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CHAPTER ONE

1.0

INTRODUCTION

Various herbal medicines derived from plant extracts are being used in the
treatment of a wide variety of clinical diseases, though relatively little knowledge
about their mechanism of actions are known. Many herbal preparations are being
prescribed widely for the treatment of inflammatory conditions and the
accompanied pain. There is a need for research and developmental work in herbal
medicine because apart from the social and economic benefits, it has become a
persistent aspect of present day healthcare in developing countries (Sofowora,
1982).
Alstonia boonei is a large deciduous evergreen tree, usually up to 45m tall and
1.2m in diameter, belonging to the family Apocynaceae consisting of about 40-60
species. A. boonei, also known as Awun among the Yorubas of southwestern
Nigeria, Egbu-ora among the Igbos of southeastern Nigeria, Ukpukunu among the
Urhobos of south central Nigeria and Ukhu in Edo, is a native of tropical and
subtropical Africa, Southeast Asia, Central America and Australia. Alstonia is
named after Dr C. Alston (1685-1760), a professor of Botany at Edinburgh
University. It is also called Devil tree in tropical and subtropical Africa, Central
America and Australia. A. boonei is a medicinal plant that is widely used across
Africa for various ailments. It has been reported to be used for the treatment of

malaria, intestinal helminthes, rheumatism, muscular pain, insomnia, and


hypertension. It has also been reported to contain phytochemicals such as saponin,
alkaloids, tannins, and steroids (Taiwo et al., 1998; Osadebe, 2002). In folk
medical practice, the stem bark of Alstonia boonei has been reported to possess
anti-inflammatory, analgesic and antipyretic activities (Olajide et al., 2000). The
stem bark extract is also used in treating painful micturition and rheumatic
conditions (Ojewole, 1984; Asuzu and Anaga, 1991). An infusion of the stem bark
and root is taken as a remedy for asthma. A liquid made from the stem bark and
leaves is drunk to treat impotence. In Ghana, it is given to assuage tooth ache and
after child delivery, to aid in expelling the placenta. In Cote d Ivoire and Burkina
Faso, it is applied topically to reduce edema and to clear suppurant sores and
exposed fractures. In Nigeria, it is used for ulcers and in Cameroon and Liberia as
remedy for snake bite and arrow poison. Anti-malaria activity of various fractions
of the stem bark extract of Alstonia boonei was reported by Bello et al., (2009);
Iyiola, et al., (2011), while Odugbemi and Akinsulire (2007), Idowu et al., (2010)
and Gbadamosi et al., (2011) confirmed indigenous medicinal usefulness of A.
boonei for malaria therapeutic usage in southwestern part of Nigeria.

Fig. 1: Pictorial view of A. boonei leaves (Sidiyasa, 1998).

Fig 2: A. boonei bole (Palla, 2005).


Inflammation is a local response of living mammalian tissues to injury. It is a body
defense reaction in order to eliminate or limit the spread of injurious agents. There
are various components to an inflammatory reaction injury. Oedema formation,
3

leukocyte infiltration and granuloma formation represent such components of


inflammation (Mitchell and Cotron, 2010). Oedema formation in the paw is the
result of a synergism between various inflammatory mediators (prostaglandins,
thromboxane and others) that increases vascular permeability and blood flow
(Lalenti et al., 1995).
Lipids (commonly known as fats) are heterogeneous group of organic substances
which are soluble in a group of organic solvents. The major lipids in human plasma
include cholesterol, triglycerides, phospholipids and non-esterified fatty acids
(NEFA). Serum lipid profile is a test that serves as an initial broad medical
screening tool for abnormalities in lipids such as cholesterol and triglycerides and
the result is used to identify certain genetic diseases, cardiovascular diseases and
rarely pancreatitis. However, cardiovascular diseases present some of the main
problems across the globe today, the major ones being coronary heart diseases,
stroke and hypertension (Alters and Shiff, 1997). Elevated plasma lipids are risk
factors in cardiovascular problems. Hyperlipidaemia and other abnormal blood
lipid profile are largely of genetic origin or due to unwholesome nutritional habits.
Lipids and other substances accumulate on arterial wall, forming plague, which
occlude the vascular lumen and obstruct the blood flow to vital organs such as the
heart, brain, liver, or kidney. Obstruction of blood supply to the heart, brain, liver
or kidney cause coronary heart diseases, stroke or kidney failure, as the case may
4

be. The important lipids whose elevations are implicated in these disease
conditions are cholesterol and triglycerides. Lipids are transported as lipid-protein
complexes called lipoproteins, which are classified based on their density and
charges. The high-density lipoprotein cholesterol (HDL-c) transports lipids out of
blood cells to the liver, while the low-density lipoprotein cholesterol (LDL-c)
mobilizes lipids against the cells and blood vessels. Triglycerides have been found
to be elevated along with total cholesterol elevation. Therefore, elevated lowdensity cholesterol, triglycerides, and total cholesterol with reduced HDL-c will
enhance the development of atherosclerosis and related cerebrovascular disorders
(Nwanjo, 2004). The clinical consequences of these disease conditions are serious;
and meaningful research efforts to improve the knowledge and understanding of
the pathogenesis is essential; in order to provide a more rational approach to their
prophylaxis and treatment. (Kritchersky, 1970; Kucera et al., 1972)

1.1

AIM OF STUDY

To evaluate the possible anti-inflammatory effects of aqueous stem bark extract of


A. boonei.

1.2 OBJECTIVES OF STUDY


To evaluate the serum lipid profile in Wistar rats.
To evaluate the relationship or association of formalin-induced inflammation
with lipid profile in Wistar rats.
To evaluate the possible anti-inflammatory properties of aqueous stem bark
extract of A. boonei.
To compare anti-inflammatory effect of aqueous stem bark extract of A.
boonei with anti-inflammatory drugs such as indomethacin.

1.3

JUSTIFICATION OF STUDY

Inflammation is generally defined as the response of living tissues to an injurious


stimulus and is recognized as a primary defense mechanism. The magnitude of the
inflammatory response is important because insufficient responses result in
immunodeficiency, while excessive responses are associated with diseases such as
rheumatoid arthritis, Crohns disease, atherosclerosis, diabetes, Alzheimers
disease, multiple sclerosis, and cerebral and myocardial ischaemia. These
inflammatory conditions are often accompanied by aches and pains which all lead
to morbidity and mortality. Certain drugs or chemical compounds are known to
control inflammation. Some plants have also been reported to possess antiinflammatory activity and these include A. boonei, Asparagus africanus, Sida
acuta, Sambulus ebulus rhizome, Vitis trifolia, and Caralluma tuberculata to
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mention but a few. Hence this study is designed to evaluate the anti-inflammatory
properties of aqueous stem bark extract of A. boonei.

1.4

SCOPE OF STUDY

Induction of chronic inflammation in Wistar rats, collection of samples from the


animals, analysis of serum for lipid profile (Triglycerides, Total cholesterol, high
density lipoprotein, low density lipoprotein).

CHAPTER TWO
2.0

LITERATURE REVIEW

A. boonei is a large deciduous evergreen tree, usually up to 45m tall and 1.2m in
diameter, belonging to the family Apocynaceae consisting of about 40-60 species.
It is a native of tropical and subtropical Africa, Southeast Asia, Central America
and Australia. Alstonia is named after Dr C. Alston (1685-1760), a Professor of
Botany at Edinburgh University. It is also called devil tree in tropical and subtropical Africa, Central America and Australia. It has been reported to be used for
treatment of malaria, intestinal helminthes, rheumatism, muscular pain, insomnia,
and hypertension. It has been reported to contain phytochemicals such as saponin,
alkaloids, tannins and steroids (Taiwo et. al., 1998; Osadebe, 2002). In folk
medical practice, an infusion of the extract of stem bark serves as anti-snake
venom and as antidote to some arrows poisons. Antimalarial activity of various
fractions of the stem bark extract of Alstonia boonei was reported by Bello et al.,
(2009); Iyiola, et al., (2011) while Odugbemi and Akinsulire (2007), Idowu et al.,
(2010) and Gbadamosi et al., (2011) confirmed indigenous medicinal usefulness of
Alstonia boonei for malaria therapeutic usage in Southwestern part of Nigeria.
Oigiangbe et al., (2010) reported insecticidal properties of an alkaloid from
Alstonia boonei De Wild, while Olajide et al., 2000, established anti-inflammatory,
antipyretic and analgesic properties of A. boonei stem bark extract. However,
potential nephrotoxicological effect, especially at high dose, was reported in
Guinea pigs ( Oze et al., 2007). The stem bark of the plant has anxiolytic properties
8

in mice (Elizabetsky and Costa-Campos, 2006) and also to contain important


minerals like calcium, phosphorous, iron, sodium, potassium and magnesium in
addition to alkaloids, tannins, saponins, flavonoids, cardiac glycoside and vitamin
C. More so, Elijah et al., 2010, reported possible and promising beneficial effect of
A. boonei for preserving palm wine.

