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TITLE PAGE
STUDIES ON ANTI-INFLAMMATORY PROPERTIES OF THE LEAF EXTRACTS OF
FICUS EXASPERATA Vahl (MORACEAE)
BY
NWUKE HENRY CHINYERE
PG/M.PHARM/08/48253.
A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY
AND TOXICOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY
OF NIGERIA NSUKKA, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR
THE AWARD OF MASTER OF PHARMACY (M.PHARM) DEGREE.
DR. C. S NWORU PROF. P.A AKAH
(SUPERVISOR) (SUPERVISOR)
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY
FACULTY OF PHARMACEUTICAL SCIENCES
UNIVERSITY OF NIGERIA,
NSUKKA
MAY, 2012.
CHAPTER ONE
INTRODUCTION
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1.1 Scientific background of study
Inflammation (coined from latin word “inflamatio”, which means to set on fire) is a complex
biological response of vascular tissues to harmful stimuli, such as pathogens, damage to cells,
or irritants. Inflammation is marked by local response to cellular injury that is associated with
capillary dilatation, leucocytes infiltration, redness, heat, pain, swelling, and often loss of
function that serves as a mechanism initiating of eliminating of noxious agents and damaged
tissues. Inflammation involves a complex array of enzyme activation, mediator release,
extravasation of fluid, cell migration, tissue breakdown, and repair (Vane and Bolting, 1995;
Perianayagam et al, 2006). The importance of inflammation and the need for novel anti-
inflammatory principles can be highlighted by the increased research interest and focus on
inflammation and antiinflammatoiry substances. Many human and animal diseases, such as
arthritic disorders, lupus erythematosus, asthma, bronchitis, inflammatory bowel disease,
ulcerative colitis, pancreatitis, ascities, hepatitis, cancer, infections are associated with
inflammation.
Despite numerous progresses made in the use or orthodox medicines in the treatment of
inflammatory conditions, there is still need for more cost-effective and improved remedies
with less gastro-erosive side effects, especially for the rural poor. In this regards, medicinal
plants and herbal remedies have been employed in Complementary and Alternative Medicine
(CAM) for the treatment of inflammation and disorders associated with inflammations.
Conventional drug treatments are limited in their effectiveness in managing the incidence and
outcome of many inflammatory diseases. They also present a significant number of side-
effects in patients. Recently, it has been shown that non-steroidal anti-inflammatory agents
may even slow down the healing process in many diseases (Ayoola et al., 2009). There is
therefore an urgent need to find safer and more effective drug treatments.
Common anti-inflammatory therapy and treatments include rest, light exercise, weight
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maintenance, stretching, and medications designed to reduce the inflammation and control
the pain. The medications include Non Steroidal Anti-Inflammatory Drugs (NSAIDs) and
steroid medications. The NSAIDs are widely used as the initial form of therapy, although
well tolerated; they can irritate the stomach and lead to ulcers. In some instances, long term
use can lead to kidney problems (Linton, 1984).
The treatment of inflammation and rheumatic disorder is an area in which the practitioners of
traditional medicine enjoy patronage and success (Akah and Nwambie, 1994). Natural
products in general and medicinal plants in particular, are believed to be an important source
of new chemical substances with potential therapeutic efficacy. Taking into account that the
many important anti-inflammatory prototypes (e.g. salicylates) were originally derived from
the plant sources, the study of plant species traditionally used as anti-inflammtory agents
would still be seen as a fruitful strategy in the search of new anti-inflammatory drugs.
The plant Ficus exasperata (Moracea) is one of the many medicinal plants used in folk
medicine to threat inflammation and inflammatory disorders by the Igede people of Benue
state, Nigeria (Igoli et al., 2005). In this study, I investigated the acute and chronic anti-
inflammatory properties of the leaf extract of Ficus exasperata in topical and in vivo rodents‟
models. The possible mechanisms of action of F. exasparata extract were also investigated in
vitro on the production of pro-inflammatory cytokines and inducible nitric oxide in cultures
of bone-marrow derived macrophages.
1.2 Mechanisms and processes of inflammation
1.2.1 Mechanisms of inflammation
Inflammation includes a sequence of reactions initially involving cytokines, neutrophils,
adhesion molecules, complement, and Immunoglobulin G (IgG). Platelets activating factors
(PAF), an agent with potent inflammatory effects, also plays a role. Later, monocytes and
lymphocytes are involved. Arterioles in the inflamed area dilate, and capillary permeability is
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increased. When the inflammation occurs in or just under the skin, it is characterized by
redness, swelling, tenderness, and pain. Elsewhere, it is a key component of asthma,
ulcerative colitis, and many other diseases. (Ross, 1999; Ganong, 2003) Evidence is
accumulating that a transcription factor, nuclear factor-κB (NF-κB) plays a key role in the
inflammatory response. NF-κB is a heterodimer that normally exists in the cytoplasm of cells
bound to IκBα, which renders it inactive. Stimuli such as cytokines, viruses, and oxidants
separate NF-κB from IκBα, which is then degraded. NF-κB moves to the nucleus, where it
binds to the DNA of the genes for numerous inflammatory mediators, resulting in their
increased production and secretion. Glucocorticoids inhibit the activation of NF-κB by
increasing the production of IκBα, and this is probably the main basis of their anti-
inflammatory action (Singer and Clark 1999; Ganong, 2003). The ability to mount an
inflammatory response is essential for survival in the face of environmental pathogens and
injury; in some situations and diseases, the inflammatory response may be exaggerated and
sustained without apparent benefit and even with severe adverse consequences. Inflammatory
responses occur in three distinct temporal phases, each apparently mediated by different
mechanisms: (1) an acute phase characterized by transient local vasodilation and increased
capillary permeability; (2) a delayed, subacute phase characterized by infiltration of
leukocytes and phagocytic cells; and (3) a chronic proliferative phase, in which tissue
degeneration and fibrosis occur.
Many mechanisms are involved in the promotion and resolution of the inflammatory process
(Kyriakis and Avruch,2001;Serhan and Chiang, 2004) . Although earlier studies emphasized
the promotion of migration of cells out of the microvasculature, recent work has focused on
adhesive interactions, including the E-, P-, and L-selectins, intercellular adhesion molecule-1
(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and leukocyte integrins, in the
adhesion of leukocytes and platelets to endothelium at sites of inflammation (Meager, 1999).
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Activated endothelial cells play a key role in "targeting" circulating cells to inflammatory
sites. Expression of the adhesion molecules varies among cell types involved in the
inflammatory response. Cell adhesion occurs by recognition of cell-surface glycoproteins and
carbohydrates on circulating cells due to the augmented expression of adhesion molecules on
resident cells. Thus, endothelial activation results in leukocytes adhesion as the leukocytes
recognize newly expressed L-selectin and P-selectin; other important interactions include
those of endothelial-expressed E-selectin with sialylated Lewis X and other glycoproteins on
the leukocyte surface and endothelial ICAM-1 with leukocyte integrins. It has been proposed
that some, but not all, NSAIDs may interfere with adhesion by inhibiting expression or
activity of certain of these cell-adhesion molecules (Diaz-Gonzalez and Sanchez-Madrid,
1998). Novel classes of anti-inflammatory drugs directed against cell-adhesion molecules are
under active development but have not yet entered the clinical arena. In addition to the cell-
adhesion molecules outlined above, the recruitment of inflammatory cells to sites of injury
involves the concerted interactions of several types of soluble mediators. These include the
complement factor C5a, platelet-activating factor, and the eicosanoid LTB4 (Burke et al.,
2006). All can act as chemotactic agonists. Several cytokines also play essential roles in
orchestrating the inflammatory process, especially interleukin-1 (IL-1) and tumor necrosis
factor (TNF) (Dempsey et al., 2003). IL-1 and TNF are considered principal mediators of the
biological responses to bacterial lipopolysaccharide (LPS, also called endotoxin). They are
secreted by monocytes and macrophages, adipocytes, and other cells. Working in concert
with each other and various cytokines and growth factors (including IL-8 and granulocyte-
macrophage colony-stimulating factor), they induce gene expression and protein synthesis in
a variety of cells to mediate and promote inflammation (Burke et al., 2006).
IL-1 comprises two distinct polypeptides (IL-1a and IL-1b) that bind to the same cell-surface
receptors and produce similar biological responses. Plasma IL-1 levels are increased in
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patients with active inflammation. IL-1 can bind to two types of receptors, an 80-kd IL-1
receptor type 1 and a 68-kd IL-1 receptor type 2, which are present on different cell types.
TNF, originally termed "cachectin" because of its ability to produce a wasting syndrome, is
composed of two closely related proteins: mature TNF (TNF-α) and lymphotoxin (TNF-β),
both of which are recognized by the same cell-surface receptors. There are two types of TNF
receptors, a 75-kd type 1 receptor and a 55-kd type 2receptor. IL-1 and TNF produce many of
the same pro-inflammatory responses. A naturally occurring IL-1 receptor antagonist (IL-
1ra), competes with IL-1 for receptor binding, blocks IL-1 activity in vitro and in vivo, and in
experimental animals can prevent death induced by administration of bacteria or LPS. IL-1ra
often is found in high levels in patients with various infections or inflammatory conditions.
Thus, the balance between IL-1 and IL-1ra may contribute to the extent of an inflammatory
response.
Other cytokines and growth factors [e.g., IL-2, IL-6, IL-8, and granulocyte/macrophage
colony stimulating factor (GM-CSF)] contribute to manifestations of the inflammatory
response. The concentrations of many of these factors are increased in the synovia of patients
with inflammatory arthritis. Certain relevant peptides, such as substance P, which promotes
firing of pain fibers, also are elevated and act in concert with cytokines at the site of
inflammation. Other cytokines and growth factors counter the effects and initiate resolution
of inflammation. These include transforming growth factor-β1 (TGF-β1), which increases
extracellular matrix formation and acts as an immunosuppressant, IL-10, which decreases
cytokine and prostaglandin E2 formation by inhibiting monocytes, and interferon gamma,
IFN-γ, which possesses myelosuppressive activity and inhibits collagen synthesis and
collagenase production by macrophages. Histamine was one of the first identified mediators
of the inflammatory process. Although several H1 histamine-receptor antagonists are
available, they are useful only for the treatment of vascular events in the early transient phase
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of inflammation (Burke, et al., 2006). Bradykinin and 5-hydroxytryptamine (serotonin, 5-HT)
also may play a role in mediating inflammation, but their antagonists ameliorate only certain
types of inflammatory response (Burke et al., 2006). Leukotriene (LT)-receptor antagonists
(montelukast and zafirlukast) exert anti inflammatory actions and have been approved for the
treatment of asthma (Burke et al., 2006). Another lipid autacoid, platelet-activating factor
(PAF), has been implicated as an important mediator of inflammation; however, inhibitors of
PAF synthesis and PAF-receptor antagonists have proven disappointing in the treatment of
inflammation (Burke et al., 2006). Intradermal, intravenous, or intra-arterial injections of
small amounts of prostaglandins mimic many components of inflammation. Administration
of prostaglandin E2 (PGE2) or prostacyclin (PGI2) causes erythema and an increase in local
blood flow. Such effects may persist for up to 10 h with PGE2 and include the capacity to
counteract the vasoconstrictor effects of substances such as norepinephrine and angiotensin
II, properties not generally shared by other inflammatory mediators. In contrast to their long-
lasting effects on cutaneous vessels and superficial veins, prostaglandin-induced vasodilation
in other vascular beds vanishes within a few minutes. Although PGE1 and PGE2 (but not
PGF2α) cause edema when injected into the hind paw of rats, it is not clear if they can
increase vascular permeability in the postcapillary and collecting venules without the
participation of other inflammatory mediators (e.g., bradykinin, histamine, and leukotriene
C4 [LTC4]). Furthermore, PGE1 is not produced in significant quantities in humans in vivo,
except under rare circumstances such as essential fatty acid deficiency. Unlike LTs,
prostaglandins are unlikely to be involved in chemotactic responses, even though they may
promote the migration of leukocytes into an inflamed area by increasing blood flow.
1.2.2 Processes of inflammation
Inflammation is characterized by the orderly occurrence of several processes:
I. Initiation of the event by a foreign substance or physical injury.
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II. Recruitment and Chemoattraction of inflammatory cells, and activation of these
cells.
III. Release of inflammatory mediators capable of damaging or killing an invading
microbe or tumour. In some instances, the inflammatory response is initiated by an
otherwise harmless foreign material (e.g., pollen). Inflammation can also result from
an autoimmune response to the host‟s own tissue, as occurs in rheumatoid arthritis.
As the result of an inflammatory response, the host tissue may undergo collateral injury, since
many of the inflammatory mediators are not specific for a particular tissue target. For
example, many of the clinical signs (fever and laboured breathing) and symptoms (shortness
of breath and cough) of pneumococcal pneumonia are the result of inflammation rather than
the invading microorganism. In most cases, the inflammatory response eventually subsides,
but if such a self-limiting regulation does not occur, the inflammatory response will require
pharmacological intervention.
1.3 Phases of inflammatory response
1.3.1 Acute phase
This rapid phase occurs within seconds to min and consists of vasodilation, increased blood
flow, edema, and pain. The acute phase is characterized by induction of inflammatory genes
by NF-_B and other transcription factors. During this phase, moderate amounts of
inflammatory mediators are produced (Craig and Stitzel, 1999)
Within min after inflammation begins, the macrophages already present in the tissues,
whether histiocytes in the subcutaneous tissues, alveolar macrophages in the lungs, microglia
in the brain, or others, immediately begin their phagocytic actions. When activated by the
products of infection and inflammation, the first effect is rapid enlargement of each of these
cells. Next, many of the previously sessile macrophages break loose from their attachments
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and become mobile, forming the first line of defence against infection during the first hour or
so. The numbers of these early mobilized macrophages often are not great, but they are
lifesaving (Guyton and Hall, 2006).Generally, acute inflammation is a reversible process
(Djukanovic et al., 1990). However, there may be serious problems when organ function is
compromised, for example in meningitis, hepatitis and asthma. The inflammatory reactions
also usually subside soon and inflammation is unlikely to cause permanent damage if treated
promptly. It is the sequelae to inflammation, that is the resolution and healing process, which
may sometimes cause permanent damage (Green et al, 1993).
1.3.2 Chronic inflammation
Chronic inflammatory diseases remain one of the world‟s major health problems (Bohlin,
1995; Yesilada et al., 1997; Li et al.,2003). The chronic phase occurs over months to years
and is marked by dramatically increased production of inflammatory mediators. The
secondary chronic phase of inflammation occurs after years of oxidative damage has
degraded blood vessels and tissues. Such chronic inflammation appears to play a role in many
disease states, such as arteriosclerosis and cancer (Craig and Stitzel 1999). Patients with
chronic inflammation associated with diseases such as rheumatoid arthritis are often treated
with glucocorticoids and may develop some of the clinical symptoms of Cushing‟s syndrome
(Guyton and Hill 2006).
Recruitment and activation of specific subsets of inflammatory and immune cells are
essential determinants of the pathologic features. In this regard, the role of activation of
regional blood vessel endothelium by pro-inflammatory cytokines (eg, tumor necrosis factor
[TNF]-α, interleukin [IL]-1) must be emphasized. Several cytokines induce the expression on
endothelial cells of ligands for the adhesion-promoting receptors of inflammatory cells
(integrins and selectins) and allow neutrophils and monocytes to adhere to the vessel wall in
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the inflamed area and migrate into the underlying tissues. The pathologic features of the
chronic inflammatory disorders reflect the combination of inflammatory damage and the
consequences of healing (Ali et al.,1997; McPhee et al., 2006).
Immune complex formation and deposition are important pathophysiologic mechanisms in
autoimmune rheumatic diseases. Any antigen that elicits a humoral immune response may
give rise to circulating immune complexes if the antigen remains present in abundant
quantities once antibody is generated. Immune complexes are efficiently cleared by the
reticuloendothelial system and are rarely pathogenic. Pathogenicity is a function of the
relative amounts of antigen and antibody and of the intrinsic features of the complex that
determine its overall composition, size, and solubility. Of particular significance in terms of
pathogenicity are immune complexes formed at slight antigen excess that are soluble, are not
effectively cleared by the reticuloendothelial system, and are of a size that allows them to
gain access to and be deposited at subendothelial and extravascular sites. Thus, if foreign
antigens (eg, drugs or infectious organisms) induce an antibody response and significant
numbers of immune complexes of the appropriate size are formed, these complexes may be
deposited (in skin, joints, kidney, blood vessel walls) where they activate several effector
pathways (eg, FcR receptor, classic complement cascade) and where they may lead to skin
rashes, arthritis, glomerulonephritis, and palpable purpura. Clinical conditions in which this
situation might arise include drug reactions, serum sickness, McPhee et al, 2006) and
infections (infective endocarditis, streptococcal skin and pharyngeal infections, and others).
