paw volume seen with HEML at 100 and 300
mg/kg (40-42%; p<0.05) and GRA (42%; p<0.05) relative to the untreated control.  No
significant reduction (p>0.05) in paw volume was observed with HEML pretreatment at 600
mg/kg over the period after serotonin challenge (Fig. 4.5B).
A Control B
Granisetron (0.1 mg/kg) *
HEML (100 mg/kg) 120
HEML (300 mg/kg)
60 HEML (600 mg/kg) 100
80
40
60
40
20 *
***
* *** 20
***
0 0
0 1 2 3 4 Control Granisetron 100 300 600
Time (h) 0.1 mg/kg HEML (mg/kg)
Fig. 4.5: Effects of pretreatment with HEML or Granisetron (GRA) on suppression of serotonin-
induced paw edema in rats with time post-serotonin challenge; (A) time-course curves and (B) area
under the curve (AUC). Data are presented as means ± S.E.M. (n=6). Value statistically significant
compared with untreated control; ** p ≤ 0.01, *** p ≤ 0.001.
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Anti-inflammatory activity
(% change in paw volume)
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4.9 LPS-INDUCED FEVER IN RATS
Injection of LPS from E. coli (i.p) caused a rise in rectal temperature of normal control animals
by 30% within 1 hour followed by a decline (18%) by the 4th hour.  That of the 300 and 600
mg/kg HEML groups followed a similar trend with 20-25% increases in rectal temperature at 1
hour followed by declines to 5.9-8.2% by the 4th hour. The 100 mg/kg HEML and
acetaminophen groups showed a peak increase of 15% at 30 minutes followed by a decline to
5.9-7.7% by the 4th hour (Fig. 4.6A). These results are depicted in the AUC in which there were
significant (p<0.05-0.01) reductions in rectal temperatures with respect to acetaminophen and the
100 and 300 mg/kg HEML (38-52%) relative to normal control with insignificant reductions in
case of the 600 mg/kg HEML group (Fig. 4.6B).
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Fig. 4.6 Effect of HEML on LPS-induced fever in rats; (A) time-course curves and (B) areas under the
curve (AUC) in arbitrary units. Data are presented as means ± S.E.M. (n=6). Value statistically significant
compared with untreated control; * p ≤ 0.05, ** p ≤ 0.01. Aceta = acetaminophen (150 mg/kg).
4.10 CELL VIABILITY OF HEML ON LPS-ACTIVATED RAW 264.7 MACROPHAGES
In order to indicate that the concentration of HEML used were not toxic to the cells, a cell
viability assay was carried out using RAW 264 cells in the presence of HEML in an MTT assay.
There was no significant difference (p > 0.05) between cells treated with LPS alone and cells
treated with HEML at concentrations of 0.62, 1.85, 5.56 and 16.67 μg/ml as shown (Fig. 4.7).
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Fig. 4.7: Effect of HEML on cell viability of LPS-activated RAW 264 cell. Data was presented as
mean ± SEM of n=3. Data generated was presented as a ratio of the optical density of treatment group to
that of the control group.
4.11 PGE2 RELEASE AND COX-2 EXPRESSION
The HEML, at 16.65 µg/ml, markedly suppressed (33.4%) LPS-induced release of PGE2 into the
culture supernatant (Fig. 4.8A), but did not inhibit the expression of COX-2 (Fig.4.8B). On the
other hand, HEML at 1.85 and 5.55 µg/ml neither affected the levels of PGE2 nor inhibited COX-
2 expression. The positive control sulforaphane (SFN), on the other hand, almost completely
suppressed PGE2 release (98%) and strongly inhibited COX-2 expression (84%).