2.1

CLASSIFICATION OF A. boonei

Alstonia comprises about 40 species and has a pan-tropical distribution. There are
about twelve species of the genus Alstonia. A. boonei De Wild belongs to the
family Apocynaceae. The species are scattered all over the world of which two are
indigenous to Africa. The plant is known locally in Ghana as Onyame dua, Osennuru, or Sinduro in Twi, Onyame dua in Fante, Sinu or Adawura in Ga-Adangbe,
Bakunin, Nyamenlebaka, Emenle, or Emie in Nzema, and Siaketekre, Nyemi dua,
or Asi atoe in Ewe (Ayiku, 1992). Elsewhere, Alstonia is known as Australian fever
bush, Australian quinine, Devil tree, Dita bark, fever bark, or palimara (Gosse,
1999).
2.2

CULTIVATION OF A. boonei

Alstonia grows into a giant tree in most of the evergreen rain forests of tropical
West Africa. The plant thrives very well in damp riverbanks. It is well known to all
the traditional healers practicing along the west coast of Africa. It occurs in
9

deciduous and fringing forest of Ghana (Gosse, 1999). Alstonia boonei De Wild is
a deciduous tree up to 35 meters high. It buttresses deep-fluted high and narrow. Its
white latexes are copious. The leaves are in whorls at nodes, oblanceolate, apex
rounded to acuminate, lateral vein prominent almost at right angle to midrib. The
flowers are white with lax terminal cymes. The fruits are paired with slender
follicle up to 16cm long with brown floss at each end.

2.3

ETHNOBOTANICAL USES OF A. boonei

The bark of Alstonia tree is one of the effective analgesic (Abbiw, 1990) herbs
available in nature. All the parts of the plant are very useful but the thick bark cut
from the matured tree is the part that is most commonly used for therapeutic
purposes. The bark of the tree is highly effective when it is used in its fresh form;
however, the dried one could equally be used. Therapeutically, the bark has been
found to possess anti-rheumatic (Abbiw, 1990), anti-inflammatory (Abbiw, 1990),
analgesic/pain-killing, anti-malaria/antipyretic, anti-diabetic (mild hypoglycaemic),
anti-helmintic, antimicrobial and antibiotic properties (Hadi et al., 2001, fakae,
2000, Kam et al., 1997). A decoction could be sweetened with pure honey and be
taken up to 4 times daily as an effective painkiller for the following conditions.
Painful menstruation (dysmenorrhoea), when associated with uterine fibroid or
ovarian cysts in women; lower abdominal and pelvic congestion associated with
10

gynaecological problems such as pelvic inflammatory diseases; to relieve the


painful urethritis common with gonococcus or other microbial infections in men.
Alstonia decoction also exerts a mild antibacterial effect in this case, relieving the
aches and pains associated with malaria fever. Alstonia is taken in the form of
preparations that exhibits anti-pyrexia and anti-malaria effects, to combat
rheumatic and arthritic pains. The decoction of Alstonia bark could be taken alone
as an effective pain-killing agent. A cold infusion made from the fresh or dried
bark of Alstonia taken orally two to three times daily exerts a mild hypoglycaemic
effect on diabetic patients. The cold infusion is also administered orally for the
purpose of expelling round worms, threadworms (Abbiw, 1990), and other
intestinal parasites in children.
The fresh bark of Alstonia could be used in preparing herbal tinctures; it is
particularly useful as an effective antidote against snake, rat, or scorpion poison. It
is also useful in expelling retained products of conception and afterbirth when
given to women. Asthma can be treated with a drink prepared from parts of Trema
orientalis and decoction of the bark of Alstonia boonei mixed with the roots and
bark of cola and fruits of Xylopia parviflora with hard potash (Abbiw, 1990). The
bark decoction of Alstonia boonei is used with other preparations in the treatment
of fractures or dislocation (Abbiw, 1990), jaundice, and for inducing breast milk.
Its latex is taken as a purgative. The hardened latex is used for the treatment of
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yaws. Alstonia boonei De Wild is regarded as one of few herbs with potential antiHIV indicators. In some African countries A. boonei is considered a sacred tree and
worshiped in the forest and hence human beings in those countries do not eat its
parts.

2.4

ANALGESIC,

ANTI-INFLAMMATORY

AND

ANTI-PYRETIC

PROPERTIES OF A. boonei
The stem bark of A. boonei tree is one of the effective analgesic herbs available in
nature (Abbiw, 1990). A. boonei, apart from being listed in the African
Pharmacopeia as an anti-malarial drug, its stem bark has analgesic, antipyretic and
anti-inflammatory properties (Olajide et al., 2000). They also reported that
methanol extract caused a significant inhibition of the carrageenan-induced paw
oedema, cotton pellet granuloma, and exhibited an anti-arthritic activity in rats.
Vascular permeability induced by acetic acid in the peritoneum of mice was also
inhibited. The extract also produced marked analgesic activity by reduction of
writhing induced by acetic acid, as well as the early and late phases of paw licking
in mice. A significant reduction in hyperpyrexia in mice was also produced by the
extract. This study has established the anti-inflammatory, analgesic and antipyretic
activities of the stem bark of A. boonei.

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2.5

HYPOLIPIDAEMIC AND ANTI-ATHEROGENIC PROPERTIES OF

A. boonei
It has been observed that the aqueous ethanolic extract of A. boonei stem bark
produced hypocholesterolaemic and anti-atherogenic effects when used at low
doses (Oze et al., 2008). This may imply that low doses of the plant extract could
be used for short periods to manage hyperlipidaemic and atherogenic conditions.
These findings agree with the current use of the plant extract by folk medicine
practitioners as antihypertensive agent (Oze et al., 2008).
This study investigated the effect of 150, 300 and 600mg/kg extract of A. boonei
stem bark (ASBE) on serum lipids for 5 days in Wistar rats. The ASBE lowered
serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C)
significantly in animals orally administered with 150 and 300mg/kg of the extract
for 5 days. The ratio of the atherogenic risk predictor indices showed that 150, 300
and 600mg/kg extract may possess anti-atherogenic effect and therefore desirable.
These results suggest possible hypolipidaemic effect of ASBE at short course of
treatment (Oze et al., 2008).
2.6

INFLAMMATION

Based on visual observation, the ancients characterized inflammation by five


cardinal signs, namely redness (rubor), swelling (tumour), heat (calor; only
applicable to the body extremities), pain (dolor) and loss of function (function
13

laesa). The first four of these signs were named by Celsus in ancient Rome (3038
B.C.) and the last by Galen (A.D 130200). More recently, inflammation was
described as "the succession of changes which occurs in a living tissue when it is
injured provided that the injury is not of such a degree as to at once destroy its
structure and vitality", or "the reaction to injury of the living micro-circulation and
related tissues. Although, in ancient times inflammation was recognized as being
part of the healing process, up to the end of the 19th century, inflammation was
viewed as being an undesirable response that was harmful to the host (Spector,
1963).
However, beginning with the work of Metchnikoff and others in the 19 th century,
the contribution of inflammation to the body's defensive and healing process was
recognized. Furthermore, inflammation is considered the cornerstone of pathology
in that the changes observed are indicative of injury and disease (Spector, 1963).
The classical description of inflammation accounts for the visual changes seen.
Thus, the sensation of heat is caused by the increased movement of blood through
dilated vessels into the environmentally cooled extremities, also resulting on the
increased redness (due to the additional number of erythrocytes passing through
the area). The swelling (edema) is the result of increased passage of fluid from
dilated and permeable blood vessels into the surrounding tissues, infiltration of
cells into the damaged area, and in prolonged inflammatory responses deposition
14

of connective tissue. Pain is due to the direct effects of mediators, either from
initial damage or that resulting from the inflammatory response itself, and the
stretching of sensory nerves due to edema. The loss of function refers to either
simple loss of mobility in a joint, due to the edema and pain, or to the replacement
of functional cells with scar tissue. Today it is recognized that inflammation is far
more complex than might first appear from the simple description given above and
is a major response of the immune system to tissue damage and infection, although
not all infection gives rise to inflammation. Inflammation is also diverse, ranging
from the acute inflammation associated with Staphylococcus aureus infection of
the skin (the humble boil), through to chronic inflammatory processes resulting in
remodeling of the artery wall in atherosclerosis; the bronchial wall in asthma and
chronic bronchitis, and the debilitating destruction of the joints associated with
rheumatoid arthritis. These processes involve the major cells of the immune
system, including neutrophils, basophils, mast cells, T-cells, B-cells, (Gilroy,
2004). However, examination of a range of inflammatory lesions demonstrates the
presence of specific leukocytes in any given lesion. That is, the inflammatory
process is regulated in such a way as to ensure the appropriate leukocytes are
recruited. These events are controlled by a host of extracellular mediators and
regulators, including cytokines, growth factors, eicosanoid (prostaglandins),
complement and peptides. In fact, it is the discovery of many of these mediators
15