Autoimmune diseases are characteristically antigen driven, but in this case the humoral
response is directed against self-antigens (eg, nucleosomes in SLE). Under conditions leading
to the liberation of significant amounts of self-antigen from host tissue (cell damage or
death), immune complex formation, Fc receptor binding, and complement activation may
result. The consequences of immune complex formation and deposition are similar whether
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caused by foreign or self-antigens (McPhee et al., 2006).
In response to inflammatory mediators (including cytokines) and T cells, cells in tissues
ordinarily unrelated to the immune response can alter their form and function to support (and
in some cases drive) a chronic inflammatory response (Lanzavecchia, 1995).
1.4 MEDIATORS OF INFLAMMATION
The more important inflammatory mediators are the eicosanoids, biological oxidants,
cytokines, adhesion factors, histamine, kinins, platelet activating factor and digestive
Enzymes (proteases, hyaluronidase, collagenase, and elastase). Only the first three of these
are therapeutic targets for anti-inflammatory drugs (McPhee et al., 2006).
1.4.1 Eicosanoids
Membrane lipids supply the substrate for the synthesis of eicosanoids and platelet-activating
factor. Eicosanoids; arachidonate metabolites, including prostaglandins, prostacyclin,
thromboxane A2, leukotrienes, lipoxins and hepoxylins are not stored but are produced by
most cells when a variety of physical, chemical, and hormonal stimuli activate acyl
hydrolases that make arachidonate available(Burke et al, 2006). Eicosanoids play a major
role in the inflammatory and immune responses, as reflected by the clinical usefulness of the
NSAIDs. While LTs generally are pro-inflammatory and lipoxins anti-inflammatory,
prostanoids can exert both kinds of activity.
LTB4 is a potent chemotactic agent for polymorphonuclear leukocytes, eosinophils, and
monocytes (Martel-Pelletier et al., 2003). In higher concentrations, LTB4 stimulates the
aggregation of polymorphonuclear leukocytes and promotes degranulation and the generation
of superoxide. LTB4 promotes adhesion of neutrophils to vascular endothelial cells and their
transendothelial migration and stimulates synthesis of pro-inflammatory cytokines from
macrophages and lymphocytes. Prostaglandins generally inhibit lymphocyte function and
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proliferation, suppressing the immune response (Rocca and FitzGerald, 2002). PGE2
depresses the humoral antibody response by inhibiting the differentiation of B-lymphocytes
into antibody-secreting plasma cells. PGE2 acts on T-lymphocytes to inhibit mitogen-
stimulated proliferation and lymphokine release by sensitized cells. PGE2 and TxA2 also may
play a role in T-lymphocyte development by regulating apoptosis of immature thymocytes
(Tilley et al., 2001). PGD2, a major product of mast cells, is a potent chemoattractant for
eosinophils and induces chemotaxis and migration of Th2 lymphocytes (Smyth and
FitzGerald, 2003). The degradation product, 15d-PGJ2, also may activate eosinophils via the
DP2 (CRTH2) receptor (Monneret et al., 2002).
Lipoxins have diverse effects on leukocytes, including activation of monocytes and
macrophages and inhibition of the activation of neutrophils, eosinophils, and lymphocytes
(McMahon and Godson, 2004).
In vasculature, locally generated PGE2 and PGI2 modulate vascular tone. PGI2, the major
arachidonate metabolite released from the vascular endothelium, is derived primarily from
COX-2 in humans (Catella-Lawson et al., 1999; McAdam et al., 1999) and is regulated by
shear stress and by both vasoconstrictor and vasodilator autacoids. Knockout studies argue
against a role for PGI2 in the homeostatic maintenance of vascular tone; PGI synthase
polymorphisms have been associated with essential hypertension and myocardial infarction
(Smyth and FitzGerald, 2003). PGI2 limits pulmonary hypertension induced by hypoxia and
systemic hypertension induced by angiotensin II. Deficiency of EP1 or EP4 receptors reduces
resting blood pressure in male mice; EP1-receptor deficiency is associated with elevated
renin-angiotensin activity. Both EP2- and EP4-receptor-deficient animals develop
hypertension in response to a high-salt diet, reflecting the importance of PGE2 in maintenance
of renal blood flow and salt excretion. PGI2 and PGE2 are implicated in the hypotension
associated with septic shock. PGs also may play a role in the maintenance of placental blood
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flow.
COX-2-derived PGE2, via the EP4 receptor, maintains the ductus arteriosus patent until birth,
when reduced PGE2 levels (a consequence of increased PGE2 metabolism) permit closure of
the ductus arteriosus (Coggins et al., 2002). The tNSAIDs induce closure of a patent ductus
in neonates. Contrary to expectation, animals lacking the EP4 receptor die with a patent
ductus during the perinatal period because the mechanism for control of the ductus in utero,
and its remodeling at birth, is absent. PGI2 specifically limits TxA2-induced smooth muscle
proliferation in vascular injury, suggesting a role for these prostanoids in vascular remodeling
(Cheng et al., 2002).
PGs and LTs are synthesized in response to a host of stimuli that elicit inflammatory and
immune responses, and eicosanoids contribute importantly to inflammation and immunity
(Tilley et al., 2001; Brink et al., 2003). Prostanoid biosynthesis is increased significantly in
inflamed tissue. Recruitment of leukocytes and the induction of COX-2 expression by
inflammatory stimuli provided a rational basis for the development of COX-2-specific
inhibitors for treatment of chronic inflammatory diseases. However, COX-1 also has a role in
inflammation: It appears that COX-1 is responsible for acute and COX-2 for sustained
prostanoid production following an inflammatory stimulus.
Prostanoids generally promote acute inflammation, although there are some exceptions, such
as the inhibitory actions of PGE2 on mast cell activation (Tilley et al., 2001). Furthermore,
deletion of COX-2 and, to a lesser extent, deletion of COX-1 are associated with greater
severity of inflammatory colitis, consistent with the exacerbation of inflammatory bowel
disease seen in patients receiving tNSAIDs. Both PGE2 and PGI2 markedly enhance edema
formation and leukocyte infiltration by promoting blood flow in the inflamed region. Both
have been associated with inflammatory pain, and both potentiate the pain-producing activity
of bradykinin and other autacoids.
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LTs are potent mediators of inflammation. Deletion of 5-LOX or FLAP reduces
inflammatory responses (Austin and Funk, 1999). Generation of BLT1-deficient mice
confirms the role of LTB4 in chemotaxis, adhesion, and recruitment of leukocytes to
inflamed tissues (Toda et al., 2002). Increased vascular permeability resulting from innate
and adaptive immune challenges is offset in mice deficient in CysLT1 or LTC4 synthase
(Kanaoka and Boyce, 2004). Deletion either of LTC4 synthase (and thus loss of CysLT
biosynthesis) or CysLT2 reduced chronic pulmonary inflammation and fibrosis in response to
bleomycin. In contrast, absence of CysLT1 led to an exaggerated response. These findings
demonstrate a role for CysLT2 in promoting, and an unexpected role for CysLT1 in
counteracting, chronic inflammation.
1.4.2 Biological oxidants
The biologically derived oxidants are potent bacterial killers but are also a major contributing
factor in tissue injury that results from the inflammatory response. These oxidants include the
superoxide anion (O2), hydrogen peroxide (H2O2), nitric oxide (NO), peroxynitrite (OONO),
hypochlorous acid (HOCl), peroxidase-generated oxidants of undefined character, probably
the hydroxyl radical (OH), and possibly singlet oxygen (O1/2). These oxidants, largely
generated by phagocytic cells such as neutrophils and macrophages, induce tissue injury
beyond that produced by digestive enzymes and eicosanoids. Inhibition of production of
these oxidants or inactivation of these substances by antioxidants is an important strategy for
the treatment of inflammatory disorders (Craig and Stitzel, 1999).
1.4.3 Cytokines
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Distinct classes of immune effector function are activated depending on the pattern of
cytokines that predominate during initiation of the inflammatory response. For example,
some cytokines (e.g. IL-12) produced by infected monocyte-macrophages skew the
lymphocyte response toward TH1 cells (which generate the TH1 cytokines IL-2, interferon-γ,
and TNF-α) that are associated with activation of macrophage killing functions. In contrast,
the presence of IL-4 during the initial response induces the differentiation of TH1
lymphocytes, which generate TH2 cytokines (e.g. IL-4, IL-5, IL-6, and IL-10). These
cytokines have their predominant function in the activation of B cells and antibody
generation. Although significant overlap exists, specific pathologic features tend to
accompany the different cytokine patterns (eg, granulomas for TH1 versus immune complex
disease for TH2). In addition, significant recent data point to an important role for type I
interferons (IFN-γ and IFN-α) in inducing novel pathways of monocyte differentiation in
patients with SLE that enhance responses to self-antigens.
1.4.4. Histamine
The release of histamine only partially explains the biological effects that ensue from
immediate hypersensitivity reactions. This is so because a broad spectrum of other
inflammatory mediators is released on mast cell activation. Stimulation of IgE receptors also
activates phospholipase A2 (PLA2), leading to the production of a host of mediators,
including platelet-activating factor (PAF) and metabolites of arachidonic acid. Leukotriene
D4, which is generated in this way, is a potent contractor of the smooth muscle of the
bronchial tree. Kinins also are generated during some allergic responses. Thus the mast cell
secretes a variety of inflammatory mediators in addition to histamine, each contributing to the
major symptoms of the allergic response. The principal target cells of immediate
hypersensitivity reactions are mast cells and basophils (Schwartz, 1994). As part of the
allergic response to an antigen, reaginic (IgE) antibodies are generated and bind to the
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surfaces of mast cells and basophils via high-affinity Fc receptors that are specific for IgE.
Increased "Capillary" Permeability;Is the effect of histamine on small vessels results in
outward passage of plasma protein and fluid into the extracellular spaces, an increase in the
flow of lymph and its protein content, and edema formation. H1 receptors on endothelial cells
are the major mediators of this response; the role of H2 receptors is uncertain.
Increased permeability results mainly from actions of histamine on postcapillary venules,
where histamine causes the endothelial cells to contract and separate at their boundaries and
thus to expose the basement membrane, which is freely permeable to plasma protein and
fluid. The gaps between endothelial cells also may permit passage of circulating cells that are
recruited to the tissues during the mast cell response. Recruitment of circulating leukocytes is
promoted by H1-receptor-mediated up-regulation of leukocyte adhesion. This process
involves histamine-induced expression of the adhesion molecule P-selectin on the endothelial
cells (Gaboury et al., 1995).
1.4.5 Kinins
A number of factors, including tissue damage, allergic reactions, viral infections, and other
inflammatory events, activate a series of proteolytic reactions that generate bradykinin and
kallidin in the tissues. These peptides contribute to inflammatory responses as autacoids that
act locally to produce pain, vasodilation, and increased vascular permeability. Much of their
activity is due to stimulation of the release of potent mediators such as prostaglandins, NO, or
endothelium-derived hyperpolarizing factor (EDHF).
A number of interesting discoveries have contributed to the elucidation of the functions of
kinins. Kinin metabolites released by basic carboxypeptidases that were formally considered
inactive degradation products are agonists of a receptor (B1) that differs from that of intact
kinins (B2), whose expression is induced by tissue injury. Kinins and their des-Arg
metabolites also release vasoactive agents and may be mediators of inflammation and pain.
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These findings may open novel avenues for therapeutic intervention in chronic inflammatory
conditions.
Kinins participate in a variety of inflammatory diseases. Plasma kinins increase permeability
in the microcirculation. The effect, like that of histamine and serotonin in some species, is
exerted on the small venules and involves separation of the junctions between endothelial
cells. This, together with an increased hydrostatic pressure gradient, causes edema. Such
edema, coupled with stimulation of nerve endings, results in a "wheal and flare" response to
intradermal injections in human beings.
In hereditary angioedema, bradykinin is formed, and there is depletion of the components of
the kinin cascade during episodes of swelling, laryngeal edema, and abdominal pain. B1
receptors on inflammatory cells such as macrophages can elicit production of the
inflammatory mediators interleukin 1 (IL-1) and tumor necrosis factor a (TNF-α) (Dray and
Perkins, 1993). Kinin levels are increased in a number of chronic inflammatory diseases,
including rhinitis caused by inhalation of antigens and that associated with rhinoviral
infection. Kinins may be significant in conditions such as gout, disseminated intravascular
coagulation, inflammatory bowel disease, rheumatoid arthritis, and asthma. Kinins also may
contribute to the skeletal changes seen in chronic inflammatory states. Kinins stimulate bone
resorption through B1 and possibly B2 receptors, perhaps by osteoblast-mediated osteoclast
activation.
1.4.6 Platelet-activating factor (PAF)
PAF is synthesized by platelets, neutrophils, monocytes, mast cells, eosinophils, renal
mesangial cells, renal medullary cells, and vascular endothelial cells. PAF is released from
monocytes but retained by leukocytes and endothelial cells. In endothelial cells, it is
displayed on the surface for juxtacrine signaling (Prescott et al., 2000). PAF increases
vascular permeability and edema in the same manner as histamine and bradykinin. The
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increase in permeability is due to contraction of venular endothelial cells, but PAF is more
potent than histamine or bradykinin by three orders of magnitude (Burke, et al., 2006). PAF
stimulates polymorphonuclear leukocytes to aggregate, to release LTs and lysosomal
enzymes, and to generate superoxide.The proinflammatory actions of PAF and its elaboration
by endothelial cells, leukocytes, and mast cells under inflammatory conditions are well
characterized. PAF and PAF-like molecules are thought to contribute to the pathophysiology
of inflammatory disorders, including anaphylaxis, bronchial asthma, endotoxic shock, and
skin diseases ( Burke et al., 2006).
1.4.7 Complement pathway
The classic complement pathway is activated when antibody binds to its specific antigen.
Activation of the complement cascade induces inflammatory cell recruitment and activation
(with all the consequences mentioned later) as well as other features of the acute
inflammatory response (e.g., increased capillary permeability).
1.5 Cells and cellular processes of inflammation
1.5.1 Myelo-monocytic cells (macrophages and neutrophils)
Although macrophages and neutrophils have numerous effector pathways that function to rid
the host of foreign invaders, some of these effector mechanisms can damage healthy tissue if
released in large amounts. These include free radical species generated during the respiratory
burst as well as a variety of secretory products contained in the granules of these
inflammatory cells. Important granule contents include a variety of proteases such as
cathepsins, elastase, and collagenase. These products are liberated into the extracellular
medium in the inflammatory locus, where they accumulate and may have damaging effects
on normal connective tissue. In addition, numerous pro-inflammatory mediators released in
this environment (including TNF-γ, IL-1, IL-6, prostaglandins, and leukotrienes) attract
further inflammatory cells to the area and amplify the potential to generate tissue damage if
19
the inflammatory response is not adequately quenched (Sercarz, 1993).
1.5.2. Lymphocyte-mediated cytotoxicity
Certain T lymphocytes are capable of killing target cells. When target cell destruction
exceeds the capacity for renewal, impaired tissue function can result. As with other
lymphocyte functions, this effector function is activated only on ligation of the T-cell
receptor by a specific peptide (bound within the cleft of a major histocompatibility complex
[MHC] molecule). On recognition of antigen on the surface of a target cell, cytotoxic T
lymphocytes induce the death of those cells, using several distinct mechanisms. One
prominent mechanism involves the Fas-Fas-ligand (FasL) pathway, whereby FasL present on
activated lymphocytes binds to the Fas receptor on target cells and activates target cell
apoptosis. The second prominent mechanism involves the release of cytotoxic T-lymphocyte
secretory granules. These granules contain at least two distinct classes of proteins. One,
called perforin, allows water, salt, and proteins (including the second class of granule protein,
the granzymes) to enter the target cell cytoplasm through mechanisms that still remain
unclear. The granzymes, a family comprising several proteases, target a number of critical
cellular substrates and activate the process of apoptosis (programmed cell death) within the
target cell (Sercarz, 1993).
1.5.3 Antibody-dependent cellular cytotoxicity
The destruction of antibody-coated target cells by natural killer cells is called antibody-
dependent cellular cytotoxicity (ADCC) and occurs when the Fc receptor of a natural killer
(NK) cell binds to the Fc portion of the surface-bound antibody. The cytotoxic mechanism
involves the release of cytoplasmic granules containing perforin and granzymes into the
cytoplasm of the antibody-coated cell (similar to cytotoxic T lymphocyte-mediated killing,
20
described previously). This mechanism has been implicated in autoantibody-mediated
syndromes, in which the autoantigen resides at the cell surface or relocates to this site after an
insult. An example of this is the photosensitive skin disease that occurs in patients with SLE
who possess the Ro autoantibody. On exposure to ultraviolet light, the Ro antigen is released
from keratinocytes and binds to their surface. Circulating Ro antibodies bind the antigen at
this site, with induction of FcR-mediated effector pathways (Sercarz, 1993; McPhee et al.,
2006).