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LPS (50 ng/ml) - + + + + +
HEML (µg/ml) - - 1.85 5.55 16.65 -
SFN (µM) - - - - - 40
COX-2 (72 kDa)
Actin (42 kDa)
120
* 100
80
60
40
20
***
*** 0
LPS (50 ng/ml)   -    +         +        +          +        +
HEML ( g/ml)    -         -       1.85    5.55  16.65     -
SFN ( M)            -        -           -         -         -         40
Fig. 4.8: Effect of HEML or sulforaphane (SFN) on PGE2 release (A) and COX-2 expression (B) in
LPS-induced RAW 264.7 macrophages. Cells were treated with varying concentrations of HEML (1.85-
16.65 μg/ml) and fixed concentration of SFN (40 μg/ml) and incubated in the presence of LPS (50 ng/ml).
Blank (without LPS and HEML/SFN) and control (with LPS but without HEML/SFN) were also
investigated. The relative density of COX-2 band intensity (B) was quantified using image J software and
normalized in the controls. Data is presented as the mean ± S.E.M (n=3). ** p ≤ 0.01, *** p ≤ 0.001
4.12 NITRIC OXIDE (NO) PRODUCTION AND iNOS EXPRESSION
HEML caused a non-concentration dependent reduction (16-36%) in NO production in culture
medium compared to 84% by SFN (Fig. 4.9A).  Expression of iNOS in the RAW 264.7 cells
(Fig. 4.9 B) was increased 100% after LPS exposure but was inhibited in cells pretreated with
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HEML at 5.55 µg/ml (40%) and 16.65 µg/ml (70%) or 40 µM SFN (100%). HEML at 1.85
µg/ml, on the other hand, showed a 20% increase in iNOS expression.
LPS (50 ng/ml) - + + + + +
HEML (µg/ml) - - 1.85 5.55 16.65 -
SFN (µM) - - - - - 40
iNOS (130 kDa)
Actin (42 kDa)
*
*
**
Fig. 4.9: Effect of HEML or sulforaphane (SFN) on NO production (A) and iNOS expression (B) in
LPS-induced RAW 264.7 macrophages. Cells were pretreated with varying concentrations of HEML
(1.85-16.65 μg/ml) and fixed concentration of SFN (40 μg/ml) and incubated in the presence of LPS (50
ng/ml). Blank (without LPS and HEML/SFN) and control (with LPS but without HEML/SFN) were also
investigated. The relative density of iNOS band intensity (B) was quantified using image J software and
normalized in the controls. Data is presented as the mean ± S.E.M. (n=3). ** p ≤ 0.01, *** p ≤ 0.001
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4.13 LPS-INDUCED CYTOKINE EXPRESSION
The cells treated with LPS alone without HEML/SFN expressed 2 to 4.8-fold increase in IL-β
and TNF-α levels but a 60% reduction in IL-10 levels compared to unstimulated cells (cells
without LPS). HEML at the three concentrations used reduced IL-1β by 19-33% (Fig 4.10 A).
TNF-α was markedly reduced by about 87% at HEML concentrations of 5.55 and 16.65 μg/ml
but was increased by 15% at 1.85 μg/ml of HEML (Fig. 4.10 B).  The standard drug SFN
markedly reduced IL-β and TNF-α by 100% and 95%, respectively. HEML caused about 7 to
10-fold increase in IL-10 levels whilst SFN caused a 10-fold increase (Fig. 4.10 C). The changes
caused by HEML in all the cytokines measured were not concentration-dependent.
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Fig. 4.10: Effect of HEML or SFN on cytokine levels of LPS-activated RAW 264 cell; IL-1β (A),
TNF-α (B) and IL-10 (C) levels. Data are presented as means duplicate determinations.
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CHAPTER FIVE
5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 DISCUSSION
The induction of paw edema by carrageenan is reported to show a biphasic pattern of
inflammation (Vinegar et al., 1969), such that mediators released at the first phase include
histamine and serotonin and then proceeds to a second phase with the release of other mediators
such as NO and prostaglandins, especially the E-series and other products of the enzyme COXs.
