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
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
1.3
JUSTIFICATION OF STUDY
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
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
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
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
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.
12
2.5
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
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
2.7
CHRONIC INFLAMMATION
19
macrophage
(mononuclear
cell)
infiltration,
tissue
destruction
by
inflammatory cells and repair with fibrosis and angiogenesis (new vessel
formation), (Hurley, 1972).
2.7.2
2.7.3
2.7.3.1
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
Features
Onset
Acute
Fast: minutes or hours
Chronic
Slow: days
Cellular infiltrate
Mainly neutrophils
Monocytes, macrophages
and lymphocytes
Usually mild and self- Often
severe
limited
Prominent
and
progressive
Less prominent; may be
subtle
(Hurley, 1972).
22
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
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
2.11.2
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
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
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
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:
33
Chylomicrons are responsible for the visual opalescence of the plasma (Alvarez,
1986).
(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
2.12.3
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
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
chylomicrons are removed, the latter are cleared from the bloodstream by the liver
(Nawaz, 2006).
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).
40
CHAPTER THREE
3.0
3.1
MATERIALS
3.1.2 APPARATUS
Spectrophotometer
Centrifuge
Gavage
41
3.2
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.
3.3
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
3.4
EXPERIMENTAL DESIGN
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.
80 mmol/L, PH 6.8
45
4-aminoantipyrine
0.25mmol/L
Phenol
6mmol/L
Peroxidase
0.5 U/mL
0.15 /mL
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
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
47
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
48
Glycerol-3-phosphate + glycerolphosphate-oxidase
dihydroacetone
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
49
REAGENT COMPOSITION
CONTENTS
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.
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
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).
CHAPTER FOUR
4.0
RESULTS
53
Table 1:
Dosage
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).
55
Variables
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
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:
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:
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:
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:
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
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
66
67
5.1
CONCLUSION
5.2
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
SELECTED REFERENCES
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Cultivated Plants, Intermediate Technology Publications, Royal Botanical
Garden, Kew, London. Pp.105.
Alters, S., and Shiff, P.W. (1997). Impact of body mass index on coronary heart
disease risk factors in men and women. Arterioscler. Thromb. Vasc. Biol. 16:
1509-15.
Alvarez, C., and Ramos, A. (1986). Lipids, lipoproteins and apolipoproteins in
serum during infection. Clin. Chem. 32:1425.
Amole, O.O., and Ilori, O.O. (2010). Antimicrobial Activity Of The Aqueous And
Ethanolic Extracts Of The Stem Bark Of Alstonia Boonei. International
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Asuzu, I.U., and Anaga, A.O. (1991). Pharmacological screening of the aqueous
extract of Alstonia boonei stem bark. Fitoter. 63:411417.
Ballantyne, C. M., Hoogeveen, R. C., and Bang, H. (2005). Lipoproteinassociated phospholipase A2, highsensitivity C-reactive protein, and risk for
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71
72
76
Nawaz, H., Comerford, B.P., Njike, V.Y., Dhond, A.J., Plavec, M., and Katz, D.L.
(2006). Repeated serum lipid measurements during the perihospitalization
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77
Olajide, O.A., Awe, S.O., Makinde, J.M., Ekhelar, A.I., Olusola, A., Morebise, O.,
and Okpako, D.T. (2000). Studies on the anti-inflammatory, antipyretic and
analgesic properties of Alstonia boonei stem bark. J Ethnopharmacol 71 (12): 179-186.
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extract of Alstonia boonei (De Wild) stem bark in Guinea pigs. The Internet
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in male rats treated with methanol extract of Alstonia boonei stem bark.
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Sahu, S., Chawla, R., and Uppal, B. (2005). Comparison of two methods of
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Friedewald estimation. Indian J. Clin. Biochem. 20: 5461.
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(1998). Activity of stem bark of Alstonia boonei de wild on human
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Bailliere-Tindall Ltd., London, pp. 553.
79
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