1.6 Antiinflammatory drugs
The overall therapeutic goals in the treatment of inflammation are: the relief of pain which is
often the presenting system and the major continuing complaint of the patient; and slowing or
in theory-arrest of the tissue damaging process. (Katzung, 1998). Anti-inflammatory drugs
are classified into steroidal anti-inflammatory drugs (glucocorticoids), non-steroidal anti-
inflammatory drugs (NSAID), disease modifying anti-rheumatic drugs (DMARDS) and
biologic response modifiers (BRMs).
1.6.1 Steroidal anti-inflammatory drugs (Glucocorticoids)
Glucocorticoids possess a wide range of effects on virtually every phase and component of
the inflammatory and immune responses, they have assumed a major role in the treatment of
a wide spectrum of diseases with an inflammatory or immune-mediated component.
Rheumatoid arthritis is the original condition for which anti-inflammatory steroids were used,
and they remain a mainstay of therapy. Intra-articular glucocorticoid injections have proven
to be efficacious, particularly in children. However, the detrimental effects of glucocorticoids
on growth are significant for children with active arthritis. Although steroids offer
symptomatic relief from this disorder by abolishing the swelling, redness, pain, and effusions,
21
they do not cure. Progressive deterioration of joint structures continues, and the disease
process may be exacerbated after steroid therapy is terminated (Craig and Stitzel, 1999).
Based on the concept that asthma is an inflammatory disease that leads to airway obstruction,
inhaled glucocorticoids are the first-line treatment for moderate to severe asthma. Steroids are
used in other collagen diseases, such as lupus erythematosus; in hypersensitivity or allergic
states, such as nephrotic syndrome, ulcerative colitis, and Crohn‟s disease; in granulomatous
disease, such as sarcoid; and in a wide range of dermatological and ophthalmological
conditions. Glucocorticoids may also be used at lower doses in combination with other drugs
for the treatment of vasculitis, lupus nephritis, and amyloidosis.
Corticosteroids are the mainstay of therapy for inflammatory demyelinating
polyneuropathies. In Guillain- Barré syndrome glucocorticoids reduce the inflammatory
attack and improve final outcome, while in chronic inflammatory demyelinating
polyneuropathy, glucocorticoids suppress the immune reaction but may not retard the
progression of the disease. Glucocorticoids are used as adjunctive therapy in Pneumo cystitis
carinii pneumonia to decrease the inflammatory response and allow time for antimicrobial
agents to exert their effects (Craig and Stitzel, 1999).
1.6.2 Non-steroidal anti-inflammatory drugs (NSAIDs)
Most currently available traditional NSAIDs (tNSAIDs) act by inhibiting the prostaglandin
G/H synthase enzymes, colloquially known as the cyclo-oxygenases. The inhibition of
cyclooxygenase-2 (COX-2) is thought to mediate, in large part, the antipyretic, analgesic, and
anti-inflammatory actions of tNSAIDs, while the simultaneous inhibition of cyclooxygenase-
1 (COX-1) largely but not exclusively accounts for unwanted adverse effects in the
gastrointestinal tract. Selective inhibitors of COX-2 are a subclass of NSAIDs that are also
discussed. Aspirin, which irreversibly acetylates cyclo-oxygenase, is discussed, along with
22
several structural subclasses of tNSAIDs, including propionic acid derivatives (ibuprofen,
naproxen), acetic acid derivatives (indomethacin), and enolic acids (piroxicam), all of which
compete in a reversible manner with the arachidonic acid (AA) substrate at the active site of
COX-1 and COX-2. Acetaminophen is a very weak anti-inflammatory drug; it is effective as
an antipyretic and analgesic agent at typical doses that partly inhibit COXs, but appears to
have fewer gastrointestinal side effects than the tNSAIDs (Burke et al., 2006).
NSAIDs are classified into:
a. Non selective cyclo-oxygenase (COX) inhibitors such as salicylates, para-
aminophenol derivatives, indole and indole acetic acids, heteroaryl acetic acids, aryl
propionic acids, fenamates, enolic acid and alkanones.
b. Selective cyclo-oxygenase (COX-2) inhibitors such as diaryl/substituted furanones
(Rofecoxib), diaryl/substituted pyrazoles (Celecoxib), indole acetic acids (Etodelac)
and sulfonanilide (Nimesulide).
As anti-inflammatory agents, NSAIDs are used to treat conditions such as muscle strain,
tendinitis, and bursitis. They are also used to treat the chronic pain and inflammation of
rheumatoid arthritis (adult onset and juvenile), osteoarthritis, and arthritic variants such as
gouty arthritis and ankylosing spondylitis. While NSAIDs used to be the sole agent of choice
for mild to moderate rheumatoid disease, they are now frequently used in conjunction with
the disease modifying antirheumatic drugs (DMARDs) early in the treatment of these
disorders (Craig and Stitzel, 1999).
1.6.2.1 Mechanism of action of NSAIDs
23
The mechanism of action of aspirin and the traditional non-steroidal anti-inflammatory drug
(tNSAIDs) was elucidated only in 1971, when John Vane and his associates demonstrated
that low concentrations of aspirin and indomethacin inhibited the enzymatic production of
prostaglandins(Vane,1971). There was some evidence that prostaglandins participated in the
pathogenesis of inflammation and fever at that time. Subsequent observations demonstrated
that prostaglandins are released whenever cells are damaged and that aspirin and tNSAIDs
inhibit their biosynthesis in all cell types. However, aspirin and tNSAIDs generally do not
inhibit the formation of other inflammatory mediators, including other eicosanoids such as
the LTs (Vane and Botting, 1995). While the clinical effects of these drugs are explicable in
terms of inhibition of prostaglandin synthesis, substantial inter- and intra-individual
differences in clinical response have been noted. At higher concentrations, NSAIDs also are
known to reduce production of superoxide radicals, induce apoptosis, inhibit the expression
of adhesion molecules, decrease nitric oxide synthase, decrease pro-inflammatory cytokines
(e.g.TNF-α, interleukin-1), modify lymphocyte activity, and alter cellular membrane
functions. However, there are differing opinions as to whether these actions might contribute
to the anti-inflammatory activity of NSAIDs (Vane and Botting, 1995) at the concentrations
attained during clinical dosing in people. The hypothesis that their anti-inflammatory actions
in humans derive from COX inhibition alone has not been rejected based on current evidence.
NSAIDs are particularly effective when inflammation has caused sensitization of pain
receptors to normally painless mechanical or chemical stimuli. Pain that accompanies
inflammation and tissue injury probably results from local stimulation of pain fibers and
enhanced pain sensitivity (hyperalgesia), in part a consequence of increased excitability of
central neurons in the spinal cord (Vane and Botting, 1995).
1.6.2.2 Adverse effects of NSAIDs
24
A number of the toxicities commonly caused by the NSAIDs result from the inhibition of
prostaglandin synthesis. The ability of NSAIDs to increase gastric acid secretion and inhibit
blood clotting can lead to GI toxicity. Mild reactions, such as heartburn and indigestion, may
be decreased by adjusting the dosage, using antacids, or administering the drugs after meals.
Occult loss of blood from the GI tract and iron deficiency anaemia are also possible. More
serious toxicity can result from prolonged NSAID therapy, including peptic ulceration and
rarely, GI haemorrhage (Singh G,1998).
NSAIDs can impair renal function, cause fluid retention, and provoke hypersensitivity
reactions, including bronchospasm, aggravation of asthma, urticaria, nasal polyps, and rarely,
anaphylactic reactions. These reactions may occur even in those who have previously used
NSAIDs without any ill effects. NSAIDs inhibit uterine contraction and can cause premature
closure of the fetal ductus arteriosus. The spectrum of toxicity produced by each NSAID is
related to its inhibition of specific COX isoforms. The earliest NSAIDs inhibit both isoforms
of COX. Certain of these drugs are more specific for COX-1, whereas others inhibit COX-1
and COX-2 with roughly equal potency. More recently developed drugs selectively inhibit
COX-2 and therefore do not elicit the GI and antiplatelet side effects common to drugs that
inhibit COX-1 (Rahme and Nedjar, 2007).
Adverse effects that are not unequivocally related to inhibition of prostaglandin synthesis
include hepatic effects (hepatitis, hepatic necrosis, cholestatic jaundice, and increased serum
aminotransferases), dermal effects (photosensitivities, Stevens-Johnson syndrome, toxic
epidermal necrolysis, onycholysis), central nervous system (CNS) effects (headaches,
dizziness, tinnitus, deafness, drowsiness, confusion, nervousness, increased
sweating, aseptic meningitis), ocular effects (toxic amblyopia, retinal disturbances), and
certain renal effects (acute interstitial nephritis, acute papillary necrosis) (Patrick JR et al.,
25
1985)
1.7 Medicinal plants with anti-inflammatory properties
Despite the progress made in medical research for the past decades, the treatment of many
serious diseases is still problematic. Inflammation has become the focus of global scientific
research because of its implication in virtually all human and animal diseases. As a result of
adverse effects such as gastric lesions caused by non-steroidal anti-inflammatory drugs
(NSAID), tolerance and dependence induced by opiates, the use of these drugs as anti-
inflammatory and analgesic agents have not been successful in all cases (Dharmasiri et
al.,2003 and Park et al.,2004 ). Therefore, new anti-inflammatory and analgesic drugs lacking
these side effects are being researched as alternatives to NSAID and opiates (Dharmasiri et
al., 2003; Kumara, 2001). Attention is being focused on the investigation of the efficacy of
plant-based drugs used in the traditional medicine because they are cheap, have little side
effects and according to WHO, about 80% of the world population still rely mainly on herbal
remedies (Kumara, 2001; Dharmasiri et al., 2003, Li et al., 2003).
Review of some medicinal anti-inflammatory plants, which is among thousands used as
folkloric medicine because of its anti-inflammatory activities.
The ethanolic extract of the leaf of Vitex leucoxylon showed significant inhibition of
carrageenin paw oedema and granulation tissue formation in rats (Makwana et al., 1994).
The aqueous suspension of dried latex of Calotropis procera (Arka) showed anti-
inflammatory property when tested in the carrageenin and formalin induced rat paw oedema
models (Kumar and Basu et al., 1994).
The roots and leaves of Butea frondosa (Palash) were evaluated for ocular anti-inflammatory
activity in rabbits. The results showed that the gel formulation of Butea frondosa leaves,
26
prepared using a commercially available, pluronic F-127, reduced the intra-ocular pressure,
decreased leucocytosis and miosis and was comparable to flubiproten gel (Mengi and
Deshpande., 1995).
The triglyceride fraction of oil of Ocimum sanctum (Tulsi) offered higher protection against
carrageenin induced paw oedema in rats and acetic acid induced writhing in mice, as
compared to the fixed oil (Singh et al., 1996).Fixed oil of Ocimum sanctum and linolenic acid
were found to possess significant anti-inflammatory activity against PGE2, leukotriene and
arachidonic acid induced paw oedema. The anti-inflammatory activity of linolenic acid
present in the fixed oil of Ocimum sanctum was probably due to blockade of both, the cyclo-
oxygenase and lipo-oxygenase pathways of arachidonic acid metabolism (Singh and
Majumdar, 1997).
Alcoholic extract of Ochna obtusata stem bark demonstrated potent anti-inflammatory effects
in the rat paw oedema and cotton pellet granuloma models (Sivaprakasam et al., 1996).
All extracts of the root of Pongamia pinnata showed significant anti-inflammatory activity
(compared to phenylbutazone) in carrageenin and PGE1 induced oedema models. Possible
mechanism of action could be prostaglandin inhibition, especially by ethanol and acetone
extract. The benzene extract was effective in carrageenin but not the PGE1 model of
inflammation. The anti-inflammatory property appears to reside mainly in the intermediate
polar constituents and not in lipophilic or extremely polar constituent (Singh and Pandey,
1996).
The petroleum ether extract and chloroform extract of the seeds of Pongamia pinnata showed
potent acute anti-inflammatory effect whereas the aqueous suspension showed pro-
inflammatory effects. Further studies have shown that maximum anti-inflammatory effect
was seen in the bradykinin induced oedema model with the direct EE (Singh et al., 1996).
27
Possible mechanism of action could be inhibition of prostaglandin synthesis and decreased
capillary permeability. Petroleum ether extract and acetone extract inhibited histamine and 5-
hydroxytryptamine induced inflammation probably by their lipophilic constituents preventing
the early stages of inflammation. However, the fractions were not effective against Freund‟s
adjuvant arthritic model. The latter finding indicates that the plant may not be effective in
rheumatoid arthritis (Singh et al., 1996).
All extracts of Abies pindrow Royle leaf showed anti-inflammatory effect in various animal
models of inflammation such as carrageenin induced paw oedema, granuloma pouch and
Freund‟s adjuvant arthritis. Chemical analysis indicated the presence of glycosides and
steroids in the petroleum ether extract and benzene extract and terpenoids and flavonoids in
the acetone extract and ethanol extract. Flavonoids and terpenoids are polar substances
effective in acute inflammation whereas glycosides and steroids are non-polar substances
effective in chronic inflammation (Singh et al., 1997).
The methanolic extracts of the flowers of Michelia champaca Linn. (Champaka), Ixora
brachiata Roxb (Rasna) and Rhynchosia cana Willd were found to possess significant anti-
inflammatory activity against cotton pellet induced subacute inflammation in rats. The latter
2 drugs showed higher activity as compared to Michelia champaca. They also reduced the
protein content, acid phosphatase, glutamate pyruvate transaminase and glutamate
oxaloacetate transaminase activities in the liver and serum. These properties are probably due
to the presence of flavonoids in the flowers of these plants (Vimala et al., 1997).
The methanolic extract of the aerial part of Sida rhombifolia (Atibala) showed significant
oedema suppressant activity in the carrageenin induced paw oedema model in rats. Probable
mechanism of action may be due to its inhibitory effects on release of mediators of
inflammation such as histamine, 5-hydroxytryptamine, bradykinin etc (Rao and Mishra SH,
28
1997).
Gmelina asiatica (Gopabhadra) root powder was effective in reducing the oedema in the
carrageenin induced rat paw oedema model of acute inflammation. When tested against the
cotton pellet granuloma model of chronic inflammation, it not only reduced the weight of the
granuloma but also the lipid peroxide content of granuloma exudate and liver and gamma-
glutamyl transpeptidase in the granuloma. It also normalised serum albumin and serum acid
and alkaline phosphatase levels. Probable mechanism of its anti-inflammatory effect may be
its anti-proliferative, anti-oxidative and lysosomal membrane stabilizing effects (Ismail et al.,
1997).
Studies have shown that, the methanol extract of Nelumbo nucifera rhizome as well as the
steroidal triterpenoid isolated from it (betulinic acid), possessed significant anti-inflammatory
activity when evaluated in the carrageenin and 5-hydroxytryptamine induced rat paw edema
models. The effects produced were comparable to that of phenylbutazone and dexamethasone
(Mukherjee et al., 1997).
The water soluble part of the alcoholic extract of Azadirachta indica exerted significant anti-
inflammatory activity in the cotton pellet granuloma assay in rats. Levels of various
biochemical parameters studied in cotton pellet exudate were also found to be decreased viz.
DNA, RNA, lipid peroxide, acid phosphatase and alkaline phosphatase suggesting the
mechanism for the anti-inflammatory effect of Azadirachta indica (Chattopadhyay, 1998).
Alcoholic extract of the roots of Clerodendron serratum showed significant anti-
inflammatory activity in the carrageenin induced paw oedema and cotton pellet granuloma
models in rats (Narayanan et al., 1998).
The aqueous extract of Gymnema sylvestre leaves showed significant anti-inflammatory
29
activity in the carrageenin induced rat paw oedema and mouse peritoneal ascitis models. It,
however, did not inhibit granuloma formation and related biochemical indices, such as
hydroxyproline and collagen, (as seen in the pith granuloma model) thus indicating that it did
not interfere in the normal healing process. In addition, the extract did not affect the integrity
of the gastric mucosa, even at high doses, thus appearing to be a less gastrotoxic anti-
inflammatory agent as compared to other non-steroidal anti-inflammatory agents (Diwan et
al., 1995).
Sandhika, an Ayurvedic drug used in the treatment of rheumatoid arthritis showed significant
anti-inflammatory activity when tested against carrageenin induced paw oedema and cotton
pellet granuloma. Possible mechanism of action could be by free radical scavenging activity
(Chaurasia et al., 1995).