The kallikrein-kinin system is relevant to the cascade and ensures the synthesis and release of
mediators (e.g. bradykinin) whose action tends to bridge these two phases (Silva et al., 2005;
Perianayagam et al., 2006). Inflammatory responses represent a principal mechanism by which
the body defends itself. This mechanism is basically controlled by the mediators NO, PGE2 and
various cytokines like that of IL-1β, IL-6, TNF-α (Dewanjee et al., 2013). For this reason,
inflammation could bypass the acute phase into the chronic state when certain levels of
mediators of the late phase of acute inflammation are sustained. A promising way to remedy the
incidence is obviously the inhibition of these mediators occurring both at the early and to some
extent late phases of inflammation either by influencing their pharmacological activities or by
interfering with their synthetic or metabolic pathways.
The current study was to assess the anti-inflammatory activity of M. lucida (HEML) and propose
its possible mechanisms of action. The LD50 of HEML has been estimated to be greater than
5000 mg/kg since it did not cause any death in the SDRs during the acute toxicity test. Hence the
doses of 100, 300 and 600 mg/kg were chosen for the study. Using a classical acute model of
inflammation, the anti-edematous effect of HEML was demonstrated in female SDRs, and its
ability to reduce the edema formation during the injury time confirmed its anti-inflammatory
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effect. It was established that HEML inhibited paw edema caused by carrageenan in an inverse
dose-dependent manner (Fig. 4.3). Thus, as the dose of HEML increased from 100 mg/kg to 600
mg/kg, anti-inflammatory activity was found to decrease (Fig. 4.3B). The significant suppression
of paw edema before and after the second hour of HEML administration in the rats indicates that,
HEML may act on both the initial and late phases of acute inflammation. HEML also reduced
significantly paw edema caused by histamine and serotonin (Figs. 4.4 and 4.5), which goes to
support its effect on the initial phase of acute inflammation in the rats.
During infection or a challenge with an inflammatory stimulus, especially at the periphery of the
body, various responses tend to be generated within the central nervous system (CNS). Other
than localized, inflammation may be systemic where the innate immune cells respond by
elevating levels of prostaglandins, cytokines, NO and complement factors in circulation, which
communicate with the brain through multiple humoral and neural routs to cause neuro-immune
activation leading to fever (Blatteis, 2007).
Fever is an adaptive defense mechanism in response to inflammation or infection. The increase
in body temperature is thought to prevent propagation of infectious microorganisms and activates
the host's immune system (Kluger et al., 1998). In mammals, body temperature is regulated by
controlling heat production to adjust to a set temperature by the hypothalamus around 37 °C. The
‘resetting’ of the set temperature above the normal threshold leads to excessive generation of
heat and prevention of heat loss, leading to increased muscle tone, shivering and peripheral
vasoconstrictions until the body temperature reaches the new set point (Cabanac, 2006). The
process is mediated by the elevation of pyretic cytokines such as TNF-α, IL-1β, and IL-6 in
circulation and consequent stimulation of PGE2 production which is considered as important
mediator to the triggering of fever (Netea et al., 2000).
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Some prominent late phase mediators produced in the carrageenan-induced paw edema include
PGE2, NO and pro-inflammatory cytokines. If HEML affects both phases (Fig 4.3), then there is
the possibility that HEML suppressed inflammation at the late phase by affecting levels of one or
combinations of or all the late phase mediators (PGE2, NO and pro-inflammatory cytokines).
LPS trigger systemic inflammation and may lead to the release of PGE2, pro-inflammatory
cytokines and NO, which results in fever or hyperthermia (Kluger, 1991). The significant
suppression of rectal temperature in rats treated with different doses of HEML as shown in the
LPS-induced fever assay (Fig. 4.6), indicates that HEML inhibited the late phase inflammatory
mediators.