over the past 20 years that has increased our understanding of the regulation of the
inflammatory process while, at the same time, revealing its complexity. These
extracellular events are matched by equally complex intracellular signaling control
mechanisms, with the ability of cells to assemble and disassemble an almost
bewildering array of signaling pathways as they move from inactive to dedicated
roles within the inflammatory response and site. Which cells and mediators come
into play depends on wide range of factors. These include: what stage the process
of inflammation is at; the initiating event, that is, type of pathogen, auto-immune,
chemical or physical injury; the tissue or organ involved; whether the inflammation
is of an acute, resolving form or chronic, non resolving or long-lasting type;
whether formation of granuloma is involved, or whether scarring results. The role
of inflammation as a healing, restorative process, as well as its aggressive role, is
also more widely recognized today. Inflammation is now considered as the full
circle of events, from initiation of a response, through the development of the
cardinal signs above, to healing and restoration of normal appearance and function
of the tissue or organ. However, in certain conditions there appears to be no
resolution and a chronic state of inflammation develops that may last the life of the
individual. Such conditions include the inflammatory disorders such as rheumatoid
arthritis, osteoarthritis, inflammatory bowel diseases, retinitis, multiple sclerosis,
psoriasis and atherosclerosis. In order to study inflammation, a multidisciplinary
16

approach is necessary. Classically, it has required the study of the immune system,
in order to understand the events involved in initiating and maintaining
inflammatory conditions. Today it is recognized that the underlying genetics and
molecular biology basis to cellular responses are also important in order to identify
genetic predisposition to inflammatory diseases, while pharmacological studies are
necessary to identify targets and develop novel treatments to bring relief from
chronic life-threatening inflammatory conditions. Thus research into inflammation
includes not only the study of immunological and cellular responses involved but
also the pharmacological process involved in drug development. Many of the drugs
used in the treatment of inflammatory conditions, predate our current
understanding of the biochemical processes involved in the disease. Traditionally,
the standard treatments for rheumatoid arthritis has been to use a non-steroidal
anti-inflammatory drug (NSAID), such as aspirin, for pain relief and to use
corticosteroids or even disease-modifying anti-rheumatic drugs in an attempt to
reduce other symptoms of the disease. For many years the pharmaceutical industry
attempted to develop NSAIDs which shared the therapeutic action of aspirin but
which did not cause the main adverse event, namely gastric ulceration. This
research led to the development of indomethacin, the fenamates, ibuprofen and
many others. However, while all these drugs had clinical utility they also eroded
the gastric mucosa. The development of NSAIDs, with reduced potential to cause
17

gastric ulcers, was finally realized with the demonstration that clinically useful
NSAIDs inhibited the enzyme cyclo-oxygenase, which was also present in the
gastric mucosa (Whelan, 2003).
The finding that cyclo-oxygenase present in inflammatory lesions (COX2) was
distinct from that found in the stomach (COX1) led to the development of selective
COX2 inhibitors, such as celecoxib. These drugs provide relief from many of the
symptoms of arthritis but have a reduced potential to cause gastric ulceration. The
differential responsiveness to these, and other, therapeutic agents and, indeed, the
induction of the inflammatory response in some patients with asthma by aspirin,
has led to the concept of pharmacogenomics to understand individual drug
sensitivities with a view to producing therapy tailored to the individual. Similarly,
glucocorticoids are widely used in the treatment of inflammation. Unlike the
NSAIDs these agents do not relieve pain but reduce inflammation by inhibiting
leukocyte function. The active ingredient responsible for the anti-inflammatory
activity of adrenal cortex extracts was discovered in the 1940s. This led to the use
of cortisol as an anti-inflammatory and the development of potent synthetic agents
typified

by

glucocorticoids

dexamethasone.
produce

their

However,

because

therapeutic

action

cortisol
at

and

synthetic

supra-physiological

concentrations, adverse effects, such as suppression of the HPA-axis and


Cushingoid changes are inevitable. Many of these adverse effects can be avoided
18

by giving glucocorticoids topically. This has led to the development of inhaled


glucocorticoids for the treatment of inflammatory diseases of the respiratory tract
and steroid containing creams for the treatment of skin inflammation. However,
applying this approach to the treatment of rheumatoid arthritis necessitates the use
of intra-articular injection. Thus, there is a clear unmet medical need for a drug that
provides relief from the symptoms of inflammation but can be given systemically.
The fact that a large number of patients with severe chronic inflammatory disease
fail to respond to conventional systemic or topical therapy resulting in a huge
clinical and socio-economic burden underlies the need to develop novel therapies.
Thus, modern research has used molecular techniques to identify which genes are
regulated by glucocorticoids receptors in an attempt to identify novel therapeutic
targets. This work has attempted to fine tune the immune system through use of
agents that inhibit specific pathways and mediators rather than to suppress immune
cell activity. In summary, inflammation can be acute or chronic. When it is acute, it
occurs as an immediate response to trauma (an injury or surgery) usually within
two hours. When it is chronic, the inflammation reflects an ongoing response to a
long-term medical condition such as arthritis (Whelan, 2003).

2.7

CHRONIC INFLAMMATION

19

Prolonged inflammation, known as chronic inflammation, leads to a progressive


shift in the type of cells present at the site of inflammation and is characterized by
simultaneous destruction and healing of the tissue from the inflammatory process
(Hurley, 1972).

2.7.1 FEATURES OF CHRONIC INFLAMMATION


Components of Chronic inflammation include the following lymphocytes, plasma
cell,

macrophage

(mononuclear

cell)

infiltration,

tissue

destruction

by

inflammatory cells and repair with fibrosis and angiogenesis (new vessel
formation), (Hurley, 1972).

2.7.2

CAUSES OF CHRONIC INFLAMMATION

Chronic inflammation is caused by the following, persistent injury or infection


(such as ulcer, tuberculosis), prolonged exposure to toxic agent (for example,
pulmonary silicosis with silica in the lungs), and auto-immune disease (selfperpetuating immune reaction that result in tissue damage) and inflammation such
as rheumatoid arthritis and multiple sclerosis.

2.7.3
2.7.3.1

CLINICAL EXAMPLE OF CHRONIC INFLAMMATION


Rheumatoid Arthritis
20

The clinical presentations of rheumatoid arthritis include chronic, progressive


inflammatory disorder characterized by auto-immune reactions and damage of
synovial joints of the hands and feet causing synositis and erosion of the cartilage
of the joints (Hurley, 1972).

2.8

ACUTE INFLAMMATION

Acute inflammation is the initial response of the body to harmful stimuli and is
achieved by the increased movement of plasma and leukocytes (especially
granulocytes) from the blood into the injured tissues. A series of biochemical
events propagates and matures the inflammatory response, involving the local
vascular system, the immune system and various cells within the injured tissue.
Vascular changes play an important role during inflammation (begins early after
injury and depends upon the severity of the injury). Examples of these changes
include vasodilation, increased vascular permeability, loss of fluid from vessels,
and leukocyte rolling, adhesion and migration (Conner, 1996).
2.8.1

CLINICAL EXAMPLES OF ACUTE INFLAMMATION

Clinical examples of acute inflammation include acute skin inflammation and


appendicitis.

2.9 ACUTE VERSUS CHRONIC INFLAMMATION


21

Features
Onset

Acute
Fast: minutes or hours

Chronic
Slow: days

Cellular infiltrate

Mainly neutrophils

Monocytes, macrophages

Tissue injury, fibrosis

and lymphocytes
Usually mild and self- Often
severe

Local and systemic signs

limited
Prominent

and

progressive
Less prominent; may be
subtle

(Hurley, 1972).

2.10 ANTI-INFLAMMATORY AGENTS


Anti-inflammation refers to the property of a substance or treatment that reduces
inflammation or swelling. Anti-inflammatory drugs make up about half of
analgesics, remedying pain by reducing inflammation as opposed to opioids, which
affect the central nervous system. Non-steroidal anti-inflammatory drugs
(NSAIDs) alleviate pain by counteracting the cyclo-oxygenase (COX) enzyme. On
its own, cox enzyme synthesizes prostaglandins creating inflammation. In whole,
the NSAIDs prevent the prostaglandins from ever being synthesized, reducing or
eliminating the pain (Vane, 1971).

2.10.1 NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs)

22

These drugs, sometimes called the aspirin-like drugs or antipyretic analgesics


are among the most widely used cyclo-oxygenase inhibitors. These drugs
provide symptomatic relief from pain and swelling in chronic joint disease such
as occurs in osteo- and rheumatoid arthritis, as well as in more acute
inflammatory conditions such as fractures, sprains, sports and other soft tissue
injuries. They are also useful in the treatment of postoperative, dental and
menstrual pain, and of headaches and migraine. Virtually all these drugs,
particularly the traditional NSAIDs can have significant unwanted effects,
especially in the elderly. Newer agents have fewer adverse actions. While there
are differences between individual NSAIDs, their primary pharmacology is
related to their shared ability to inhibit the fatty acid COX enzyme, thereby
inhibiting the production of prostaglandins and thromboxane. There are two
common isoforms of this enzyme, COX-1 and COX-2. These two enzymes are
closely related (60% sequence identity) and catalyze the same reaction, it is
clear that there are important differences between the expression and role of
these two isoforms. COX-1 is a constitutive enzyme expressed in most tissues,
including blood platelets. It has a housekeeping role in the body, being
involved in tissue homeostasis, and is responsible for the production of
prostaglandins involved in, for example, gastric cytoprotection, platelet
aggregation, renal blood flow and auto-regulation and the initiation of
23

parturition. In contrast, COX-2 is induced in inflammatory cells when they are


injured, infected or activated by, for example, the inflammatory cytokines
interleukin (IL)-1 and tumour necrosis factor (TNF)-. Thus the COX-2
isoform is mainly responsible for the production of the prostanoid mediators of
inflammation (Vane and Botting, 2001). Most traditional NSAIDs inhibit both
COX-1 and COX-2, although they vary in the degree to which they inhibit each
isoform. It is believed that the anti-inflammatory action (and probably most
analgesic and antipyretic actions) of NSAIDs are related to inhibition of COX2, while their unwanted effects, particularly those affecting the gastro-intestinal
tract are largely a result of their inhibition of COX-1 (Trease, 1986).