Table 1: Review of some medicinal plants with anti-inflammatory activities
S/No Plant (Family) Parts of plant
used
Properties reported References
1 Gymnema sylvestre
(Asclepiadaceae)
Aqueous leaf
extract
Inhibits acute and
chronic inflammation
Diwan et al.,
1995
2. Clerodendron
serratum
(Verbenaceae)
Alcoholic root
extract
Inhibits acute and
chronic inflammation
Narayanan et
al., 1998
3 Azadirachta indica Alcoholic
extract of water
Inhibits chronic Chattopadhyay
30
(Meliaceae) soluble parts inflammation 1998
4 Nelumbo nucifera
(Nelumbonaceae)
Methanolic
extractsof
rhizome
Inhibit oxidants
induced inflammation
Mukherjee et
al., 1997
5 Gmelina asiatica
Gopabhadra
(Verbenaceae)
Root powder Inhibit chronic
inflammation and
possesses anti-
proliferative, anti-
oxidative and
lysosomal membrane
stabilizing effects
Ismail et al.,
1997
6 Sida rhombifolia
Atibala
(Malvaceae)
Methanolic
extract of aerial
part
Rao and Mishra
1997.
7 Michelia champaca
Linn. Champaka
(Magnoliaceae)
Methanolic
extract of the
flowers
Inhibits Subacute
inflammation
Vimala et al.,
1997
8 Ixora brachiata Roxb
Rasna( Rubiaceae)
Methanolic
extract of the
flowers
Inhibits Subacute
inflammation
Vimala et al.,
1997
9 Rhynchosia cana Methanolic
extract of the
Inhibits Subacute Vimala et al.,
31
Willd (Leguminosae) flowers inflammation 1997
10 Abies pindrow Royle
(Pinaceae)
All leaf extracts
(petroleum,
chloroform,
acetone and
ethanol)
Inhibits both acute and
chronic inflammation
Singh and
Pandey 1997
11 Pongamia pinnata
(Fabaceae)
Petroleum and
chloroform seed
extract
Potent inhibitor of
acute inflammation via
inhibition of
prostaglandin synthesis
Singh and
Pandey 1996
12 Ochna obtusata
(Ochnaceae)
Alcoholic stem
extract
Inhibits both acute and
chronic inflammation
Sivaprakasam
et al., 1996
13 Ocimum sanctum
Tulsi (Lamiaceae)
triglyceride
fraction of oil
Inhibits acute and sub-
acute inflammation
Singh and
Majumdar
1997; Singh et
al., 1996
14 Butea frondosa
Palash( Fabaceae)
Gel form of
leaf and stem
Inhibits acute
inflammatory process
Mengi and
Deshpande.,
1995
15 Calotropis procera
Arka( Apocynaceae)
Aqueous
suspension of
dry latex
Inhibits both acute and
chronic inflammation
Kumar and
Basu et al.,
1994
32
16 Vitex leucoxylon
(Verbenaceae)
Ethanolic leaf
extract
Inhibits both acute and
sub-acute inflammation
Makwana et al.,
1994
17 Justicia pectoralis
var. Stenophylla
(Acanthaceae)
Hydro-alcoholic
extract of laef
Inhibit acute
inflammation
Lino et al.,
1997
18 Cordyline
dracaenoides
(Agavaceae)
Dil.ethanol
extract of Dried
rhizome
Inhibit Acute
inflammation
Calixto et al.,
1990
19 Pfaffia paniculata
(Amaranthaceae)
20% ethanol
extract of root
Inhibit acute
inflammation and sub-
acute inflammation
Mazzanti, et al.,
1993
20 Echinodorus
grandiflorus
(Alismataceae)
Methanol dried
rhizome extract
Inhibit acute
inflammation
Dutra.,et al
2006
21 Pfaffia glomerata
(Amaranthaceae)
Dilute ethanol
extract of root
Inhibit acute
inflammation
Teixeira., et al
2006
22 Anacardium
occidentale
Bark Adsorbed
in shell ,
Inhibit acute
inflammation, sub-
acute and chronic
Mota et al.,
1985
33
(Anacardiaceae ) Isopropanol-
H2O extract
(1:1)
inflammation
23 Astronium urundeuva
(Anacardiaceae)
Bark extracted
with ethanol
and its tannin
fraction
Inhibit both acute and
sub-acute inflammation
Viana., et al
2003
24 Spondias mombin
(Anacardiaceae)
Ethanol extract
of dried bark
Inhibit acute
inflammation
Abad et al.,
1996
25 Bonafousia longituba
(Apocynaceae )
Ethanol and
methelene
chloride extracts
of dried parts
Inhibit acute
inflammation
de Las Heras et
al., 1998.
Ortega, et al.,
1996
26 Ervatamia coronaria
(Apocynaceae)
Ethanol and
aquoues extracts
of dried stem
Inhibit acute
inflammatory response
Henriques et
al., 1996
27 Himatanthus sucuuba
(Apocynaceae)
Latex extracted
with n-hexane
Inhibit acute
inflammation
de Miranda et
al., 2000
28 Mandevilla velutina
(Apocynaceae)
Aqueous/
alcoholic and
dil. Ethanol
extracts of
Inhibit acute sub-acute
and chronic
inflammation
Calixton et al.,
1986,
Calixto et al.,
1991,
34
rhizome Henriques et
al., 1991
29 Peschiera vanheurckii
(Apocynaceae)
Ethanolic
extract of dry
stem bark
Inhibit acute
inflammation
Dunstan et al.,
1997
30 Hedera helix
(Araliaceae)
Ethanolic
extract of dried
leaf
Inhibit acute
inflammation used in
human being
Fazio et al.,
2009
31 Orbignya phalerata
(Arecaceae )
Chloroform
extract of dried
fruit
Inhibit both acute and
sub-acute inflammation
Maia and rao
1989
32 Aristolochia
triangularis
(Aristolochiaceae)
Methanol
methelene
chloride and
aqueous extracts
of dried root
Inhibit both acute and
sub-acute inflammation
Muschietti et
al., 1996
33 Marsdenia
cundurango
(Asclepiadaceae)
Methylene
chloride extract
of whole plant
Inhibit acute
inflammation
Ortega et al.,
1996
34 Achyrocline
satureioides
Aqueous (hot
and cold) and
Inhibit both acute and
sub-acute inflammation
Simões et al
.,1988
35
(Asteraceae)
ethanol extracts
of dried
inflorescence
35 Ageratum conyzoides
(Asteraceae)
Hydro-alcohol
extract of dried
leaf
Inhibit sub-acute and
chronic inflammation
Magalhães, et
al., 1997,
Moura, et al.,
2005, Viana et
al., 1998
36 Ambrosia tenuifolia
(Asteraceae)
Aqueous,
methylene
chloride and
methanol
extracts of dried
arial parts
Inhibit both acute and
chronic inflammation
Muschietti et
al., 1996
37 Artemisia copa
(Asteraceae)
Hot aqueous,
methylene
chloride extracts
of whole plants
Inhibit both acute and
chronic inflammation
Perez et al.,
1995 and Miño
et al., 2005
38 Baccharis decussata
(Asteraceae)
Methanol
extract of dried
leaf
Inhibit acute
inflammation
Salama, et al.,
1987
39 Baccharis incarum Methylene
chloride extract
Inhibit acute Perez, et al.,
36
(Asteraceae) of entire plant inflammation 1995
40 Baccharis medullosa
(Asteraceae)
n-hexane extract
of aerial parts
Inhibit acute
inflammation
Cifuente, et al.,
2001
1.8 Ficus exasperata
Since ancient times of civilization, people have been relying on plants as either prophylactic
or therapeutically arsenal to restore and maintain healthy, and plants are well known as an
important source of many biologically active compounds. Rates (2001) reported that there
has been a growing interest in plants as a significant source of new pharmaceuticals. Ficus
exasperata belongs to the family Moraceae, with 800 species occurring in the warmer part of
the world, chiefly in Indomalaya and Polynesia (Odunbaku et al., 2008). The Nigeria are
replete with over 45 different species of Ficus (Keay and Onochie, 1964), such as Ficus
glomosa, Ficus lecardi, Ficus goliath, Ficus capensis, Ficus ingens and F. elastica, which
can be found in the Savannah, rainforest, besides rivers and streams.
F. exasperata is commonly known as sand paper tree and is widely spread in West Africa in
all kinds of vegetation and particularly in secondary forest re-growth.
37
1.8.1 Taxonomy of Ficus exasperata
Domain Eukaryote
Kingdom Plantae
Phylum Tracheophyta
Class Magnoliopsida
Order Urticales
Family Moraceae
Tribe Ficeae
Genus Ficus
Species Exasperata
Specific epithet Exasperata vahl
1.8.2 Common names/synonyms
Region Common and Vernacular Name of F.
exasparata
English Sand paper plant
South-East Nigeria (Central plain of Igbo) Anwurinwa (Nsukka)
South Western Nigeria Eepin, „sampaper‟ (local English)
Ovia North East, Edo State Sand paper plant (English), amenmen (Bini),
ipin (Yoruba)
Benue state Uhuo (Igede)
(Prelude Medicinal Plants Database:www. Africanconservation.org/medicinal plant)
38
1.8.3 Botanical descriptions of Ficus exasperata:
Ficus exasperata is a Deciduous trees up to 18 m tall. The trunk and Bark is pale greenish
and lenticellated. The branches are terete with stout white scabrid hairs. It expresses
profusely watery latex. The leaves are arranged in simple, alternate stipule in hairs, lateral
and caduceus with scar. The petiole is about 1-6 cm long. The lamina is about 5.5-17x3.0-7.5
cm. The leave base is rounded or acute-cuneate, the margin denticulated. The leaf is
trespassed with both secondary and tertiary nerves (3-6 in numbers) the inflorescence is
syconia and the flower appears unisexual with its peduncles up to 1.5cm. The shape of the
fruit is oblong and is 1.5cm long, yellow or purple when ripped (Hyde et al., 2012).
1.8.4 Geographical distribution of Ficus exasparata :
Ficus exasperata is widely distributed in West Africa, East Africa, India, Arabia and Sri
Lanka; in the Western_Ghats- South, Central and Maharashtra Sahyadris (Hyde et al., 2012)..
1.8.5. Ethnomedicinal uses of Ficus exasperata
In Nigeria, young leaves of F. exasperata are prescribed as a common anti-ulcer remedy.
Various pharmacological actions such as anti-diabetic, lipid lowering and antifungal activities
have been reported for F. exasperata (Sonibare et al., 2006). Ijeh and Agbor (2006) reported
the use of F. exasperata for treating several problems like difficult child birth, bleeding and
diarrhoea in traditional medicine. The leaf extract from F. exasperata reported to have
diverse use such as in treating hypertension (Ayinde et al., 2007), heamostatic opthalmia,
coughs and heamorrhoid (Odunbaku et al., 2008).
The whole plant is known to have several medicinal properties in African traditional
medicine. The leaf extract has been used to treat high blood pressure, rheumatism, arthritis,
intestinal pains and colics, epilepsy, bleeding and wounds (Irvine et al.,1961). The roots are
39
also used to manage asthma (Chhabra et al., 1990), dyspnoea (Chhabra et al.,1990)
and
venereal diseases. Previous work on F. exasperata shows that an aqueous leaf extract
produced a dose-related reduction in mean arterial blood pressure (Ayinde et al., 2007) as
well as significant anti-ulcerogenic effect in aspirin-induced ulcerogenesis (Akah et al.,1998).
Macfoy demonstrated that the methanolic and hot and cold aqueous extracts of F. exasperata
were inactive against three Gram-negative and three Gram-positive bacteria species:
Aerobacter aerogenes, Agrobacterium tumefaciens, Bacillus subtilis, Clostridium
sporogenes, Escherichia coli and Staphylococcus aureus and another work reported
antimicrobial activities of leaf, stem and root of Ficus exasperata (Adebayo et al., 2009).
Other industrial uses of sand paper leaves are for polishing woods (Cousins and Michael,
2002), stabilization of vegetable oils, suppression of foaming, supplement as food stock and
antimicrobial (Odunbaku et al., 2008). The activities of leaf extract of F. exasperata against
some pathogenic organisms have been extensively investigated (Ayinde et al., 2007;
Odunbaku et al., 2008).
Recently, the anti-inflammatory, antipyretic and antinociceptive effects were also established
(Woode et al., 2009).
1.9 Aim and scope of study
Ficus Exasperata is reportedly used to treat skin disorders and to reduce joint inflammation
in arthritis patients (Igoli et al., 2005). This project work is aimed at assessing the effect of
the leaf extracts of Ficus exasperata on acute and chronic inflammation using in vitro and in
vivo models. The purpose of the work is to establish the scientific basis, if any, of this
practice, to identify and isolate the phytochemical constituent(s) responsible for any anti-
inflammatory effects. The work is also aimed at establishing the possible anti-inflammatory
mechanism(s) and the effects of the extracts on some of the mediators of inflammation.
40
CHAPTER TWO
MATERIALS AND METHODS
2.1 Chemicals, Solvents, and Reagents
(i) Extraction Solvents: methanol (BDH), n-hexane (BDH), ethyl acetate (BDH), chloroform
(BDH), and methylene chloride (BDH).
(ii) Reagents for phytochemical tests: ferric chloride, iodine solution, ethanol (96%), dilute
ammonia solution, Tetraoxosulphate (VI) (Sigma Aldrich), Naphthol solution in ethanol,
Potassium mercuric iodide solution(Sigma Aldrich), Bismuth potassium iodide(BDH), Iodine
in potassium iodide solution, Fehling‟s solution, Million‟s reagent, Picric acid solution.
(iii) Phosphate buffered solution: Sodium chloride, Sodium phosphate, Potassium chloride
and Potassium phosphate and Distilled water.
(iv) Reagents for pharmacological studies: Agar, Tween85, Xylene, Formaldehyde,
Diclofenac potassium (pure sample, ChemIndustry,Monrovia), Indomethacin (pure sample,
Sigma Aldrich), Distilled water, reagent (Applichem, Darmstadt, Germany), NaNO2
(Applichem, Germany)
(v) Cell culture medium: RPMI 1640 medium (Gibco, Germany) supplemented with 5%
heat-inactivated foetal calf serum (FCS), 50 μM 2-mercaptoethanol (Gibco, Germany), 1% L-
glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100
μg/ml streptomycin, and 10 ng/ml of recombinant murine colony-stimulating factor (rmCSF-
1; Immunotools, Friesoythe, Germany)
(v) Elisa kits: CommercialIL-1β and TNF-α ELISA kits (PeproTech, Hamburg, Germany)
2.2 Equipment
Vacuum evaporator (Mac, Delhi, India), oven (Mac, Delhi, India), Cork borer, Petri dishes
(Pyrex,Germany), spatula, separating funnel (Pyrex,Germany), stop clock, blender, filter
41
paper, Electronic balance, test tubes (Pyrex,Germany),, autoclave (Mac, Delhi, India),
refrigerator(LG, China), measuring cylinder, CO2 incubators, Laminar flow chamber, Cell
culture flasks, Inverted microscope, water bath, -20 °C and -80°C freezers, multiwell
microtiter plate reader (Tecan, Grödig, Austria).
2.3 Animals
Adult Swiss albino rats (150–250 g) and mice (15–25 g) of both sexes were obtained from the
Laboratory Animal Facility of the Department of Pharmacology and Toxicology, Faculty of
Pharmaceutical Sciences, University of Nigeria, Nsukka (UNN). Balb/c mice (20-25 g) were
obatined from Janvier, France and maintained in the animal facility of Ruhr University,
Bochum, Germany. Animals were housed in steel cages within the facility under standard
conditions and allowed free access to standard pellets and water. Prior to their use in the
different experiments, they were allowed 10 days for acclimatization.
2.4 Collection of plant material
Fresh leaves of Ficus exasperata were collected in March 2009 from Nsukka, Enugu State,
Nigeria and authenticated by Mr. A. Ozioko of Bioresources Development and Conservation
Program (BDCP) Centre, Nsukka where a voucher Specimen is maintained. The leaves were
cleaned by hand picking and air dried for 48 h and pulverized to coarse powder using slow
speed electronic blender.