To examine which specific late phase mediator was affected, in vitro assays were conducted
using LPS-induced RAW 264.7 cells. At the concentrations of HEML used in these studies no
cytotoxic effects were observed (Fig. 4.7). HEML significantly reduced PGE2 levels in LPS
activated RAW 264.7 cells without affecting COX-2 expression (Fig. 4.8). This suggests that,
HEML does not affect the de novo synthesis of COX-2 but rather interferes with its catalytic
activity, which may explain why PGE2 levels reduced in culture supernatant of cells treated with
HEML. COX-2 is inducible and its over-expression has been associated with inflammation and
cancer (Maier et al., 1990) due to increased PGE2 production. Inhibiting PGE2 overexpression
may provide a promising remedy to solving such pathophysiological conditions (Masferrer et al.,
1994; Oshima et al., 1996). Due to the standard limitations of the conventional NSAIDs, newer
COX-2 specific inhibitors are being sought after (Vane, 1994), while available ones (e.g.
Celecoxib and Rofecoxib) have still not lived up to the claim that they are devoid of adverse
effects (Celotti and Laufer, 2001).
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Data gathered from clinical studies indicates that, some COX-2 specific inhibitors accelerate
vascular events leading to cardiovascular related problems such as heart attack or non-fatal
myocardial infarction, stroke and even death; hence the withdrawal of these drugs from the
market (Kearney et al., 2006; Antman et al., 2007). The control of PGE2 levels in a biological
system, without affecting homeostasis provides a key concept in dealing with inflammation,
where the available conventional NSAIDs and COX-2 specific inhibitors happen to fail. This
may account for some of these adverse side effects associated with their treatment.  As a result,
the reduction in PGE2 levels by HEML without affecting COX-2 expression may denote an
effective approach of curbing the numerous problems associated not only with treatment of
inflammation by conventional NSAIDs but also COX-2 specific inhibitors. However, more data
is needed to establish this claim.
Nitric oxide is another potent inflammatory mediator and excess generation is due to higher
expressions of iNOS, which promote tumour formation by transforming cells with normal
growth pattern into malignant tumour (Ohshima and Bartsch, 1994). As a result, the interference
of iNOS activity or its complete inhibition may be a reasonable approach to controlling
inflammation. In the current study, HEML was shown to lower NO levels in culture supernatant
by downregulating the expression of iNOS (Fig.4.9) suggesting that it may suppress the de novo
synthesis of iNOS.
Interestingly, some group of researchers (Mollace et al., 2005) have indicated a cross-
relationship between iNOS and COX-2. They explained that, during stimuli, inflammatory cells
such as macrophages generate ROS and increase iNOS expression, which consequently increases
NO levels. The NO produced combines with superoxide anion (a specific form of ROS) to form
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peroxy-nitrite (ONOO-). Although ONOO- generated has important role in parasitic and bacterial
infection, it also has the ability to activate the NF-κB pathway which leads to the activation and
transcription of COX-2 and iNOS genes. On the other hand, when the NF-κB pathway was
studied at lower levels of endogenous NO concentration in cells, the pathway was kept inactive
(Togashi et al., 1997).  Thus, HEML by its ability to reduce NO levels and as a result of its
oxyradical scavenging activity (Fig. 4.2) may significantly reduce ONOO, which in turn may
prevent the co-activation of the inducible COX-2 and thus reduce inflammation. The antioxidant
activity of HEML may be due to its phenolic content, particularly flavonoids (Table 4.1; Fig.
4.2). By means of extrapolations, HEML may have the ability to stabilize superoxide anion and
other free radicals including NO generated by the immune cells, thereby controlling
inflammation to some extent. It is, however, possible that some of the phyto-constituents present
in HEML antagonize the actions of other components responsible for its anti-inflammatory effect
at higher concentrations.  This may explain why HEML at 100 mg/kg showed significant anti-
inflammatory activity but did not show such significant activity at 300 and 600 mg/kg (Fig. 4.3).
This observation could be resolved with the isolation of the active component(s) of HEML
responsible for its anti-inflammatory activity.
As part of the immuno-regulatory process, inflammatory response proceeds with increasing
levels of circulating cytokines, where pro-inflammatory cytokines predominate initially, but their
levels are later regulated by increasing anti-inflammatory cytokines (Zhang and An, 2007).