2.10.2

Cyclo-oxygenase Inhibitors

These drugs have three major therapeutic actions, stemming the suppression of
prostanoid synthesis in inflammatory cells through inhibition of the cyclooxygenase (COX)-2 isoform of the arachidonic acid COX. They are as follow:
An anti-inflammatory action: the decrease in prostaglandin E2 and
prostacyclin reduces vasodilatation and indirectly oedema.
24

An analgesic effect: decrease prostaglandin generation means less


sensitization of nociceptive nerve endings to inflammatory mediators
such as bradykinin and 5-hydroxytryptamin. Relief of headache is
probably a result of decreased prostaglandin-mediated vasodilatation
An antipyretic effect: Interleukin-1 releases prostaglandins in the central
nervous system, where they elevate the hypothalamic set point for
temperature control, thus causing fever. NSAIDs prevent this. Some
important NSAIDs are aspirin, ibuprofen, naproxen, indomethacin,
piroxicam and paracetamol. Newer agents with more selective inhibition
of COX-2 (and thus fewer adverse effects on the gastro-intestinal tract)
include celecoxib and etoricoxib (Trease, 1989).

2.10.3 MECHANISM OF ACTION OF NSAIDs


Vane and colleagues established in 1971 that the main actions of NSAIDs were
brought about through inhibition of arachidonic acid oxidation by the fatty acid
cyclo-oxygenase. These are bi-functional enzymes, having two distinct catalytic
activities. The first, dioxygenase step incorporates two molecules of oxygen
into the arachidonic (or other fatty acid substrate) chain at C11 and C15, giving
rise to the highly unstable endoperoxide intermediate PGG2 with a hydroperoxy
25

group at C15. A second, peroxidase function of the enzyme converts this to


PGH2 with a hydroxyl group at C15, which can then be transformed in cellspecific manner by separate Isomerase, reductase or synthase enzymes into
other prostanoids. Both COX-1 and COX-2 are haem-containing enzymes that
exist as homodimers attached to intracellular membrane. Structurally, the
isoforms are similar; both contain a hydrophobic channel into which the
arachidonic or other substrate fatty acids dock so that the oxygenation reaction
can proceed (Vane, 1971).

2.10.4 ANTI-INFLAMMATORY EFFECTS OF NSAIDs


Many mediators coordinate inflammatory and allergic reactions. The NSAIDs
reduce mainly those components of the inflammatory and immune response in
which prostaglandins, mainly derived from COX-2 play a significant part.
These include:
Vasodilatation (by reducing the synthesis vasodilator prostaglandins)
Oedema (by an indirect action: the vasodilatation facilitates and
potentiates the action of mediators such as histamine that increase the
permeability of prostcapillary venules (Vane, 1971).

2.10.5 UNWANTED EFFECTS OF NSAIDs

26

Overall, the burden of unwanted side effects is high, probably reflecting the fact
that NSAIDs are used extensively in the more vulnerable elderly population, and
often for extended periods of time. When used for joint diseases (which usually
necessitates fairly large doses and long-continued use), there is a high incidence of
side effects particularly in the gastro-intestinal tract but also in the liver, kidney,
spleen, blood and bone marrow (Vane, 1971).

2.10.6 INDOMETHACIN
DRUG SUMMARY: A very potent aryl-acetic acid NSAID derivative.
Because of its high potential to cause side effects when use in high doses, it
should be carefully considered for active disease unresponsive to adequate
trials with salicylates. It has equal or little superior action than naproxen, but
higher incidence of side effects. This medication will enable reduction of
steroid doses in severe forms of rheumatoid arthritis. (In this case reduce
steroid dose slowly).
INDICATION: Use for treatment of inflammatory conditions such as
rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, tendinitis,
bursitis, acute gout, and acute painful shoulder (Vane, 1971).
2.11 LIPIDS
Lipids are heterogeneous group of organic substances which are insoluble in water
and are soluble in a group of organic solvents. The common fats solvents are
27

chloroform, ether, benzene, hexane and light petroleum (Ochei and Kolhaktar,
2008). Lipid comprises a large number of diverse chemical substances. The major
lipids in human plasma include cholesterol, triglycerides, phospholipids and nonesterified fatty acids (NEFA). Lipoproteins are lipids modified for their transport in
plasma and tissues (Alvarez, 1986).

2.11.1

INDICATIONS

The medical community recognizes lipid testing as appropriate for evaluating


atherosclerotic cardiovascular disease. Conditions in which lipid testing may be
indicated include assessment of patients with atherosclerotic cardiovascular
disease, evaluation of primary dyslipidaemias, diagnostic evaluation of diseases
associated with altered lipid metabolism such as nephrotic syndrome, pancreatitis,
hepatic disease and hypo and hyperthyroidism, secondary dyslipidaemia including
diabetes mellitus, disorders of gastro-intestinal absorption, chronic renal failure,
and as follow up to the initial screen for coronary heart disease (Bang et al., 2005)

2.11.2

FACTORS (PRE-ANALYTICAL) WHICH MAY AFFECT LIPID

COMPONENTS
Factors which may affect lipid components include prolonged tourniquet
application (2-5minutes) can increase cholesterol from 5 to 15%, cholesterol is
slightly higher in winter than in summer and the opposite is true for triglycerides,
28

disease conditions such as nephrotic syndrome increase total cholesterol, LDL-C


and VLDL-C and hyperthyroidism increases LDL-C and total cholesterol.
Infection and inflammation may decrease total cholesterol and HDL-C and
increase triglycerides, and finally lipids are altered following myocardial infarction
and these changes may persist for several weeks. That is why it is better to do lipid
profile in such patients within 24 hours of myocardial infarction (Chawla et al.,
2005).

2.11.3 CHOLESTEROL
Cholesterol is unsaturated steroid alcohol. About 70 to 80% of cholesterol in
plasma is esterified with long chain saturated and unsaturated fatty acids. About
20-30% exists as free unesterified cholesterol (Ochei and Kolhaktar, 2008). The
major function of cholesterol is as a metabolic precursor for the biosynthesis of
bile acids and steroid hormones which include male and female sex steroids
(androgens and oestrogens) and adrenal steroid hormones (aldosterones and
corticosterones). Liver, ovaries, testes and adrenal gland synthesize these hormones
using cholesterol as the main precursor (Ochei and Kolhaktar, 2008). Cholesterol is
also an important structural component of cell membranes. Being insoluble in
water, cholesterol can control the flow of water soluble products in and out of cells.
In humans 60-70% of cholesterol is transported by low density lipoproteins (LDL),
29

20-35% by high density lipoproteins (HDL) and 5-12% by very low density
lipoprotein (VLDL) (Ochei and Kolhaktar, 2008). It is important to maintain
healthy levels of the two major kinds of lipoproteins (HDL and LDL) that transport
cholesterol throughout the body.
LDL cholesterol is sometimes called bad cholesterol because it carries
cholesterol from the liver to the arteries.
High LDL cholesterol leads to build up of cholesterol in the lumen of
arteries. The higher the LDL levels in the blood, the greater the chance of
developing coronary heart disease (CHD).
HDL cholesterol is sometimes called good cholesterol because it carries
cholesterol from cells to the liver where it is either used for synthesis of bile
acids or excreted. The higher the HDL cholesterol level, the lower the
chance of developing coronary heart disease (Ochei and Kolhaktar, 2008).
Too much cholesterol in the blood can build up in the walls of the arteries
resulting in plague formation. Over time, plague can cause narrowing of the
arteries which may eventually result in atherosclerosis or hardening of the
arteries. Narrowing of the coronary arteries due to plague can stop or slow
down the flow of blood to the heart. When the arteries narrow, the amount of
oxygen-carrying blood is decreased. This is called coronary heart disease
(CHD). Large plague formation can lead to chest pain called angina. Angina
happens when the heart does not receive enough blood and the oxygen it
30

carries. Angina is a common sign of CHD. Some plagues may rupture


thereby releasing fat and cholesterol into the bloodstream. This may cause
the blood to clot thereby occluding blood flow. Lowering cholesterol levels
decrease the chance of having a plague burst and developing heart attack.
Normal values of cholesterol in plasma or serum is about 5.17mmol/l to
6.20mmol/l (Tietz, 1990).

2.11.4

CLINICAL SIGNIFICANCE

Serum cholesterol rises with age and the rise is more marked in men than in
women. In middle age it may rise up to 300mg/dl (7.0mmol/l). In pregnancy, there
may be about 20 to 25% rise in cholesterol levels (Ochei and Kolhaktar, 2008).
Increase in cholesterol occurs in nephrotic syndrome, diabetes mellitus, obstructive
jaundice, angina pectoris, and hypothyroidism.
Cholesterol level may be normal in chronic glomerulonephritis. In acute
glomerulonephritis increased levels are seen in larger stages.
Decrease in serum or plasma cholesterol levels may occur in pernicious anaemia,
haemolytic

anaemia,

mal-absorption

hyperthyroidism (Mora, 2008).