2.5 Extraction of plant material
About 2.0 kg of dry leaf powder of Ficus exasperata was extracted with 50:50 ratios of
methylene chloride (1L) and methanol (1L) by cold maceration for 48 h to obtain the
methylene chloride/methanol filtrate. The filtrate was concentrated in vacuo to get methylene
chloride/ methanol extracts (MME). The MME was adsorbed on silica gel and eluted in
42
succession with n-hexane, chloroform, ethyl acetate, and methanol to obtain n-hexane,
chloroform, ethyl acetate, and methanol fractions respectively. The fractions were
concentrated in vacuo. Aqueous extract of Ficus exasperata was obatined by macerating
250g of the leaf powder for 2 h in 400ml of warm sterile distilled water with intermittent
agitations. The extract was filtered and lyophilised to give the dried extract (FE). All the
extract and fractions were stored in the refrigerator between 0-4⁰c until used (Okoye and
Osadebe, 2009).
2.6 Phytochemical analysis of extracts
Phytochemical analyses of the extracts were done using the method of Harborne (1984).
I. Test for saponin
To 0.2ml each of the plant extracts in 100ml beaker, 20 ml of water was added and boiled
gently in a hot water bath for 2 min. The mixture was filtered hot and allowed to cool and the
filtrate tested as follows:
About 5ml of the filtrate was diluted with 20ml of water and shaken vigorously and observed
on standing.
II. Test for acidic compounds
Each extract (2 ml) was mixed with sufficient water, warmed in a hot water bath and then
cooled. A piece of water-wetted litmus paper was dipped into the filtrate and colour change
on the litmus paper observed.
III. Test for protein
Picric acid test
A few drops of picric acid were added to 2 ml of the extract and the precipitate observed.
43
IV. Test for tannins.
Ferric chloride test:
The extracts (1.0 ml) were boiled with 50 ml of water, filtered and used for the ferric chloride
test. To 3 ml of each extract filtrate, few drops of ferric chloride were added and the colour of
the resulting precipitate observed.
V. Test for carbohydrates
Iodine Test
To 0.5 ml of each of the extracts was mixed with a drop of iodine solution. A blue-black
colour indicates the presence of starch.
VI. Tests for reducing sugar
Fehling’s Test
The extracts (1.0 ml) was shaken vigorously with 5ml of distilled water and filtered. The
filtrate was used in the Fehling‟s test as follows:
To 1.0ml portion of the filtrate was added equal volumes of Fehling‟s solution A and B
boiled on water bath for few min. A brick-red precipitate indicates the presence of reducing
sugar.
VII. Test for resins
Precipitation test:
Each of the extract (5 ml) was extracted with 15 ml of 96% ethanol. The alcoholic extract
was then poured into 20 ml of distilled water in a beaker. A precipitate occurring indicates
the presence of resins.
44
VIII. Test for oil
General tests:
The extracts (0.1 ml) were dropped on filter paper and observed translucency of the filter
paper indicates the presence of oil.
IX. Test for glycosides
Modified Borntrager’s test.
About 5ml of dilute sulphuric acid and ferric chloride solution was added to 2ml of each of
the filtrate boiled for 5 min, cooled and filtered into a 50 ml separatory funnel. The filtrate
was shaken with an equal volume of carbon tetrachloride and the lower organic layer
carefully separated into a test tube. Ammonia solution (5 ml) was then added to the test tube
containing each filtrate and then shaken. A rose pink to red colour in the ammoniacal layer
shows the presence of anthraquinone glycoside.
X. Test for flavonoids
Ammonium test
Ethyl acetate (10 ml) was added to 2 ml of each plant extract and heated on a water bath for 3
min. The mixture was cooled, filtered and the filtrate subjected to ammonium test thus:
About 4ml of filtrate was shaken with 1 ml of dilute ammonium solution. The sugars were
allowed to separate and the yellow colour in the ammoniacal layer indicates the presence of
flavonoids.
XI. Test for alkaloids (General test)
About 20 ml of 5% sulphuric acid in 50% ethanol was added to 1ml of the plant extracts and
45
heated on a boiling water bath for 10 min, cooled and filtered. 2ml of the filtrate was treated
with a few Meyer‟s reagent, Wagner‟s reagent and picric acid solution (1%).
The remaining filtrate in 100 ml separating funnel and made alkaline with dilute ammonia
solution, the aqueous alkaline solution was separated and extracted with two 5ml portions of
dilute sulphuric acid. The extract was tested with a few drops of Meyer‟s reagent, Wagner‟s
and dragendoff‟s reagent. Alkaloids give milky precipitate with one drop of Meyer‟s reagent,
reddish-brown precipitate with one drop of picric acid solution and brick-red precipitate with
one drop of Dragendoff‟s reagent.
XII. Test for Steroids and Terpenoids
Ethanol (9 ml) was added to 1ml of the plant extract, refluxed for a few min and filtered. The
filtrate was concentrated to 2.5 ml on a boiling water bath and 5ml of hot water were added.
The mixture was allowed to stand for one hour and the waxy matter filtered off. The filtrate
was further extracted with 2.5 ml of chloroform using funnel. To 0.5ml of the chloroform
extract in a test tube was carefully added 1ml of concentrated sulphuric acid to form a lower
layer. A reddish-brown interface shows the presence of steroids. Another 0.5 ml of the
chloroform extract was evaporated to dryness in a water bath and heated with 3ml of
concentrated sulphuric acid for 10 min in a water bath. A grey colour indicates the presence
of terpenoids.
2.7. Pharmacological studies
2.7.1 Acute toxicity (LD 50) test
The median lethal dose of the crude extract was determined in mice using the method
described by Lorke (1983) using the oral and intraperitoneal routes respectively. The test
46
was divided into two stages.
Stage one: determination of the toxic range of the extracts
Mice were divided into 3 groups of 3 animals. Each group received a dose (10, 100 and 1000
mg/kg) of the methanol/methylene chloride extracts (MME) suspended in 2% v/v Tween 85.
The doses were administered orally or intraperitoneally and the treated animals observed for
24 h for number of deaths.
Stage two: determination of lethality.
The doses used in the stage were determined from the number of deaths per dose recorded in
the stage one test. Since no death occurred in the stage one test, three different higher doses:
1600, 2900 and 5000 mg/kg were administered to another group of animals at one dose per
animal. The treated animals were monitored for number of deaths for 24h.The LD50 in this
test is determined by calculating the geometric mean of the least and most toxic doses.
2.7.2 Effect of extract and fractions on acute inflammation induced by xylene in the
mouse ear.
The effect of extracts or fractions on acute topical inflammation was evaluated by a
modification of the methods of Tubaro et al, 1985 and Atta and Alkofahi, 1998. For each
experiment, adult Swiss albino mice (15–25 g) of either sex were divided into seven groups
of 8 animals. The treatment groups received of extract or fractions (50 µl in 2% Tween/ear)
applied on the anterior surface of the right ear. Topical inflammation was instantly induced
on the posterior surface of the same ear by application of xylene (0.03 ml). Control animals
received either 2% Tween or indomethacin (50 µl in 2% Tween/ear).Two hours after
induction of inflammation, mice were euthanized by ether anesthesia and both ears removed.
Circular sections (4 mm diameter) of both the right (treated) and left (untreated) ears were
47
punched out using a cork borer, and weighed. Edema was quantified as the weight difference
between the two ear plugs. The anti-inflammatory activity was evaluated as percentage of
oedema reduction/inhibition in the treated animals relative to control animals (Tubaro et al.,
1985, Asuzu et al., 1999) using the relation:
100*)(
)(1(%)/
LcRc
LtRtInhibitionredutionEdema
Where;
Rt = mean weight of right ear plug of treated animals;
Lt = mean weight of left ear plug of treated animals;
Rc = mean weight of right ear plug of control animals;
Lc = mean weight of left earplug of control animals.
2.7.3 Effect of extract and fractions on agar-induced systemic acute oedema in rat
The rat paw edema method (Winter et al., 1962) was used. Acute inflammation was
measured in terms of change in volume of the rat hind paw (Backhouse et al., 1996) induced
by subplantar injection of agar (Okoli et al., 2007, Iwueke et al., 2006). Animals (n =
5/group) received 200 or 400 mg/kg of extracts or fractions administered orally. Edema was
induced one hour later with agar (0.1 ml) injected into the subplantar region of the right hind
paw of the rats. The volume of distilled water displaced by the treated paw was measured
before and 1, 2, 3, 4, and 5 h after induction of edema. Control groups received either
equivalent volume of the vehicle (distilled water in 2% Tween) or Diclofenac potassium (50
mg/kg). Inflammation was assessed as the difference between the zero time volume of the
treated paw (Vo) and the volume at the various times (Vt) after the administration of the
phlogistic agent.
Percentage inhibition of edema (Ahmed et al 1993, Perez, 1996) was calculated using the
relation:
48
100*1(%)
yb
xaoedemaofInhibition
Where;
a = mean paw volume of treated rats at various time after agar injection;
x = mean paw volume of treated rats before agar injection;
b = mean paw volume of control rats at various time after agar injection;
y = mean paw volume of control rats before agars injection.
2.7.4 Effect of extract and fractions on formaldehyde-induced arthritis in rats
The effect of the extract or fractions on chronic inflammation was assessed using arthritis
induced by formaldehyde (Seyle, 1949) in rats. On day one, adult Swiss albino rats of either
sex received the aqueous extract (200 or 400 mg/kg) administered orally. One hour later,
arthritis was induced by subplantar injection of 0.1 ml of 2.5% formaldehyde solution and
repeated on day 3. Arthritis was assessed by measuring the volume of distilled water
displaced by the paw before induction of arthritis and once every day for ten days, starting
from day one, after induction of arthritis.
Extracts or fractions administration was continued once daily for ten days. Control animals
received either indomethacin (50 mg/kg) or equivalent volume of vehicle (2% Tween). The
global edematous response was quantified as the area under the curve (AUC) of the time-
course of the arthritic event. The AUC was calculated using the trapezoidal rule. The level of
inhibition of arthritis was calculated using the relation:
100*1(%)
AUCcAUCt
arthritisofInhibition
Where AUCc = AUC of the control group; AUCt = AUC of the treated group
49
2.7.5 Effect of extract on membrane stabilization
(I) Preparation of erythrocyte suspension
Fresh whole healthy human blood that has kept away from drugs for at least 2 weeks (10 ml)
was collected, transferred to heparinized centrifuge tubes, centrifuged at 3000 rpm for 5 min,
and The supernatants (plasma and leucocytes) were carefully removed while the packed red
blood cell was washed in fresh normal saline (0.9% w/v NaCl). The process of washing and
centrifugation were repeated five times until the supernatants were clear. The volume of the
blood was measured and reconstituted as a 40% (v/v) suspension with isotonic buffer solution
(10 mM sodium phosphate buffer pH 7.4). The composition of the buffer solution (g/L) was
NaH2PO4 (0.2), Na2HPO4 (1.15) and NaCl (9.0) (Shinde et al., 1999).
(II) Heat induced haemolysis
The isotonic buffer solution (5 ml) containing 200, 400 and 800μg/ml of the Methylene
chloride/Methanolic extracts were put in 4 sets (per concentration) of centrifuge tubes.
Control tubes contained 5 ml of the vehicle or 5 ml of 50 and 100 µg/ml of hydrocortisone.
Erythrocyte suspension (0.05 ml) was added to each tube and gently mixed. A pair of the
tubes was incubated at 54°C for 20 min in a regulated water bath. The other pair was
maintained at 0–4°C in a freezer for 20 min. At the end of the incubation, the reaction
mixture was centrifuged at 1300 g for 3 min and the absorbance (OD) of the supernatant
measured at 540 nm using Spectronic 2ID (Milton Roy) spectrophotometer. The level of
inhibition of hemolysis was calculated using the relation (Shinde et al., 1999).
100*
1312
1(%)
ODOD
ODODhaemolysisofInhibition
Where OD1 = absorbance of test sample unheated;
50
OD2 = absorbance of test sample heated;
OD3 = absorbance of control sample heated
2.7.6 Effect of extract on In-vivo leucocytes mobilisation
The effect of MME on leukocyte migration in-vivo was studied in rats using the method
described by Ribeiro et al., 1991. Adult albino rats (140–240 g) of either sex were used. The
extract was administered orally at 200, 400, or 800 mg/kg to animals (n = 5/dose). One hour
after drug administration, animals received intraperitoneal injection of 1 ml of 2.8% w/v agar.
Four hours later, the animals were sacrificed and the peritoneal cavities washed with 5 ml of
phosphate buffer saline containing 0.5 ml of 10% EDTA. Total and differential leukocyte
counts in the peritoneal wash were taken and the level of inhibition (%) or otherwise of
neutrophil and lymphocyte migration was calculated.
% inhibition of leucocyte migration = 100 × (1 – T/C)
where, C is the total leucocyte in control group and T is the total WBC in treated group.
2.8 In-vitro studies on the effects of F. exasparata aqueous extract on pro-inflammatory
mediators
2.8.1 Isolation and culture of bone marrow-derived macrophages (BMDMs)
Murine BMDMs were generated from the BM cells of the tibia, humerus, and femur of
BALB/c donor mice by a modification of the methods previously described (Lin et al., 2001;
Weischenfeldt and Porse, 2008). BM cells were harvested and cultured in DC-medium
containing RPMI 1640 medium (Gibco, Germany) supplemented with 5% heat-inactivated
foetal calf serum (FCS), 50 μM 2-mercaptoethanol (Gibco, Germany), 1% L-glutamine, 1%
non-essential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml
streptomycin, and 10 ng/ml of recombinant murine colony-stimulating factor (rmCSF-1;
51
Immunotools, Friesoythe, Germany) in T-75 cell culture flasks. The cells were incubated at
37°C and 5% CO2 for 24 h to adhere and remove stromal cells and mature BM resident
macrophages. Non-adherent cells, which are mainly progenitors, were recovered after 24 h of
incubation and further incubated in cell culture flasks to expand and differentiate the cells
under the influence of the rmCSF-1. After 7 days of culture, non-adherent cells were removed
and the adherent cells were washed and harvested using a cell scraper. The viability of the
generated macrophages was assessed by trypan blue exclusion. The BMDMs generated were
plated and used for the in vitro studies of the effects of FE on LPS-induced NO and pro-
inflammatory cytokines production by macrophages.
2.8.2 Viability of FE-treated BMDM assessed using an MTT assay
The viability of the BMDM after treatment with FE extract was determined using cellular
respiration as an indicator. Cell viability was determined on the basis of mitochondrial
dependent reduction of MTT to formazan (Mosmann, 1983). BMDM were cultivated in 96-
well plates (1 × 105 cells/well) for 24 h. The cells were then treated with various
concentrations (0, 5, 25, and 100 μg/ml) of FE in a fixed volume of 100 μl. After 24 h of
incubation at 37°C, the medium in each well was discarded; the cells were then incubated
with fresh medium containing 5 mg MTT/ml for 4 h. The formazan blue that formed in the
cells was then dissolved by addition of 150-μl dimethyl sulfoxide to each well and the optical
density of the solution in the well was measured at 550 nm in a multiwell microtiter plate
reader (Tecan, Grödig, Austria).
52
2.8.3 Measurement of NO concentration by Griess reagent
BMDM were cultivated in 48-well plates (5 × 105 cells/well) at 37°C in a 5% CO2 incubator
for 24 h. Thereafter, the cells were pretreated with various concentrations (0, 5, 25, and 100
μg/ ml) of FE. After 2 h of incubation, the cells were then treated with 10 μg/ml of LPS or
vehicle (PBS) only. Supernatants were collected after 24 h of incubation and stored at −80°C.
Nitrite levels in aliquots of each supernatant were measured in 96-well microtiter plates by
mixing 100 μl of cell-free culture supernatant with an equal volume of Griess reagent
(Applichem, Darmstadt, Germany) and then incubated at room temperature for 10 min. The
Griess reagent contains equal volumes of 0.1% naphthylethylenediamine dihydrochloride and
1% sulphanilamide in 5% phosphoric acid. The NO concentration was determined at 550 nm
in a multiwell microtiter plate reader (Tecan) by extrapolation from a standard curve
generated using NaNO2 standards that had been included in each measurement plate.
2.8.4 Determination of pro-inflammatory cytokine secretion in culture supernatant
BMDMs (5 × 105 cells/well) was cultured in 48-well plates for 24 h. The cells were then
pretreated with various concentrations (0, 5, 25, and 100 μg/ml) of FE for 2 h. After this
period of incubation, the cells were treated with 10 μg LPS/ml or vehicle. After 24-h
incubation at 37°C, the cell-free medium in each well was collected and the concentrations of
IL-1β and TNF-α in the culture supernatant were determined using commercially available
ELISA kits, according to the manufacturer‟s instructions (PeproTech, Hamburg, Germany)
2.9 Statistical analysis
The data obtained was analyzed by Analysis of variance using SPSS Version 14. Difference
between means were accepted as significant at P<0.05. The results are presented as mean ±
SEM.