Based on this, a good anti-inflammatory agent should have the ability of suppressing pro-
inflammatory cytokines but proportionally elevate the levels of anti-inflammatory cytokines, just
to maintain a balance. HEML reduced the levels of IL-1β and TNF-α (pro-inflammatory
cytokines) whilst boosting IL-10 (anti-inflammatory cytokine) levels markedly (Fig.4.10). This
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is consistent with the inferences from the initial animal studies which showed that HEML
resolved acute inflammation.
5.2 CONCLUSIONS
The hydroethanolic extract of M. lucida (HEML) contains groups of phytochemicals such as
saponins, phenolics, flavonoids and reducing sugars. HEML has antioxidant activity, which may
be due to the presence of phenolic compounds particularly flavonoids. It showed anti-
inflammatory activity by affecting both early and late phases of acute inflammation. The
potential mechanism of action of HEML as an anti-inflammatory agent may be through (1) the
down regulation of iNOS expression leading to reduction in NO formation, (2) the reduction in
PGE2 production without directly affecting COX-2 expression, but probably through inhibition
of its catalytic activity, (3) elevation of anti-inflammatory cytokine (IL-10) levels and reduction
in pro-inflammatory cytokine (IL-1β and TNF-α) levels and (4) mopping up of ROS. These
findings provide scientific evidence for the anecdotal use of M. lucida in the treatment of
inflammation.
5.3 RECOMMENDATIONS
Further data may be needed to explain and improve on the knowledge acquired so far. It is
therefore recommended that;
 A dose response assay be carried out to determine the effective dose at 50 (EC50) which
will help to establish the therapeutic dose of HEML.
 The effect of HEML on the promotor region of COX-2 gene and COX-2 enzyme activity
should be examined in order to explain why PGE2 levels reduced without affecting COX-
2 protein expression.
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 Detailed signaling mechanisms should be conducted to investigate the effect of HEML on
iNOS transcription and protein expression.
 The toxicity profile of HEML be established pre-clinically using both acute and chronic
modules.
 The active component responsible for the anti-inflammatory effect should be isolated and
its structure elucidated.
 The effect of HEML on the inherent COX-1 expression in gastro-intestinal and renal cells
especially should be assessed. This will provide scientific evidence to indicate that
treatment of inflammation with the extract is not accompanied with side effects like that
of conventional NSAIDs.
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APPENDICES
Appendix I: Standard Curve for Gallic Acid
0.14
y = 0.0005x + 0.0017
0.12 R² = 0.9969
0.1
0.08
0.06
0.04
0.02
0
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0
)
Appendix II: Standard Curve for Quercetin
2.0000
1.6000 y = 0.0316x + 0.0151
R² = 0.9975
1.2000
0.8000
0.4000
0.0000
0 20 40 60
Quercetin Concentration (ug/ml)
80
Absorbance at 415 (nm)
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Appendix III: Standard Curve for Sodium Nitrite
2.5
y = 0.0225x + 0.0104
2 R² = 0.9998
1.5
1
0.5
0
0 20 40 60 80 100 120
Soduim Nitrate Conc (uM)
Appendix IV: Standard Curve for Bovine Serum Albumin (BSA)
0.5
y = 0.4402x + 0.0204
R² = 0.9716
0.4
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2
BSA (mg/ml)
81
Absorbance at 550 (nm)
Absorbance at 590 (nm)
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Appendix V: Standard Curve for PGE2
1.2
1
y = -0.3911x + 1.6475
R² = 0.9082
0.8
0.6
0.4
0.2
0
0 1 2 3 4
log conc. (pg/ml)
Appendix VI: Standard Curve for IL-1Β
IL-1β (pg/ml)
l)
82
OD at 450 (nm)
absorbances at 450/540 (nm)
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Appendix VII: Standard Curve for TNF-α
TNF-α (pg/ml)
Appendix VIII: Standard Curve for IL-10
IL-10 (pg/ml)
83
OD at 450 ((nnmm)) OD at 450 (nm)
ml)