2.11.5 TRIGLYCERIDES
31

syndrome,

acute

infection,

and

Triglycerides are long chain fatty acids esterified to glycerol. Usually there are
three different fatty acids present in a molecule. Triglycerides are the primary
constituents used for long chain storage of energy. One molecule of triglyceride
produces twice the amount of energy as compared to polysaccharides such as
glycogen. Hence triglyceride metabolism is a key component which derives energy
for many body functions including that of the heart. The primary source of
triglycerides is from the diet. Triglycerides are transported in plasma mostly in the
form of chylomicrons and VLDL but are also present in LDL-C and HDL-C in
small amount, with a reference value of about 1.71mmol/l (150mg/dl) to
2.29mmol/l (200mg/dl) (Tietz, 1990).

2.11.6

CAUSES OF HYPERGLYCERIDAEMIA

Increased levels of serum triglyceride have been identified as risk factors related to
arteriosclerosis which causes thickening of the walls of larger blood vessels. This
may lead to heart attack (Ochei and Kolhaktar, 2008). Other conditions in which
raised triglycerides are observed include liver disease, nephrotic syndrome,
hypothyroidism, diabetes mellitus, pancreatitis, and acute myocardial infarction.

2.12

LIPOPROTEINS

32

Lipoproteins are spherical particles that carry lipids, particularly cholesterol and
triglyceride, in the plasma. There is a well-established association between
dyslipidaemias, or disorders of lipoprotein metabolism, and coronary heart disease
(CHD): elevated levels of blood cholesterol, especially low density lipoprotein
cholesterol (LDL-C), and low levels of high-density lipoprotein cholesterol (HDLC) increase risk for CHD. The relationship between triglyceride levels and CHD is
controversial, although it is known that some triglyceride-rich lipoproteins can be
atherogenic. Specific proteins called apolipoproteins which have both hydrophobic
and hydrophilic charges on their surface are associated with lipoproteins.
Depending on their density, plasma proteins can be separated into several fractions
according to their rates of sedimentation or by Svedberg flotation index (S f).
Usually the sedimentation rate increases with the molecular weight of protein but
lipoprotein are exception to this rule. In lipoproteins, the lipid fractions attached to
the apoproteins are lighter than water and therefore, lipoproteins are high
molecular weight, low density, and large size molecules. Ultracentrifugation
separates lipoproteins on the basis of their density into the following:

Low density lipoprotein (LDL-C)


Very low density lipoprotein (VLDL)
Intermediate density lipoprotein (IDL)
High density lipoprotein (HDL-C), with the exception of chylomicrons.

33

Chylomicrons are responsible for the visual opalescence of the plasma (Alvarez,
1986).

2.12.1 HIGH DENSITY LIPOPROTEIN


High density lipoprotein (HDL) is one of the five major groups of lipoproteins.
HDL is the smallest of the lipoprotein particles. It is the densest because it contains
the highest proportion of protein to lipids. Its most abundant apolipoproteins are
apo A-I and apo A-II. The liver synthesizes these lipoproteins as complexes of
apolipoproteins and phospholipids, which resemble cholesterol-free flattened
spherical lipoprotein particles; the complexes are capable of picking up cholesterol
carried internally from cells by interaction with the ATP-binding cassette
transporter A1

(ABCA1). A plasma

enzyme

called

lecithin-cholesterol

acyltransferase (LCAT) converts the free cholesterol into cholesteryl ester (a more
hydrophobic form of cholesterol), which is then sequestered into the core of the
lipoprotein particle, eventually causing the newly synthesized HDL to assume a
spherical shape. HDL transports cholesterol mostly to the liver or steroidogenic
organs such as ovary, adrenal and testes by both direct and indirect pathway. HDL
is removed by HDL receptors such as scavenger receptor BI (SR-BI), which
34

mediate the selective uptake of cholesterol from HDL. In humans, the most
relevant pathway is the indirect one, which is mediated by cholesteryl ester transfer
protein (CETP) (Hirano et al., 2008).

2.12.2

CAUSES OF INCREASED HDL LEVELS

Causes of increased HDL levels include decreased intake of simple carbohydrates,


weight loss, niacin, smoking cessation, red wine consumption (Emokpae, et al.,
2003), addition of soluble fibres to diet and consumption of omega-3 fatty acids
such as fish oil or flax oil (Hausenloy and Yellon, 2008).

2.12.3

LOW DENSITY LIPOPROTEIN

Low density lipoprotein (LDL) is one of the five major groups of lipoproteins
which in order of molecular size, largest to smallest, are chylomicrons, very low
density lipoprotein (VLDL), intermediate density lipoprotein (IDL), LDL and
HDL. LDL particles (composed of thousands of various molecules) are often called
bad cholesterol because they can transport their content of many fat molecules into
artery walls, attract macrophages and thus enhance atherosclerosis (Dashty et al.,
2014).

35

2.12.4

FACTORS THAT AFFECT LOW DENSITY LIPOPROTEIN

Heredity: Genes influence how high the LDL-C is by affecting how fast LDL is
made and removed from the blood. One specific form of inherited high cholesterol
that affects one in 500 people is familiar hypercholesterolaemia, which often leads
to early heart disease. But even if there is no specific genetic form of high
cholesterol, genes play a role in influencing LDL-C level.
Food: Two main nutrients in food make LDL-C level go up. Saturated fat, a type
of fat found mostly in foods that come from animals, and cholesterol, which comes
only from animal products. Saturated fats increases LDL-C level more than
anything else in the diet. Eating too much saturated fat and cholesterol is the main
reason for high levels of cholesterol and a high rate of heart attacks. Reducing the
amount of saturated fat and cholesterol in the food we eat is a very important step
in reducing blood cholesterol levels.
Weight: Excess weight tends to increase LDL-C level. Weight loss may help lower
LDL-C level. Weight loss also helps to lower triglycerides and increase HDL-C
levels (Alvarez, 1986).

2.12.5

LIPOPROTEIN METABOLISM

36

Lipoprotein metabolism can be divided into two groups. The exogenous


metabolism deals with lipids and lipoproteins derived from external sources (foods
and nutrients).
The endogenous metabolism involves lipids and lipoproteins produced in the body
to synthesize complex lipids in the liver and other tissues (Alvarez, 1986).

2.12.6 EXOGENOUS METABOLISM


As the food passes the stomach, pancreatic enzymes (amylase, lipase and
peptidase) are released and act on the complex molecule in the food to break them
to metabolites which are readily absorbed. Triglycerides are hydrolyzed by lipases
to mono-glycerides and free fatty acids. The monoglycerides and fatty acids are
absorbed through the intestinal mucosa and are converted again to triglycerides and
phospholipids. The newly formed lipids are packaged into chylomicron
(triglyceride-rich lipoproteins) which passes into the lymph. Chylomicrons in the
lymph enter the bloodstream through the thoracic duct. This absorption of
triglycerides into the blood can occur within 30 to 90 minutes from the intake of
food. The walls of the blood vessels contain lipoprotein lipase which hydrolyzes
triglycerides in the chylomicrons to fatty acids and glycerides. These are absorbed
by the cells of the capillary wall. The hydrolyzed products are re-synthesized to
triglycerides which are stored for energy. When all the triglycerides from
37

chylomicrons are removed, the latter are cleared from the bloodstream by the liver
(Nawaz, 2006).

2.12.7 ENDOGENOUS METABOLISM


The liver is the principal organ of metabolism of lipids and synthesis of
lipoproteins. Triglycerides and cholesterol in the liver cells are packed as VLDL
and secreted into circulation. The enzyme lipoprotein lipase acts on the VLDL in
the same manner as on the chylomicrons. Hydrolyses of triglyceride from VLDL
results in the formation of IDL with high cholesterol content. IDL is rapidly
processed to LDL-C which transports cholesterol to peripheral tissues. LDL-C
binds to the cell receptor on the cell surface and enters the cell. The cholesterol
content of LDL-C is added to the intracellular pool. By a feedback mechanism, as
the pool increases, the rate of synthesis is reduced and vice versa. HDL from the
liver and intestine is released in circulation in the form known as nascent form
which is not spherical in shape. Thus it can absorb cholesterol as cholesterol ester
and regulates its movement from peripheral. However it can absorb lipid from
other lipoproteins to attain a spherical shape. Thus it can absorb cholesterol ester
38

and regulate its movement from peripheral tissue back to the liver, acting as a
reverse cholesterol transport system. HDL-C can also exchange cholesterol esters
with triglyceride from VLDL. Thus VLDL, LDL-C and HDL-C interact to
maintain the intracellular lipid levels. The lipoprotein transport lipids from the area
of synthesis to areas of storage, metabolic needs or catabolism (Nawaz, 2006).