53
CHAPTER THREE
RESULTS
3.1 Extraction
The extraction process yielded 62.80g (3.14%, w/w) of the methylene chloride/ methanol
extract (MME). After exhaustive and successive fractionation with solvents of graded
polarity, 50 g of MME yielded 15.33 g (38.33%, w/w) of n-hexane fraction (HF), 11.27 g
(29.30%, w/w) of chloroform fraction (CF), 8.50 g (21.25%, w/w) of ethyl acetate fraction
(EF) and 9.29 g (22.23%, w/w) of the methanol fraction (MF). The aqueous extraction
yielded 20.68 g (8.27% w/w) of the lyophilised extract.
3.2 Phytochemical analysis
The methanol/methylene chloride extract (MME) tested positive for carbohydrate, alkaloids,
flavonoids, resins, proteins, oil and terpenoids (Table 2). The n-hexane fraction (HF) tested
positive for carbohydrate, alkaloids, flavonoids, resins, proteins, oil, steroids terpenoids and
acidic compounds. The Chloroform fraction (CF) tested positive for carbohydrate, alkaloids,
flavonoids, proteins, oil, steroids and terpenoids. The ethyl acetate fraction (EF) tested
positive for alkaloids, flavonoids, resins, proteins, oil and acidic compounds. The last
fraction, methanol fraction (MF) tested positive for alkaloids, glycoside, saponin, resin,
protein, oil and terpenoids. The result of phytochemical analysis together with the relative
abundance of the various constituents in the extracts and the solvent fractions is as shown in
Table 2.
54
Table 2. Result of phytochemical screening and the relative abundance of
phytoconstituents in extract and fractions of Ficus exasperata
Phytoconstituent
Extract/Fractions
MME
HF
CF
EF
MF
FE
CARBOHYDRATE ++ ++ ++ - - +
ALKALOIDS ++ + + ++ + +
GLYCOSIDE - - - - ++ ++
SAPONINS - - - - ++ ++
TANNINS - - - - - -
FLAVONOIDS + ++ + ++ - +
RESINS ++ + - ++ + +
PROTEINS + ++ + + + +
OIL +++ +++ +++ + + -
STEROIDS - ++ + - - -
TERPENOIDS + ++ ++ - ++ ++
ACIDIC COMPOUNDS - + - + - +
Legend: MME = methylene chloride/methanolic extract; HF = n-hexane fraction; CF =
Chloroform fraction; EF = Ethylacetate fraction; Methanol fraction
Absent = - ; Low in concentration = + ; Medium concentration = ++;
High concentration = +++
55
3.3 Acute toxicity test
The acute toxicity testing carried out for the extract (MEE) using the oral and intraperitoneal
routes, showed neither lethality nor observable signs of acute intoxication in the mice after a
24 h observation in the two stages of the study (Table 3). This implies that the LD50 of the
test extract is greater than 5 g/kg body weight (Lorke, 1983) in mice for both routes.
3.4 Effect of extract and fractions on topical (acute) inflammation.
The extract and fractions significantly (P<0.05) inhibited the xylene-induced ear oedema in
mice (Table 4). The n-hexane fraction (HF) showed the greatest inhibition of ear oedema by
as much as 46.7% compared to the mean oedema of the untreated group; this was followed
by the methanol fraction (MF) with an inhibition of 32.0%; the ethyl acetate fraction (EF)
with mean oedema inhibition of 30.0%, then the chloroform fraction (CF) with mean oedema
inhibition of 22.0% and then the crude extract (MME) with mean oedema inhibition of
21.2%.. The inhibition of inflammation produced by the HF (46.7%) was comparable
(P>0.05) to the level of inhibition produced by indomethacin (50.8%) used as a standard anti-
inflammatory agent in the study (Table 4).
3.5 The systemic effect of the extract and fractions on agar-induced acute
inflammation of the rat paw
The extract and its fractions significantly (P<0.05) inhibited rat paw oedema to varying
degrees. The methanol extract and the ethyl acetate fraction showed better levels of inhibition
compared to other extracts. The highest levels of inhibition were produced at the third hour
after the administration of phlogistic agent (Figures 1a and 1b). The chloroform fraction (CF)
showed the least inhibition of paw oedema. At a dose of 200 mg/kg, the group that received
56
ethyl acetate fraction (EF) showed an inhibition levels higher than those of the standard
agent, Diclofenac K (50 mg/kg). The degree of activity is rated thus:
EF>CF>MME>MF>HF. At a dose of 400mg/kg, the group that received methanol fraction
(MF) showed an inhibition levels higher than those of the standard agent, Diclofenac K (50
mg/kg). Various extract at 400mg/kg and fractions produced varying degrees of paw
oedema inhibition and can be ranked thus: EF>MF>HF>MME>CF.
TABLE 3. Result of acute toxicity (LD50) test of the crude extract of F. exasparata.
STAGES DOSE (mg/kg) MORTALITY
Stage 1 10 0/3
100 0/3
1000 0/3
Stage 2
1600 0/1
2900 0/1
5000 0/1
57
Table 4. Effect of extract and fractions of F. exasparata on xylene-induced edema
Extract
fractions
Mean weight ± SEM (mg)
of the extracts and fractions
Inflammation (% ) Inhibition (%)
MME 7.88 ± 1.456 78.80 21.20
HF 5.33 ± 1.085a 53.30 46.70
CF 7.80 ± 1.641 78.00 22.00
EF 7.00 ± 0.817 70.00 30.00
MF 6.80 ± 0.833 68.00 32.00
Indomethacin 4.92 ± 1.027b 49.20 50.80
Control 10.00 ± 1.647 - -
a= P<0.05; b=P<0.01; c=P<0.001,
58
3.6 Effect of extract and fractions on formaldehyde-induced arthritis in rat
The extracts of F. exasparata caused significant dose-dependent inhibition of formaldehyde-
induced arthritis in rats. The highest inhibition of level of 22% was produced by MME and
this was comparable (P>0.05) to that produced by 50mg/kg Indomethacin (26.88%) used as a
standard anti-inflammatory agent. n-hexane fraction (HF) ranked next after MME in terms of
inhibition of chronic inflammation and the methanol fraction (MF) is the least active (Table
6).
3.7 Effect of extract on membrane stabilization (heat-induced haemolysis)
The extract did not protect the erythrocyte against heat-induced haemolysis and had no
protection on cold-induced erythrocyte haemolysis (Table 7).
3.8 Effect of the extract on cell migration
The extract MME (200 - 800 mg/kg) inhibited the mobilisation of leucocytes in response to
inflammatory stimulus (Table 8). The extract showed higher levels of inhibition of
lecucocytes migration in lower doses than indomethacin. There was little or no change in the
neutophils counts compared to the control treatment except in 800mg/kg (Table 8).
59
Table 5: Effect of extract and fractions of F. exasparata on acute systemic
inflammation of the rat paw
Extract
/Fractions
Dose
(mg/kg)
Oedema volume (cm3)
1 h 2 h 3 h 4 h 5 h
MME
200 0.28±0.02 0.20±0.03 0.18±0.02a 0.34±0.04 0.40±0.02
400 0.32±0.04 0.25±0.02 0.30±0.04 0.35±0.03 0.37±0.04
Hexane
200 0.30±0.03 0.23±0.03 0.25±0.025 0.30±0.03 0.40±0.04
400 0.34±0.04 0.17±0.04 0.16±0.02 0.17±0.02a 0.26±0.02
Chloroform
200 0.30±0.04 0.25±0.02 0.15±0.02 0.30±0.02 0.40±0.04
400 0.33±0.07 0.27±0.04 0.30±0.05 0.37±0.05 0.40±0.05
Ethyl acetate
200 0.10±0.01a 0.15±0.02
a 0.25±0.02 0.30±0.04 0.35±0.02
400 0.33±0.02 0.28±0.05 0.20±0.03 0.23±0.05 0.36±0.05
Methanol
200 0.30±0.03 0.35±0.02 0.30±0.04 0.40±0.03 0.54±0.05
400 0.26±0.05 0.13±0.02 0.20±0.03 0.17±0.03a 0.23±0.07
Diclofenac K 50 0.32±0.06 0.24±0.04 0.28±0.07 0.36±0.08 0.42±0.07
Control (2%
Tween)
0.42±0.04 0.36±0.02 0.44±0.06 0.52±0.06 0.54±0.05
a= P<0.05
60
Figure 1: Effect of extract and fraction of F. exasparata (200 mg/kg) on acute systemic
inflammation of the rat paw
200 mg/kg
1 h
2 h
3 h
4 h
5 h
0
20
40
60
80MME (200 mg/kg)
HF (200 mg/kg)
CF (200 mg/kg)
EF (200 mg/kg)
MF (200 mg/kg)
Diclofenac K (50 mg/kg)
Negative control
Time intervals (h)
Paw
oed
em
a i
nh
ibit
ion
(%)
61
Figure 2: Effect of extract and fraction of F. exasparata (400 mg/kg) on acute systemic
inflammation of the rat paw
400 mg/kg
0 1 2 3 4 5 6
0
20
40
60
80MME (400 mg/kg)
HF (400 mg/kg)
CF (400 mg/kg)
EF (400 mg/kg)
MF (400 mg/kg)
Diclofenac K (50 mg/kg)
Negative control
Time intervals (h)
In
hib
itio
n o
f P
aw
oed
em
a (
%)
62
Table 6. Effect of extract and fractions of F. exasparata on formaldehyde-induced
arthritis
Treatment AUC±SEM Inhibition (%)
200 mg of the extracts
MME 1.6950 ± 0.30 22.00
HF 1.9158 ± 0.17 11.85
MF 1.9700 ± 0.09 9.35
400 mg of the extracts
MME 1.8417 ± 0.57 15.26
HF 2.0183 ± 0.23 7.13
MF 2.0418 ± 0.20 6.05
Indomethacin 50mg/kg 1.5892 ± 0.13 26.88
Control 2.1733 ± 0.31 -
63
Table 7. Effect of the extract of F. exasparata on membrane stabilization
Treatment Dose
(µg/ml)
Heat Abs. % Inhibition Cold
Abs.
% Inhibition
Hydrocortisone 100 0.160 - 0.055 48
Indomethacin 50 0.088 14.6 0.070 34.7
100 0.117 - 0.099 7.3
MME 200 0.225 - 0.137 -
400 0.353 - 0.171 -
800 0.408 - 0.188 -
Control 0.103 - 0.101 -
64
Table 8: Effect of Extract on Leucocytes Migration Count In Vivo
Drug/Extract Dosage (mg/kg) TLC±SEM×103
(cells/ml)
Neutrophils±SEM
(%)
MME 200 3.23 ± 0.54 51.6 ± 3.01
400 2.45 ± 0.14 51.4 ± 1.80
800 4.30 ± 0.58 41.5 ± 3.84
INDOMETHACIN 100 3.85 ± 0.63 54.3 ± 0.47
CONTROL 5.20 ± 0.83 56.5 ± 1.34
TLC=Total Leucocytes Count
65
Figure 3. Effect of FE on the viability of bone marrow-derived macrophages (BMDMs)
0 10 50 100
200
50
75
100
125
150
175
Concentration of FE (g/ml)
Via
bil
ity (
%)
66
Figure 4. Effect of FE on LPS-induced TNF-α production in vitro
Values shown are the mean (±SEM) of triplicate values. Value is significantly different (P <
0.05) compared with that of the ‘LPS alone’ treatment.
Unst
imula
ted
LPS o
nly
g/ml)
LPS +
FE (5
g/m
l)
LPS +
FE (2
5 g/m
l)
LPS +
FE (1
00
0
1
2
3
4
Treatment
TN
F
(n
g/m
l)
*
*
*
67
Figure 5. Effect of FE on LPS-induced IL-1β production in vitro
*Value is significantly different (P < 0.05) compared with that of the ‘LPS alone’ treatment.
Unst
imula
ted
LPS o
nly
g/ml)
LPS +
FE (5
g/m
l)
LPS +
FE (2
5 g/m
l)
LPS +
FE (1
00
0.0
0.2
0.4
0.6
Treatment
IL-1
(n
g/m
l)
*
*
*
68
Figure 6. Effect of FE on LPS-induced NO production
*Value is significantly different (P < 0.05) compared with that of the ‘LPS alone’ treatment.
Unst
imula
ted
LPS o
nly
g/ml)
LPS +
FE (5
g/m
l)
LPS +
FE (2
5 g/m
l)
LPS +
FE (1
00
0
10
20
30
40
Treatment
NO
co
nc.
in
M/1
05cells
* *
69
CHAPTER FOUR
DISCUSSION AND CONCLUSION
4.1 DISCUSSION
Inflammatory diseases include different types of rheumatic disorders such as rheumatic
fever, rheumatoid arthritis, ankylosing spondylitis, polyarthritis nodosa, systemic lupus
erythematosus and osteoarthritis. Inflammation results in the liberation of endogenous
mediators like histamine, serotonin, bradykinin, prostaglandins, cytokines, and nitric
oxide (McPhee et al., 2006). Array of drugs are available to treat these disorders but only
very few are free from toxicity. Gastrointestinal problems associated with the use of anti-
inflammatory drugs are still an enduring dilemma of medical world. It is very important
that profound research with ethnobotanical plants possessing anti-inflammatory and
analgesic properties can definitely open up new vistas in inflammatory disorders. Purified
natural compounds from plants can serve as template for the synthesis of new generation
anti-inflammatory drugs with low toxicity and higher therapeutic value (Newman et al.,
2003).
In this project, the anti-inflammatory properties of Ficus exasperata was investigated in
several models of inflammation and the possible mechanisms of actions studied in-vitro.
Ficus exasperata belongs to the family Moraceae and is used in traditional medicine
practice to treat inflammation (Igoli et al, 2005) and disorders with associated
inflammatory components. The anti inflammatory properties of the various leaf extracts
of Ficus exasperata were evaluated for anti-inflammatory activities.
In the study, the crude methylene chloride/methanol extract (MME) as well as the
fractions showed a significant (P<0.05) inhibition of both acute and chronic
inflammation.
70
Bioassay-guided fractionation of the crude MME gave rise to three fractions; n-hexane,
methanol and ethyl acetate fractions respectively. The fractions were found to inhibit
early phase of acute inflammation which is associated with the release of some pro-
inflammatory mediators (Damas et al., 1990; Ialenti et al., 1992, White., 1999). It is
possible that the extract may have either inhibited the release or antagonize the action of
these inflammatory mediators. Ethyl acetate and methanol fractions showed a
comparably higher inhibition of chronic inflammation in the rats. Chronic inflammation is
invariably associated with the release of macrophages and other leucocytes at the site of
inflammation (Lanzavecchia, 1995).
Previously, the leaf extract of F. exasparata was shown to contain alkaloids (Ijeh and
Ukwemi, 2007), flavonoids, tannins, and saponins (Ayinde et al., 2007) which may
account for the pharmacological effects. In this work, phytochemical investigation of the
MME and the three active fractions (n-hexane, methanol, and ethyl acetate) showed that
they contained flavonoid, terpenoids (volatile oil, triterpenes and steroids), alkaloids and
saponin. The presence of flavonoid indicates the natural occurring phenolic compound,
with beneficial effects in the human diet as antioxidants and neutralizing free radicals
(Adebayo et al., 2009). Anti-inflammatory activities of many plants have been attributed
to their high sterol/triterpene (Ahmad et al., 1983) or flavonoids contents (Parmar and
Ghosh, 1978; Silva et al., 2005).Various mechanisms of anti-inflammatory effects have
been postulated for some of these phytochemical constituents (Shinde et al., 1999, Mills
and Bone., 2000, Umukoro and Ashorobi., 2006 ).
Edema is a multimediated phenomenon that liberates diversity of mediators. It is
believed to be biphasic; the first phase (1h) involves the release of serotonin and
histamine while the second phase (over 1h) is mediated by prostaglandins, the
cyclooxygenase products, and the continuity between the two phases is provided by
71
kinins (Perianayagam et al., 2006, Asongalem et al., 2004, Silva et al., 2005).
Development of edema induced by agar is commonly correlated with early exudative
stage of inflammation (Silva et al., 2005, Ozaki, 1990). In this study, n-hexane fraction,
ethyl acetate fraction and MME exhibited a non-dose related effect and may possibly act
at the first phase of oedema formation thereby inhibiting the histamine and serotonin
production. Though, its maximum activity was noticed on the third hour of edema
formation. n-hexane fraction will however, be more effective topically, since the
lipophylic constituents will easily permeate the lipoidal layers of skin.