2.13 EFFECT OF INFLAMMATION ON LIPID PROFILE


Several pieces of evidence indicate that rheumatoid arthritis (RA) is a proatherogenic disease associated with increased cardiovascular (CV) mortality.
Besides genetic and traditional CV risk factors, chronic inflammation has emerged
as a pivotal component implicated in the development of this process. While the
risk of developing atherosclerosis increases progressively with increasing LDLcholesterol levels and declines with increasing levels of HDL-cholesterol in
healthy individuals, the presence of a pro-inflammatory state leads to a decrease of
total cholesterol, HDL-cholesterol and LDL-cholesterol in patients with RA.
Paradoxically, anti-inflammatory therapies increase total cholesterol, HDLcholesterol and LDL-cholesterol to variable degrees in patients with RA. Chronic
inflammation leads to oxidative changes that alter HDL structure and reduce
apolipoproteins A-1 in patients with active RA. Levels of para-oxonase-1, an antioxidant enzyme associated with HDL, are lower in patients with RA compare with
39

healthy controls. Therefore, because of inflammation there is an impairment of the


normal anti-inflammatory, antioxidant and cardio-protective function of HDLcholesterol that turns to be pro-inflammatory.

2.14 MECHANISM OF FORMALIN-INDUCED INFLAMMATION


Administration of formalin produced an elevated level of lipid peroxidation (LPO),
which may be due to the free radicals and is responsible for damaging cell
membranes thereby further intensifying inflammatory damage (Telang et al.,
1990). The inflammatory tissue damages could be due to the liberation of reactive
oxygen species from phagocytes invading the inflammation sites (Conner and
Grisham, 1996).

40

CHAPTER THREE
3.0

MATERIALS AND METHODS

3.1

MATERIALS

3.1.1 REAGENTS, DRUGS AND CHEMICALS


The reagents used for this research were of high analytical grade. They include:
Double distilled water
Indomethacin (St Louis, MO, U.S.A).
Fomaldehyde (Vardhman Exports India).
Chloroform
Lipid profile estimation kit (Randox Laboratories LTD, U.K.)

3.1.2 APPARATUS
Spectrophotometer
Centrifuge
Gavage
41

3.2

Digital weighing balance (METLER MT-5000D)


Pestle and Mortar
Glass pipettes
Beakers
Measuring Cylinder (Pyrex, England)
Conical flask (Pyrex, England)
Heater
Syringes
Micropipettes
Cuvette
Pasteur pipette
Test tubes
Test tube rack
Rat cages
Feeding troughs
Drinking troughs
Refrigerator
Dissecting sets
Cotton wool
Water bath
Gloves
Vernier caliper

PLANT SPECIMEN COLLECTION AND AUTHENTICATION

The stem bark Of A. boonei was collected from the growing tree inside University
of Benin, Benin City, in June 2015. It was identified and authenticated by Dr A. O.
Akinibosun (Plant Taxonomist), Department of Plant Biology and Biotechnology
(PBB), University of Benin, Benin City. The sample was washed, air dried and
42

pulverized using plant milling machine (Kenwood UK LTD) and the stem bark was
stored in air tight containers prior to extraction.

3.2.1 AQUEOUS EXTRACT PREPARATION


The powdered stem bark (400g) was boiled in 4 Litres of distilled water for 15
minutes to obtain the aqueous extract. The extract was filtered and then
concentrated under pressure in a rotarvapour at 68 C and dried in an oven at 50 C
for 48 h (yield 5.5%) and the dried extract was stored in an air-tight clean glass
container at 4 C until use.

3.3

ANIMAL MODELS

Thirty female Wistar rats weighing between 150-230g were obtained from the
animal house of the Department of Pharmacology and Toxicology, University of
Benin, Benin City. The animals were stabilized for two weeks in the animal house
of the Department of Pharmacology, University of Benin, Benin City. The animals
were fed with standard rodent cubes obtained from Ladokun feed Ltd. (Ibadan,
Nigeria) and had free access to feed and water ad libitum. All animals were fasted
overnight before the beginning of each experiment. Animals were exposed to
natural lighting condition and were handled according to standard experimental

43

protocols approved by the Faculty of Pharmacy Animal Ethics Committee,


University of Benin.

3.4

EXPERIMENTAL DESIGN

Formalin-induced arthritic inflammation


The animals were divided into 6 groups comprising of 5 animals in each
group. They were fully described, weighed, identified and marked, prior to the
experiment.
Inflammation was induced by subaponeurotic injection of 0.1mL of 2% w/v
formalin in normal saline in the right hind paw of the rats on the first and third day.
Group 1: This is the control group. This group was fed with normal food and
water only.
Group 2: This group received 3ml/kg of distilled water once a day for five days.
Neither plant extract nor indomethacin was administered to the animals in this
group.
Group 3: This test group received 150mg/kg, p.o of the extract once a day for five
days.
Group 4: This test group received 300mg/kg, p.o of the extract once a day for five
days.

44

Group 5: This test group received 600mg/kg, p.o. of the extract once a day for five
days.
Group 6: This is the reference group. This group received indomethacin (10mg/kg,
p.o.) once a day for five days.
The rat paw thickness was measured daily for five days using Vernier caliper. The
percentage inhibition of the mean increase in the paw edema of each group was
calculated on the 5th day and compared with the control. At the end of the
experiment all the animals were anaesthesized using cotton wool soaked in
chloroform. They were dissected using dissecting set. Blood was collected from
abdominal aorta and directly from the heart using a 5mL syringe into a plain
container and then allowed to clot. It was then centrifuged at 4000rpm for 10
minutes. The serum were collected into plain sterile bottles and used for total
cholesterol, triglycerides and high density lipoprotein-cholesterol analysis.

3.5 ESTIMATION OF SERUM TOTAL CHOLESTEROL LEVEL


METHOD: Cholesterol CHOD-PAP Assay (RANDOX Laboratories Limited).
REAGENT COMPOSITION
Pipes buffer

80 mmol/L, PH 6.8
45

4-aminoantipyrine

0.25mmol/L

Phenol

6mmol/L

Peroxidase

0.5 U/mL

(E. C. 1. 11. 1.7, +250C)


Cholesterolesterase

0.15 /mL

(E. C. 1. 1.3.6. Nocardia, 370C)

0.10 /mL

PRINCIPLE
Total serum cholesterol is measured enzymatically in a series of coupled reactions
that hydrolyze cholesteryl esters and oxidize 3-OH group of cholesterol. One of the
reaction by-products, hydrogen peroxide (H2O2) is measured quantitatively in a
peroxidase catalyzed reaction that produces a colour (pink). Absorbance is read at
500nm. The intensity of the colour is proportional to the cholesterol concentration.
The reaction sequence is as follows:
Cholesterol ester + H20

cholesteryl ester hydrolase

cholesterol + fatty acids

Cholesterol + 02 cholesterol oxidase Cholest-4-ene-3-one + H202


2H202 peroxidase

4-aminophenazone + 4H20 + 4-(p-benzoquinone-monoimino)-

phenazone.

PROCEDURE

46

The frozen plasma is thawed and kept at 370C prior to analysis. The procedure is as
follows:
BLANK

Blank

Standard

Test

TUBES

(l)

(l)

(l)

Reagent
1000
1000
1000
Sample
10
Standard
10
Distilled H20
10
It was mixed gently and incubated for 10 minutes at room temperature.
The absorbance was read at 500nm against water blank.

Calculation:
The concentration of cholesterol (mmol/L) in the sample is calculated using this
formula:
=

3.6

Absorbance of test
Concentration of standard
Absorbance of standard

ESTIMATION OF SERUM TRIGLYCERIDE (TRIGS)

47

METHOD: GPO-PAP (RANDOX Laboratories Limited).


Pipes buffer

40mmol/L, PH 7.6

4-chloro-phenol

5.5mmol/L

Magnesium ions

17.5mmol/L

4-aminophenazone

0.5mmol/L

ATP

1.0mmol/L

Lipases

150U/mL

Glycerol-kinase

0.4U/mL

Glycerol-3-phosphate oxidase

1.5U/mL

Peroxidase

0.5U/mL

PRINCIPLE
Triglycerides are measured enzymatically in plasma using a series of coupled
reactions in which triglycerides are hydrolyzed to produce glycerol. Glycerol is
then oxidized using glycerol oxidase and hydrogen peroxide (H 202), one of the
products of the reaction is measured as described for cholesterol. Absorbance is
measured at 500nm. The reaction sequence is as follows:
Triglycerides + 3H20

lipase

glycerol + fatty acids

Glycerol + ATP glycerol kinase glycerol-3-phosphate + ADP

48

Glycerol-3-phosphate + glycerolphosphate-oxidase

dihydroacetone

phosphate (DHAP) + H202


Peroxidase + H202 + 4-aminophenazone + 4-chlorophenol

4-(p-

bezoquinone-monoimino)-phenazone + 2H20
PROCEDURE
The frozen plasma is thawed and kept at 370C prior to analysis.
The procedure is as follows:
BLANK

Blank

Standard

Test

TUBES
(l)
(l)
(l)
Reagent R1
1000
1000
1000
Sample
10
Standard
10
Distilled water H20
10
It was mixed gently and incubated for 10 minutes at room temperature.
The absorbance was read at 500nm against water blank.
Calculation:
The concentration of triglycerides (mmol/L) in the sample was calculated using the
formula below:
=

3.7

Absorbance of test
Absorbance of standard

concentration of standard

ESTIMATION OF HIGH DENSITY LIPOPROTEIN (HDL-C)

METHOD: Enzymatic end point method (RANDOX Laboratories Limited).

49

REAGENT COMPOSITION
CONTENTS

CONCENTRATIONS IN THE TEST

R1 Phosphotungstic Acid

0.55mmol/L

Magnesium chloride

25mmol/L

PRINCIPLE
Lipoproteins present in the specimen (LDL, VLDL and CHYLOMICRONS) are
precipitated quantitatively by the addition of phosphotungstic acid in the presence
of magnesium ions. This renders them non-reactive and effectively excluded from
the assay. Thus, after centrifugation, only HDL-cholesterol is detected.

PROCEDURE:
Stage 1: Precipitation
Precipitate into centrifuge tubes
Macro(l)
Semi micro(l)
Sample/standard
500
200
Precipitant (R1)
1000
Diluted precipitant (R1)
500
It was mixed and allowed to stand at room temperature after which it was then
centrifuged for 10 minutes at 4,000rpm.
50

Separate off the supernatant within two hours and determine the cholesterol
content by the CHOD-PAP method.

Stage 2: CHOLESTEROL (CHOD-PAP) ASSAY


BLANK

Blank (l)

Standard (l)

Test (l)

TUBES
Reagent
1000
1000
1000
Sample
100
Standard
100
Distilled H20
100
It was mixed gently and incubated for 10 minutes at room temperature.
The absorbance was read at 500nm against water blank.
Calculation:

51

The concentration of HDL (mmol/L) in the sample was calculated using the
formula bellow:
=

Absorbance of test
concentrationof standard
Absorbance of standard

3.8

LOW DENSITY LIPOPROTEIN CHOLESTEROL(LDL-C)

LDL-cholesterol is calculated from the measured values of total cholesterol,


triglycerides and HDL-cholesterol according to the relationship:
TG
LDL-c = TC ( HDL+ 5 )

This is referred to as the Freidewald equation (Freidewald et al., 1972).

3.9

ATHEROGENIC INDICES

Cardiac risk ratios (CRR), atherogenic co-efficient (AC) and atherogenic index of
plasma (AIP) were calculated as shown below:

AIP =

TG
HDLc
()
log

CRR =

TC
HDLc

AC =

TC HDLc
HDLc
52

TG, HDL and LDL were expressed in molar concentrations (Dobiasova and
Frohlich, 2001).

3.10 STATISTICAL ANALYSIS


The data obtained were statistically analyzed using Statistical Package for Social
Sciences (SPSS) version 20.0. Values obtained in this research were represented as
mean Standard error of the mean (SEM) for both test and controls. Analysis of
variance (ANOVA) was used to compare data at 95% confidence interval (P
0.05

CHAPTER FOUR
4.0

RESULTS

The results obtained from the study are presented below:


Acute toxicity studies showed that all the doses (150, 300 and 600 mg/kg) of the A.
boonei extract used for the study were non-toxic.
In the formalin-induced paw edema (table 4.1), the aqueous extract of A. boonei
(300 mg/kg) significantly (p < 0.05) inhibited paw edema at the 3rd hour compared
with the control animals which received distilled water. This effect was

53

comparatively less than indomethacin which showed significantly (p 0.01) higher


inhibition compared to the control animals.

Table 1:

Effect of aqueous extract of A. boonei on formalin-induced

inflammation in Wistar rats.


Treatment

Dosage

Paw Thickness on day

Inhibition

Controls
A. boonei

(mg/kg)
3ml/kg
150

5(MM)
8.630.11
8.100.12

(%)
6.14

300

7.800.21

9.62

600

8.120.26

5.92

7.630.14

11.59

Indomethacin

54

Data are the mean SEM values for five rats in each group. AB A. boonei, ID
indomethacin *p < 0.05, **p 0.01 as compared to the control (n = 5 for each
group).

Figure 3: Effect of aqueous extract of A. boonei on formalin-induced paw edema in


rats.

55

Table 2: Independent t-test showing comparism between control and Group 1.

Variables

MEAN S.E.M (mmol/L)


Controls
Group 1

t-value

P-Value

TC mmol/L

n=5
2.230.25

n=5
2.560.02

3.64

0.30

TRIG

1.200.48

1.390.14

1.27

0.00

mmol/L
HDL-C

0.660.24

1.320.07

1.42

0.00

mmol/L
LDL-C

1.000.22

1.290.09

2.51

0.60

mmol/L
The above table shows the mean concentration of lipid profile in serum samples of
control Group and Group one. Lipid profile levels were significantly higher
compared to the control group.

56

Table 3: Independent t-test showing comparism between control and


150mg/kg extract.
MEAN S.E.M (mmol/L)
Controls
Group 2
Variables

t-value

P-Value

n=5

n=5

TC mmol/L

2.230.25

2.020.15

1.50

0.01

TRIG

1.200.48

1.530.22

0.58

0.22

mmol/L
HDL-C

0.660.24

1.160.09

1.86

0.74

mmol/L
LDL-C

1.000.22

0.560.15

2.69

0.37

mmol/L
The above table shows the mean concentration of lipid profile in serum samples of
control Group and Group treated with 150mg/kg extract. Serum total cholesterol
was significantly lower compared to the control.

57

Table 4:

Independent t-test showing comparism between control Group

and Group treated with 300mg/kg extract.


MEAN S.E.M (mmol/L)
Controls
Group 3
Variables

t-value

P-Value

TC

n=5
2.230.25

n=5
2.280.19

3.40

0.04

mmol/L
TRIG

1.200.48

1.320.16

2.45

0.82

mmol/L
HDL-C

0.660.24

1.790.24

0.73

0.02

mmol/L
LDL-C

1.000.22

0.280.25

1.63

0.05

mmol/L

The above table shows the mean values of lipid profile in serum samples of control
Group and Group treated with 300mg/kg extract. Low-density lipoprotein
cholesterol levels were significantly lower compared to the control.
58

Table 5:

Independent t-test showing comparism between control Group

and Group 4.
MEAN S.E.M (mmol/L)

59

Variables

Controls

Group 4

t-value

P-Value

n=5

n=5

TC

2.230.25

2.340.06

1.54

0.02

mmol/L
TRIG

1.200.48

1.630.12

2.87

0.92

mmol/L
HDL-C

0.660.24

1.220.11

3.82

0.64

mmol/L
LDL-C

1.000.22

0.790.09

2.15

0.98

mmol/L

The table above shows the mean concentration of lipid profile in serum samples of
control Group and Group treated with 600mg/kg extract. Serum total cholesterol
levels were significantly higher compared to the control.

60

Table 6:

Independent t-test showing comparism between control Group

and Group 5.
MEAN S.E.M (mmol/L)
Controls
Group 5
Variables

t-value

P-Value

N=5

n=5

TC

2.230.25

2.860.09

2.95

0.05

mmol/L
TRIG

1.200.48

3.310.72

2.23

0.00

mmol/L
HDL-C

0.660.24

0.890.09

3.21

0.90

61

mmol/L
LDL-C

1.000.22

1.310.11

3.79

0.59

mmol/L

The table above shows the mean concentration of lipid profile in serum samples of
control Group and the Group treated with 10mg/kg standard drug (Indomethacin).
Serum total cholesterol and triglycerides levels were significantly higher compared
to the control.

62

Table 7:

One way ANOVA showing comparism between control and all

Groups. Values are MEAN S.E.M (mmol/L), (n=5).

Variables
TC mmol/L

Controls
2.230.2

Group 1
2.560.0

Group 2
2.020.1

Group 3
2.280.19

F-value
3.29

P-Value
0.05

TRIG

5
1.200.4

2
1.390.1

5
1.530.2

1.320.16

1.47

0.26

mmol/L
HDL-c

8
0.660.2

4
1.320.0

2
1.160.0

1.790.24

3.90

0.03

mmol/L
LDL-c

4
1.000.2

7
1.290.0

9
0.560.1

0.280.25

3.77

0.03

mmol/L

63

The above table shows the mean concentration of lipid profile in serum samples of
all test Groups and control Group. 150, 300 and 600mg/kg extract showed a
significant decrease in low-density lipoprotein cholesterol compared to the control.

64

Table 8:

One way ANOVA for all groups in Atherogenic Indices. Values are

MEAN S.E.M (mmol/L), (n=5).


PVariable

Control

Group 1

Group 2

Group 3

Group 4

Group 5

Value

s
AIP

0.090.0

0.050.0

0.110.0

1.361.2

0.130.0

0.510.1

0.18

mmol/L
CRR

2
1.370.1

4
1.960.1

5
1.760.1

0
1.340.1

6
1.990.2

0
3.260.2

0.00

mmol/L
AC

4
0.070.1

2
0.960.1

1
0.790.1

6
0.340.1

0
0.990.2

2
2.270.2

0.00

mmol/L

KEY: AIP=Atherogenic

Index

of

Plasma,

AC=Atherogenic Co-efficient.