The later phase of acute inflammatory response (acute cellular response) involves the
migration of neutrophils to the site of inflammatory stimulus (Insel, 1990, Cotran et al.,
1999, Guyton, 2006). Investigated is the effect of the extract on in vivo leucocytes
migration. Leucocytes usually migrate to the site of inflammation in response to
chemotactic stimulus (Wagner and Roth, 2000). This plays a pivotal role in the
pathogenesis of inflammatory disorders of both acute and chronic types. During
phagocytosis, the activated leucocytes release superoxide radicals and other cytoplasmic
contents at the site of inflammation; this can further cause tissue damage and
inflammation (Weissmann et al., 1980, Perez and Weismann, 1981). Inhibiting the
migration of leucocytes to the site of inflammation may be the important mechanism of
action of the anti-inflammatory constituent in MME. MME has no effect on membrane
stabilization (cold). Release of inflammatory mediators by degranulated mast cells causes
edema .Flavonoids in previous studies, has shown that it stabilizes cell membrane (Chaika
and Lal., 1977). It is not very clear why MME was unable to stabilize the cell membrane
since it does not contain saponins which have hemolytic properties. Though, one of the
fractions of MME (methanol) possesses saponins in mild quantity.
The effect of extracts and fractions on formaldehyde-induced arthritis was investigated.
72
Formaldehyde is a potent edematous agent and produces inflammation through the
release of several inflammatory mediators including prostaglandins (Tjolsen et al., 1992).
The ability of these extracts/fractions to inhibit the global edematous response induced by
formaldehyde suggests that they contain chemical agents that which can be very useful in
the management of chronic arthritis. MME extracts showed the highest inhibition while
methanol expressed the least inhibition.
It has been reported that macrophages recruited to sites of inflammation are less mature,
retain proliferative capacity, and display phenotypic changes characteristic of activated
cells such as an enhanced respiratory burst and an enhanced ability to restrict the growth
of intracellular parasites (Bursuker and Golden, 1983; Gordon et al., 1984; Walker and
Yen, 1984; Lepay et al., 1985). Phagocytic capacity, cytotoxicity, expression of
transferring receptor, chemotactic responses, and the production of various molecules
associated with inflammation (plasminogen activator, inhibitors of fibrinolysis,
complement factor C2, and interferon) have all been shown to be expressed maximally at
specific stages of macrophage differentiation (Neuman and Sorg, 1980; Sorg 1982; Alpert
et al., 1983). The possible anti-inflammatory mechanism of action of Ficus exasperata
was elucidaated by the action of the aqueous extract on bone marrow-derived
macrophages (BMDM) on the release of pro-inflammatory meditors (TNF-αand IL-1β).
TNF-α is a member of the pro-inflammatory cytokines family and can stimulate the
recruitment of neutrophils and monocytes to sites of infection. Increased plasma TNF-
αlevels during sepsis contribute to host lethality. It has been found that host treatment
with TNF-α-neutralizing antibodies yields significant protective effects during an episode
of acute sepsis that is usually associated with the release of several pro-inflammatory
mediators (Wheeler and Bernard, 1999; Raza, 2000). The inhibition of iNO release by
activated macrophages by the extract of FE is also an important mechanism in its anti-
73
inflammatory effect. NO is produced by NO synthase (NOS) (Korhonen et al., 2005);
after exposure to LPS, inducible NOS (iNOS) is induced quantitatively (Duval et al.,
1996).
The role of NO in host defense against microorganisms and tumor cells is well
recognized. Nevertheless, excess production of NO is also associated with several
diseases, e.g., arthritis, autoimmune diseases, septic shock, as well as in several chronic
inflammatory diseases. In these disorders, NO is known to contribute to the inflammation
cascade by increasing vascular permeability, extravasations of fluid and proteins at
inflammatory sites (Moncada et al., 1991; Snyder and Bredt, 1992; Guzik et al., 2003).
As such, the inhibition of high-output NO production has been a therapeutic strategy
increasingly used for the treatment of various inflammatory diseases. With respect to the
effects on inducible TNF-α and NO production, the observed suppressive effects were not
due to cytotoxicity or systemic toxicity of the extract.
Specifically, the acute toxicity studies in mice did not suggest severe untoward effect
after oral/intraperitonial administration at doses up to 5,000 mg/kg. The implication is
that for all practical purposes, the crude extract of the F. exasparata is considered safe
(Lorke, 1983) for use in the animals.
Furthermore, in-vitro, the viability of the macrophages was not affected by FE extract at
the concentrations used in the study; this reaffirms its safety profile. The inhibition of
inducible TNF-α and NO production seen here to have been caused by the extract of FE
provides clues to possible mechanism(s) that could be used to explain the reported
efficacy of the extract in its traditional use against disorders that are characterized by
inflammation. Some phytoconstituents, such as flavonoids that are present in FE might be
responsible for these anti-inflammatory properties. Flavonoids and other phenolic
compounds that were found to be present in the extract of FE have been shown to possess
74
anti-inflammatory activities through an inhibition of the generation of pro-inflammatory
arachidonic acid derivatives and cytokines (Williams et al., 1995).
4.2 CONCLUSION
The results of this study provide a rationale for the ethnomedicinal uses of the leaf of
Ficus exasperata in the management of both acute and chronic inflammatory disorders. The
extracts of Ficus exasperata displayed anti-inflammatory properties against agar-induced
paw oedema, xylene-induced ear oedema, as well as in formaldehyde-induced chronic
oedema in rodents. Aqueous extract of Ficus exasperata inhibited LPS-induced nitric oxide
(iNO) and tumour necrosis factor-α (TNF-α) formation in vitro in cultures of bone marrow
derived macrophages. These results lend credence to the ethno-medicinal uses of Ficus
exasperata leaf extracts against inflammatory disorders and suggest the involvement of a
suppression of pro-inflammatory mediators (such as NO and TNF-α) as a possible
mechanism underlying these effects.
75
REFERENCES
Abad MJ, Bermejo P, Carretero E, Martinez Acitores C, Noguera B and Villar A (1996).
anti-inflammatory activity of some medicinal plant extracts from
Venezuela.J.Ethnopharmacol. 55, 63–68.
Adebayo EA, Ishola OR, Taiwo OS, Mayolagbe ON and Adekeye BT (2009).
Evaluations of the methanol extract of Ficus exasperata stem, leaf and
antimicrobial activities. African J. Plant Sc. 283-287.
Ahmad MM, Quresh S, Shah A, Qazi NS,Rao RM, Albakiri M (1983). Anti-
inflammatory activity of Caralluma tuberculata alcoholic extract. Fitoterapia. 46,
357-360.
Ahmed MM, Qureshi S, Al-bekairi AM, Shah AH, Rao RM , Qazi NS (1993). Anti-
inflammatory Activity of Caralluma tuberculata alcoholic extract. Fitoterapia ,
64:359-362.
Akah PA, Nwambie AI (1994). Evaluation of Nigerian traditional medicines: plants used
for rheumatic disorder. J. Ethnopharmacol., 42: 179-182.
Akah PA, Orisakwe OE, Gamaniel KS , Shittu A(1998). Evaluation of Nigerian traditional
medicines: II. Effects of some Nigerian folk remedies on peptic ulcer. J
Ethnopharmacol 62:123-7.
Ali H, Haribabu B, Richardson RM, Snyderman R (1997). Mechanisms of
inflammation and leukocyte activation. Med Clin North Am. 81:1-28.
Alpert SE, Auerbach HS, Cole FS, Colten HR (1983). Macrophage heterogeneity occurs
through a developmental mechanism, J. Immunol. 130, 102-107
Asongalem EA, Foyet HS, Ekoo S, Dimo T, Kamtchouing P (2004). Anti- inflammatory, lack
of central analgesia and antipyretic properties of Acanthusmontanus (Ness) T.
76
Anderson. J. Ethnopharmacol 95, 63-68.
Asuzu IU, Sosa S, Della LR (1999). The anti-inflammatory activity of Icacina trichantha
tuber. Phytomed 6:267-272.
Atta AH, Alkohafi A (1998): Antinociceptive and anti-inflammatory effects of some
Jondanian medicinal plants extracts. J Ethnopharmacol, 60:117-124.
Austin SC, Funk CD (1999). Insight into prostaglandin, leukotriene, and other eicosanoid
functions using mice with targeted gene disruptions. Prostaglandins Lipid Mediat.,
58:231-252.
Ayinde BA, Omogbai EK , Amaechina FC (2007). Pharmacognosy and hypotensive
evaluation of Ficus exasperata Vahl (Moraceae) leaf. Acta Pol. Pharm 64:543-6.
Ayoola GA, Akpanika GA, Awobajo FO, Sofidiya MO, Osunkalu VO, Coker HAB ,
Odugbemi TO ( 2009). Anti-inflammatory properties of the fruits of
Allanblanckia floribunda oliv (Guttiferae) Bot. Res. Intl. 2 (1): 21-26.
Backhouse N, Delporte C, Negrete R, Salinus P, Pinto A, Aravena S and Cassels BK
(1996). Anti-inflammatory and antipyretic activities of Cuscuta chilensis, Cestrum
parqui, and Psolarea glandulosa. Int J Pharmacog , 34:53-57.
Bohlin L (1995). Structure-activity studies of natural products with anti-inflammatory
effects. In: Hostettmann, K. (Ed.), Phytochemistry of Plants used in Traditional
Medicine. Clarendon Press, Oxford. pp. 137-161.
Brink C, Dahlen SE , Drazen J (2003). International Union of Pharmacology: XXXVII.
Nomenclature for leukotriene and lipoxin receptors. Pharmacol. Rev., 55:195-227.
Burke A, Smyth E, FitzGerald G A (2006). Goodman and Gilman‟s, the pharmacological
basis of therapeutics, Analgesic-Antipyretic Agents; Pharmacotherapy Of
77
Gout; 11th
ED Mc Graw-Hill companies;London .
Bursuker I & Goldman R (1983) J. Reticuloendothel. Soc. 33; 207-220.
Calixto JB, De Lima TCM, Morato GS, Nicolau M, Takahashi RN, Valle RMR, Schmidt
CC, Yunes RA (1990). Chemical and pharmacological analysis of the crude
aqueous/alcoholic extract from Cordyline dracaenoides. Phytother. Res. 5, 167–
171.
Calixto JB, Zanini JC, Cruz AB, Yunes RA , Medeiros YS (1991). Extract and compounds
obtained from Mandevilla velutina inhibit arachidonic acid- induced ear
oedema in mice, but not rat stomach contraction. Prostaglandins 41, 515–526.
Calixton JB, Nicolau M, Trebien H, Henrique MGO, Weg VB, Cordeiro RSB , Yunes RA
(1986). Antiedematogenic actions of a hydroalcoholic crude water- alcohol
extract of Mandevilla velutinabraz. Braz. J. Med. Biol. Res. 19, 4–5.
Catella-Lawson F, McAdams B, Morrison, BW(1999). Effects of specific inhibition of
cyclooxygenase-2 on sodium balance, hemodynamics, and vasoactive
eicosanoids. J. Pharmacol. Exp. Ther., 289:735-741.
Chaika LA, lal khadzhai (1977). Membrane stabilizing effect of medicinal substances used
for the treatment of chronic venous insufficiency. Farmkol toksikol; 40: 306-
309.
Chattopadhyay RR (1998). Possible biochemical mode of anti-inflammatory action of
Azadirachta indica A. Juss in rats. Indian J Exp Biol. 36:418-20.
Cheng Y, Austin SC, Rocca B (2002). Role of prostacyclin in the cardiovascular response to
thromboxane A2. Science, 296:539-541.
Chhabra SC, Mahunnah RL, Mshiu EN (1990). Plants used in traditional medicine in
78
eastern Tanzania. IV. Angiosperms (Mimosaceae to Papilionaceae). J
Ethnopharmacol; 29:295-323.
Cifuente DA, Simirgiotis MJ, Favier LS, Rotelli AE, Pelzer E (2001). Anti-
inflammatory activity from aerial parts of Baccharis medullosa, Baccharis
rufescens and Laennecia sophiifolia in mice. Phytother. Res. 15, 529–531.
Coggins KG, Latour A, Nguyen MS (2002). Metabolism of PGE2 by prostaglandin
dehydrogenase is essential for remodeling the ductus arteriosus. Nature Med.,
8:91-92.
Cotran RS, Kumar V, CollinsT(1999). Robbin‟s pathological basis of disease. 6th
ed.
W.B. Saunders, Philadelphia. pp 50-111.
Cousins ON, Micheal AH (2002). Medicinal properties in the diet of Gorillas. An ethno-
pharmacological evaluation. Afr. Stud. Monogr. 23(2): 65-89.
Craig CR and Stitzel RE (1999). Modern pharmacology with clinical applications 5th
ed.
Lippincott Williams & Wilkins., New York; p 434.
Damas J, Bourdon V, Remeade-volon G, Adams A (1990). Kinins and peritoneal exudates
induced by carregeenan and zymosan. Br. J Pharmacol. 211: 81-86.
De Las Heras B, Slowing K, Benedi J, Carretero E, Ortega T, Toledo C Bermejo P,
Iglesias I, Abad MJ, Gomez-Serranillos P, Liso PA, Villar A , Chiriboga X
(1998). Anti-inflammatory and antioxidant activity of plants used in traditional
medicine in Ecuador. J. Ethnopharmacol. 61, 161–166.
Dempsey PW, Doyle SE, He JQ, Cheng G (2003). The signaling adaptors and pathways
activated by TNF superfamily. Cytokine Growth Factor Rev., 2003, 14:193-
209.
79
Dharmasiri JR, Jayakody AC, Galhena G, Liyanage SSP and Ratnasooriya WD (2003).
Anti-inflammatory and analgesic activities of mature fresh leaves of Vitex
negundo. J Ethnopharmacol 87,199-206.
Diaz-Gonzalez F , Sanchez-Madrid F (1998). Inhibition of leukocyte adhesion: an
alternative mechanism of action for anti-inflammatory drugs. Immunol. Today,
19:169-172.
Diwan PV, Margaret I, Ramakrishna S (1995). Influence of Gymnema sylvestre on
inflammation. Inflammopharmacology, (U.K). 3: 271-277.
Djukanovic R, Roche WR, Wilson JW, Beasley CRW, Twentyman OP, Howarth PH,
Holgate ST (1990). Mucosal inflammation in asthma Am. Rev. Resp. Dis.
142:434-457
Dray A, Perkins M (1993). Bradykinin and inflammatory pain. Trends Neurosci., 16:99-104.
Dunstan CA, Noreen Y, Serrano G, Cox PA, Perera P, Bohlin L (1997). Evaluation of
some samoan and peruvian medicinal plants by prostaglandin biosynthesis and
rat ear oedema assays. J. Ethnopharmacol. 57, 35–56.
Dutra RC, Tavares CZ, Ferraz SO, Sousa OV , Pimenta DS(2006) Investigation of
analgesic and anti-inflammatory activities of Echinodorus grandiflorus
Rhizomes methanol extract. Rev. Bras. Farmacogn., 16, 469–474.
Duval DL, Miller DR, Collier J, Billings RE (1996). Characterization of hepatic nitric oxide
synthase: identification as the cytokine-inducible form primarily regulated by
oxidants. Mol. Pharmacol. 50:277–284.
Fazio S, Pouso J, Dolinsky D, Fernandez A, Hernandez M, Clavier G, Hecker M
(2009).Tolerance, safety and efficacy of Hedera helix extract in inflammatory
80
bronchial diseases under clinical practice conditions: a prospective, open,
multicentre postmarketing study in 9657 patients. Phytomedicine 16, 17–24.
Gaboury JP, Johnston B, Niu XF, Kubes P (1995). Mechanisms underlying acute mast cell-
induced leukocyte rolling and adhesion in vivo. J. Immunol., 154:804-813.
Ganong WF (2003). Review of medical physiology, Cardiovascular Homeostasis in
Health & Disease. 21ST
edition: Mc Graw-Hill companies.london.
Gordon S, Hirsh S, Ezekowitz RAB (1984). In Mononuclear Phagocyte Biology, ed.
Volkman, A. (Dekker, New York), pp. 301-316.
Green RJ, Harris ND (1993). Pathology and Therapeutics for Pharmacists: A basis for
clinical pharmacy practice. 1st edition. Chapman and hall. London. pp 31-38
Guyton CA, Hall JE (2006). Textbook of Medical Physiology 11TH
ED W.B Saunders
company, Philadelphia. pp 434-435.
Guzik TJ, Korbut R, Adamek-Guzik T (2003). Nitric oxide and superoxide in
inflammation and immune regulation. J. Physiol. Pharmacol. 54:469–487.
Harbone, JB (1984). Phytochemical methods. A guide to modern technique of plant
analysis. 2nd
edition. Chapman and hall ltd. London. p.282.
Henriques AT, Melo AA, Moreno PRH, Ene LL, Henriques JAP, Schapoval EES (1996).