65

CRR=Cardiac

Risk

Ratio,

CHAPTER FIVE
DISCUSSION
5.0

DISCUSSION

In the present study, the anti-inflammatory activity of the aqueous extract of A.


boonei has been evaluated in chronic inflammatory models. The inhibition of
formalin-induced inflammation in rats is an established model for evaluating antiinflammatory drugs, which has been used frequently to assess anti-edematous
effect of natural products. The development of formalin-induced edema is biphasic
(Vinegar, et al., 1987); the first phase occurs within one hour of formalin
inflammation and is attributed to the release of cytoplasmic enzymes, histamine
and serotonin, from the mast cells. The second phase (> 1.0 hour) is mediated by
an increased release of prostaglandins in the inflammatory area and continuity
between the two phases is provided by kinins. Since the extract significantly
inhibited paw edema induced by formalin in the second phase, this finding
suggests a possible inhibition of cyclo-oxygenase synthesis by the extract, because
the formalin inflammatory model basically reflects the actions of prostaglandins
(Di Rosa, et al., 1971; Ferreira, et al., 1974). This effect is similar to that produced
by non-steroidal anti-inflammatory drugs such as indomethacin, whose mechanism

66

of action is inhibition of the cyclo-oxygenase enzyme, which catalyses the


synthesis of cyclic endoperoxides important in the formation of prostaglandins
Lipid profile refers to some routinely done biochemical tests to assess the
atherogenic status of individuals at risk of coronary heart disease (CHD). It
includes serum triglycerides (TG), serum total cholesterol (TC) and its subfractions like HDL-c and LDL-c. Atherogenicity with subsequent cardiovascular
manifestation is one of the major causes of death and morbidity in the world (Raju
and Binda, 2005). Various studies indicate that high serum cholesterol levels are
strongly related to coronary atherosclerosis and increased risk of cardiovascular
diseases. Clinical studies have also shown that lowering levels of serum cholesterol
using diet or drugs decreases the incidence of coronary heart disease (Steiner and
Li, 2000, Treasure, et al., 1995).
Increased low-density lipoprotein cholesterol with decreased high-density
lipoprotein cholesterol usually increases the plasma total cholesterol. This is
because the plasma clearance of cholesterol is often impaired in the presence of
low HDL-c. Triglycerides levels have also been found to increase with increase in
plasma cholesterol. Atherogenicity therefore develops when LDL cholesterol,
triglycerides, and total cholesterol are elevated relative to plasma HDL-c. Elevated
HDL-cholesterol improves the transportation of cholesterol from the plasma to the

67

liver for biotransformation and excretion, thereby preventing atheroma formation


and blood vessel occlusion (Ojiako and Nwanjo, 2005).
On evaluation of lipid ratios in the current study, we observed that Atherogenic
Index of Plasma (AIP) was significantly higher in tests as compared to controls.
AIP is a ratio calculated as (logTG)/HDL-c). Studies have shown the inverse
relationship that exist between triglycerides and high-density lipoprotein
cholesterol and that the ratio of TG to HDL-c is a strong predictor of infarction.
AIP is being used by some practitioners as a significant predictor of
atherosclerosis. It has been suggested that AIP values of -0.3 to 0.1 are associated
with low, 0.1 to 0.24 with medium, and above 0.24 with high cardiovascular risk.
So from this study as TG and HDL-c are both deranged, we therefore expect AIP to
be significantly raised. We observed AIP ratio of 1.36 in the test and 0.09 in
controls which are in concordance with the suggested cut-offs.
The administration of 150, 300 and 600mg/kg of A. boonei aqueous extract in rats
did not produce any significant weight changes in the animals. The extract
however produced hypolipidaemic and anti-atherogenic effect after five days of
treatment with 150mg/kg, which significantly (P 0.05 reduced total cholesterol
and LDL-c ratios of atherogenic indices to desirable levels.
Echitamine, the major constituent of the plant extract has been reported to be
responsible for most of the pharmacological activities observed in the plant extract.
68

Echitamine is an alkaloid with anti-oxidative and free radical scavenging


properties (Elisabetsky and Costa- campos, 2006). Anti-oxidants prevent the
oxidative modification of lipoproteins before their incorporation into the fatty
streaks of the arterial wall. Studies have shown that oxidation of lipids increases
their deposition on arterial walls, hence atherosclerosis and atherogenicity.
However, the exact mechanism by which anti-oxidants lower blood cholesterol is
not properly established. Galton and Krone (1991) suggested that it could be by
promoting the stimulation of cholesterol excretion in the faeces via its
biotransformation to bile acids.
However, Beliga et al., (2004) in a study on A. scholaris (which also contains
echitamine) reported that the alkaloid lowered serum cholesterol. The study also
showed that the alkaloid exhibited a dose and time dependent cholesterol antiperoxidative effect. This supports the current findings that the extract appeared to
lose its hypolidaemic activity at higher doses and prolonged administration. The
aqueous extract of A. boonei stem bark produced hypo-cholesterolaemic and antiatherogenic effects after five days treatment with 150mg/kg, which was not
observed at 300 and 600mg/kg. This may imply that low doses of the plant extract
could be used for short periods to manage hyperlipidaemic and atherogenic
conditions. These findings agree with the current use of the plant extract by folk
medicine practitioners as anti-hypertensive agent.
69

5.1

CONCLUSION

In conclusion, the aqueous extract of A. boonei has been shown to be effective


against chronic inflammation (formalin-induced paw edema) in a dose related
manner. This present study supports the claim in the use of the extract of A. boonei
in traditional medicine for the treatment of inflammatory conditions. The current
study also shows that A. boonei stem bark extract caused hypolipidaemic effect at
the tested doses.

5.2

RECOMMENDATIONS FOR FURTHER STUDIES

Further studies are suggested to elucidate the mechanisms of A. boonei stem bark
extract on blood lipids. Also further research should be designed in a dosedependent manner so as to accurately define the exact dose that exerts optimal
therapeutic and/or toxic effect.

70

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80

APPENDIX
CALCULATION OF MEAN S.E.M VALUES OF PAW THICKNESS IN
FORMALIN-INDUCED PAW EDEMA IN WISTAR RATS.
CONTROL
1
2
3
4
5
N
MEAN
S.E.M
150mg/kg
1
2
3
4
5
N
MEAN
S.E.M

Paw
size
Day 1
Day 2
Day 3
Day 4
Day 5
4.61
6.35
7.81
7.05
8.25
8.43
5.1
7.05
7.94
6.95
9.17
8.84
5.23
6.79
8.28
7.34
8.98
8.68
4.8
6.29
8.1
6.83
8.32
8.33
4.98
6.46
9.26
8.05
8.75
8.87
5
5
5
5
5
5
4.944
6.588
8.278
7.244
8.694
8.63
0.1094 0.1442 0.2578 0.2184 0.1800 0.1082
81
36
06
4
72
13
Paw
size
Day 1
Day 2
Day 3
Day 4
Day 5
5.24
7.3
10.33
8.67
8.52
7.99
5.51
7.09
7.8
6.79
7.94
8.25
5.33
7.15
8.78
7.42
8.58
8.2
4.99
6.31
8.63
7.79
8.52
8.37
5.54
7.69
8.7
7.25
6.97
7.71
5
5
5
5
5
5
5.322
7.108
8.848
7.584
8.106
8.104
0.0999 0.2252 0.4104 0.3154 0.3070 0.1160
7
2
07
93
44
86

81

300mg/kg
1
2
3
4
5
N
MEAN
S.E.M
600mg/kg
1
2
3
4
5
N
MEAN
S.E.M
Indomethac
in
1
2
3
4
5
N
MEAN
S.E.M

Paw
size
Day 1
Day 2
Day 3
Day 4
Day 5
4.47
6.27
8.3
7.61
8.85
7.18
5.19
7.15
8.77
7.59
8.47
7.79
4.77
6.59
9.05
8.13
8.22
7.85
5.24
6.94
8.73
7.77
7.88
7.67
5.44
6.83
9.12
7.41
8.78
8.5
5
5
5
5
5
5
5.022
6.756
8.794
7.702
8.44
7.798
0.1758 0.1513 0.1450 0.1212 0.1798 0.2114
81
8
03
6
05
1
Paw
size
Day 1
Day 2
Day 3
Day 4
Day 5
5.25
6.69
8.27
7.42
8.53
8.5
5.14
6.98
8.81
7.24
8.01
7.1
5.34
6.74
8.36
7.7
8.02
8.24
5.17
6.8
8.57
8.12
8.79
8.53
5.77
6.06
8.89
7.36
8.12
8.23
5
5
5
5
5
5
5.334
6.654
8.58
7.568
8.294
8.12
0.1143 0.1563 0.1211 0.1573 0.1561 0.2626
94
84
61
02
6
21
Paw
size
Day 1
Day 2
Day 3
Day 4
Day 5
4.83
6.23
8.89
7.03
7.73
7.27
5.52
6.83
8.93
7.65
7.58
7.37
5.03
6.58
8.7
6.91
7.97
7.66
5.24
6.93
9.07
8.32
8.45
7.89
5.74
6.75
9.23
8.14
9.33
7.96
5
5
5
5
5
5
5.272
6.664
8.964
7.61
8.212
7.63
0.1636 0.1227 0.0889 0.2839 0.3159 0.1368
82

28

03

72

89

83

81

58