Ervatamia coronaria: Chemical constituents and some pharmacological
activities. J. Ethnopharmacol. 50, 19–25.
Henriques MGMO, Fernandes PD, Weg VB, Yunes RA, Cordeiro RSB, Calixto JB (1991).
Inhibition of rat paw oedema and pleurisy by the extract from Mandevilla
velutina. Agents Actions 33, 272–278.
81
Hyde, M.A., Wursten, B.T. & Ballings, P. (2012). Flora of Zimbabwe: Species information:
Ficus exasperata.
www.zimbabweflora.co.zw/speciesdata/species.php?speciesid120280, retrieved 12
may 2012
Ialenti A , Ianaro A, Moncada S , Di Rosa M (1992). Modulation of acute inflammation by
endogenous nitric oxide. Eur J Pharmacol. 211: 177-182.
Igoli JO, Ogaji OG, Tor-Anyiin TA
, Igoli N P
(2005) Traditional Medicine Practice
amongst the Igede People of Nigeria. PART II, Afr. J. Trad. CAM 2 (2): 134 –
152
Ijeh I I, Agbor C (2006). Body and organ weight changes following administration of
aqueous extracts of Ficus exasperata Vahl on white albino rats. J. Animal Veterin.
Adva. 5(4): 277-279.
Ijeh II, Ukweni AI (2007). Acute effect of administration of ethanol extracts of
Ficus exasperata vahl on kidney function in albino rats. J. Medi. Plants Res. Vol.
1(2), pp. 027-029.
Insel PA (1990). Analgesic-antipyretics and anti-inflammatory agent; drugs employed in
the treatment of rheumatoid arthritis and gout. In Goodman L S, Gilman A,eds.
New York : Pergamon Press, 638-681.
Irvine FR (1961). Woody plants of Ghana. 1 ed. London: Oxford University Press; London
: xcv, 868
Ismail TS, Gopalakrishnan S, Hazeena BV (1997). Biochemical modes of action of Gmelina
asiatica in inflammation. Indian J Pharmacol. 29:306-9.
Iwueke AV, Nwodo OFC, Okoli CO (2006). Evaluation of the anti-inflammatory and
82
analgesic activities of Vitex doniana leaves. Afr J Biotech, 5(20):1929-1935.
KanaokaY, Boyce JA (2004). Cysteinyl leukotrienes and their receptors: Cellular distribution
and function in immune and inflammatory responses. J. Immunol.,173:1503-1510.
Katzung BG, Furst DE (1998) Non steroidal anti inflammatory drugs, disease modifying
anti rheumatic drugs. In: Basic and Clinical Pharmacology. (Bertram G. Katzung
ed.) 7th
edition. Appleton and Lange USA. ;Pp 584.
Keay RWJ , Onochie CFA (1964). Nigeria Trees. Dept. For. Res. 1&2:
389-390.
Korhonen R, Lahti A, Kankaanranta H , Moilanen E( 2005). Nitric oxide production and
signaling in inflammation. Curr. Drug Targets. Inflamm. Allergy. 4:471–479.
Kumara NKVMR (2001). Identification of strategies to improve research on medicinal
plants used in Sri Lanka. In: WHO Symposium. University of Ruhuna, Galle, Sri
Lanka. Ethnopharmacol; 85, 25-32.
Kumar VL, Basu N (1994). Anti-inflammatory activity of the latex of Calotropis procera.
Journal of Ethnopharmacology 44, pp. 123–125.
Kyriakis JM, Avruch J (2001). Mammalian mitogen-activated protein kinase signal
transduction pathways activated by stress and inflammation. Physiol.
Rev., 81:807-869.
Lanzavecchia A (1995). How can cryptic epitopes trigger autoimmunity? J Exp Med.
181:1945.
Lepay DA, Steinman RM, Nathan CF, Murray HW, Cohn ZA (1985). J. Exp. Med. 161,
1503-1512.
Li RW, Myers SP, Leach DN, Lin GD, Leach G (2003). A cross-cultural study: anti-
83
inflammatory activity of Australian and Chinese plants. J Ethnopharmacol. 85(1):
25-32
Lin H, Chen C, Ben DM (2001) Resistance of bone marrow-derived macrophages to
apoptosis is associated with the expression of X-linked inhibitor of apoptosis protein
in primary cultures of bone marrow cells. Biochem. J. 353, 299±306
Lino CS, Taveira ML, Viana GSB , Matos FJA (1997). Analgesic and anti-inflammatory
activities of Justicia pectoralis Jacq and its main constituents: Coumarin and
umbelliferone. Phytother. Res. 11, 211–215.
Linton LA (1984). Adverse effects of NSAIDs on renal function. Can Med Assoc J.131(3):
189–191
Lorke D (1983). A new approach to practical acute toxicity testing. Arch Toxicol. 53:275-289
Macfoy CA, Cline EI (1990). In vitro antibacterial activities of three plants used in
traditional medicine in Sierra Leone. J Ethnopharmacol. 28:323-7.
Magalhães JFG, Viana CFG, Aragão AGM Jr Moraes VG, Ribeiro RA, Vale MR
(1997). Analgesic and antiinflammatory activities of Ageratum conyzoides in
rats. Phytother. Res. 11, 183–188.
Maia MBS, Rao VS (1989). Anti-inflammatory activity of Orbignia phalerata in rats.
Phytother. Res. 3, 170–174.
Makwana HG, Ravishankar B, Shukla VJ (1994). General pharmacology of Vitex leucoxylon
Linn leaves. Indian J Physiol Pharmacol.38:95-100.
Martel-Pelletier J, Lajeunesse D, Reboul P, Pelletier JP (2003). Therapeutic role of
dual inhibitors of 5-LOX and COX, selective and non-selective non-steroidal
anti-inflammatory drugs. Ann. Rheum. Dis. 62:501-509.
Mazzanti G, Braghiroli L, Tita B, Bolle P, Piccinelli D (1993). Anti-inflammatory
84
activity of Pfaffia paniculata (Martius) Kuntze and Pfaffia stenophylla (Sprengel)
Stuchl. Pharmacol. Res. 27, 91–92.
McAdam BF, Catella-Lawson F, Mardini IA (1999). Systemic biosynthesis of
prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective
inhibitor of COX-2. Proc. Natl. Acad. Sci. U.S.A., 96:272-277.
McMahon B, Godson C (2004). Lipoxins: Endogenous regulators of inflammation. Am.
J. Physiol. Renal Physiol., 286: F189-201.
McPhee SJ, Ganong WF (2006). Pathophysiology of Disease: An Introduction to Clinical
Medicine, 5th Edition. McGraw-Hill, pp. 677-679
Meager A (1999). Cytokine regulation of cellular adhesion molecule expression in
inflammation. Cytokine Growth Factor Rev., 10:27-39.
Mengi SA, Deshpande SG. Evaluation of ocular anti-inflammatory activity of Butea
frondosa. Indian J Pharmacol 1995;27:116-9.
Mills S , Bone K (2000). principles and practice of phytotherapy- modern herbal medicine.
New York: Churchill livingstone.
Miño J, Moscatelli V, Hnatyszyn O, Gorzalczany S, Acevedo C and Ferraro G (2004)
Antinociceptive and antiinflammatory activities of Artemisia copa extracts.
Pharmacol. Res. 50, 59–63.
Miranda AL, Silva JR, Rezende CM, Neves JS, Parrini SC, Pinheiro MLB, Cordeiro
MC, Tamgborini E , Pinto AC (2000). Anti-inflammatory and analgesic activities
of the latex containing triterpenes from Himatanthus sucuuba. Planta Med. 66,
284–286.
Moncada S, Palmer RM , Higgs EA (1991). Nitric oxide: physiology, pathophysiology,
and pharmacology. Pharmacol. Rev. 43:109–142.
85
Monneret G, Li H,Vasilescu J (2002). 15-Deoxy-12, 14-prostaglandins D2 and J2 are
potent activators of human eosinophils. J. Immunol., 168:3563-3569.
Mosmann T (1983). Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Meth. 65 (1–2): 55–63
Mota MLR, Thomas G, Barbosa Filho JM (1985). Anti-inflammatory actions of tannins
isolated from the bark of Anacardium occidentale L. J. Ethnopharmacol. 13,
289–300.
Moura ACA, Silva ELF, Fraga MCA, Wanderley AG, Afiatpour P, Maia MBS (2005).
Antiinflammatory and chronic toxicity study of the leaves of Ageratum
conyzoides L. in rats. Phytomedicine, 12, 138–142.
Mukherjee PK, Saha K, Pal M, Saha BP (1997). Studies on the anti-inflammatory activity of
rhizomes of Nelumbo nucifera (letter). Planta Med. 63:367-9.
Muschietti L, Martino V Ferraro G, Coussio J, Segura L, Cartana C, Canigueral S ,
Adzet T (1996). The antiinflammatory effect of some species from South America.
Phytother. Res. 10, 84–86.
Narayanan N, Thirugnanasambantham P, Viswanathan S (1998). Antinociceptive
antiinflammatory and antipyretic effects of ethanol extract of Clerodendron serratum
roots in experimental animals. J Ethnopharmacol (accepted for publication).
Neumann C , Sorg C (1980). Sequential expression of functions during macrophage
differentiation in murine bone marrow liquid cultures. Eur. J. Immunol. 10, 834-840.
Newman DJ , Cragg GM , and Snader KM. (2003) Natural Products as Sources of New
Drugs over the Period 1981−2002. J. Nat. Prod., 66 (7), pp 1022–1037.
Odunbaku OA, Ilusanya OA , Akasoro KS (2008). Antibacterial activity of ethanolic
leaf extract of Ficus exasperata on Escherichia coli and Staphylococcus
albus. Sci. Res. Essay. 3(11): 562-564.
86
Okoli CO, Akah PA, Nwafor SV, Anisiobi AI, Ibegbunam IN , Erojikwe O (2007)
Anti-inflammatory activity of hexane leaf extract of Aspilia africana C.D. Adams. J
Ethnopharmacol. 109: 219-225.
Okoye FBC, Osabebe PO (2009). Study on the mechanism of anti-inflammatory activity of
the extract and fractions of Alchorea floribunda leaves. Asia Pac J Trop Med.2 (3): 7-
14.
Ortega T, Carretero ME, Pascual E, Villar AM , Chiriboga X (1996). Anti- inflammatory
activity of ethanolic extracts of plants used in traditional medicine in Ecuador.
Phytother. Res. 10, S121–S122.
Ozaki Y (1990). Anti-inflammatory effects of Curcuma xanthorrhiza Roxb, and its
active principle. Chem Pharm Bull. 38, 1045-1048.
Park JH, Son KH, Kim SW, Chang HW, Bae K, Kang SS, Kim HP (2004). Anti-
inflammatory activity of Synurus deltoids. Phytother Res. 18, 930-933.
Parmar NS , Ghosh MMN (1978). Current trends in flavonoid research. Indian J Pharm. 12:
213-228.
Patrick JR, Walter FK, Yasmin B K, and Murray RA (1985). Adverse effects of NSAIDs.
Can Med Assoc J. 132(6): 617.
Perez F, Marin E , Adzet T (1995). The antiinflammatory effect of several Compositae from
South America extracts in rats. Phytother. Res. 9, 145–146.
Perez GRM (1996). Anti-inflammatory activity of Ambrosia artemisiaefoli and Rheo
spathaceae. Phytomed. 3:163-167
87
Perez HD, Weissmann G (1981). Lysozymes as mediators of inflammation. In: keller W.
textbook of rheumatology.1st edition. Philadelphia: W.B. Saunder, 179-194.
Perianayagam JB, Sharma SK, Pillai KK (2006). Anti-inflammatory activity of
Trichodesma indicum root extract in experimental animals. J Ethnopharmacol.
104, 410-414.
Prelude Medicinal Plants Database, http//www. Africanconservation.org/medicinal
plant
Premack BA, Schall TJ (1996). Chemokine receptors: Gateways to inflammation and
infection. Nat Med. 2:1174.
Prescott SM, Zimmerman GA, Stafforini DM, McIntyre TM (2000). Platelet-activating factor
and related lipid mediators. Annu. Rev. Biochem., 69:419- 445.
Rahme E and Nedjar H(2007). Risks and benefits of COX-2 inhibitors vs non-selective
NSAIDs: does their cardiovascular risk exceed their gastrointestinal benefit? A
retrospective cohort study. Rheum. 46(3):435-8.
Rao KS, Mishra SH (1997). Anti-inflammatory and hepatoprotective activities of Sida
rhombifolia Linn. Indian J Pharmacol. 29:110-6.
Rates SMK (2001). Plants as source of drugs. Toxicon 39, 603–613.
Raza A (2000). Anti-TNF therapies in rheumatoid arthritis, Crohn‟s disease, sepsis,
and myelodysplastic syndromes. Microsc. Res. Tech. 50:229–235.
Ribeiro RA, Flores CA, Cunha FQ, Ferreira SH (1991). IL-8 causes in vivo neutrophil
migration by a cell dependent mechanism. Immunol 73:472-477.
Rocca B, FitzGerald GA (2002). Cyclooxygenases and prostaglandins: Shaping up the
88
immune response. Int. Immunopharmacol., 2:603-630.
Ross R (1999). Atherosclerosis an inflammatory disease. N Engl J Med 340:115- 126.
Salama AM, Polo NA, Contreras CR, Maldonado L (1987). Preliminary phytochemical and
pharmacological analysis of Baccharis decussata leaves. Rev. Colomb Cienc.
Quim. Farm. 16, 45–50.
Schwartz LB (1994). Mast cells: Function and contents. Curr. Opin. Immunol., 6:91- 97.
Sercarz EE (1993). Dominance and crypticity of T cell antigenic determinants. Ann Rev
Immunol. 11:729.
Serhan CN, Chiang N (2004). Novel endogenous small molecules as the checkpoint
controllers in inflammation and resolution: entree for resoleomics. Rheum. Dis. Clin.
North Am., 30:69-95.
Seyle H (1949). Further studies concerning the participation of adrenal cortex in the
pathogenesis of arthritis. Brit Med J. 2:1129-1135.
Shinde UA, Phadke AS, Nari AM, Mungantiwar AA, Dikshit VJ, Saraf MN (1999)
Membrane stabilization activity – a possible mechanism of action for the anti-
inflammatory activity of Cedrus deodora wood oil. Fitoterapia 70:251-257.
Singh G (1998). Recent considerations in nonsteroidal anti-inflammatory drug gastropathy.
American Journal of Medicine; 105(1B): 31S-38S.
Singh S, Majumdar DK (1997). Evaluation of anti-inflammatory activity of fatty acids of
Ocimum sanctum fixed oil. Indian J Exp Biol. 35:380-3.
Singh RK, Pandey BL (1996). Anti-inflammatory activity of seed extracts of Pongamia
pinnata in rat. Indian J Physiol Pharmacol. 40:355-8.
Singh RK, Pandey BL (1997). Further study of anti-inflammatory effects of Abies pindrow.
Phytother Res. 11:535-7.
89
Singh S, Majumdar DK, Rehan HMS (1996). Evaluation of anti-inflammatory potential of
fixed oil of Ocimum sanctum (Holy basil) and its possible mechanism of action. J.
Ethnopharmacol., 54: 19-26.
Sivaprakasam P, Viswanathan S, Thirugnanasambantham P, Reddy MK, Vijayasekaran V
(1996). Pharmacological screening of Ochna obtusata. Fitoterapia 67:117-20.
Silva GN, Martins FR , Matheus ME (2005). Investigation of anti-inflammatory and
antinociceptive activities of Lantana trifolia. J Ethnopharmacol.100, 254- 259.
Simões CMO, Schenkel EP, Bauer L, Langeloh A (1988). Pharmacological
investigations on Achyrocline satureioides (Lam). DC., Compositae. J.
Ethnopharmacol. 22, 281–293.
Singer AJ, Clark RAF(1999). Cutaneous wound healing. N Engl J Med .341:738
Smyth EM, FitzGerald GA (2003). Prostaglandin mediators. In, Handbook of Cell
Signaling. (Bradshaw, R.D., ed) Academic Press, San Diego, pp. 265-273
Snyder SH, Bredt DS (1992). Biological roles of nitric oxide. Sci. Am.
266:68–71, 74.
Sonibare MO, Isiaka AO, Taruka MW, Williams NS, Soladoye M, Emmanuel O (2006).
Constituents of Ficus exasperata leaves. Natural product communications. 23-26.
Sorg C (1982). Heterogeneity of macrophages in response to lymphokines and other
signals. Mol. Immunol. 19, 1275-1278.
Teixeira CGL, Piccoli A, Costa P, Soares L , da Silva-Santos JE (2006). Involvment of the
nitric oxide/soluble guanylate cyclase pathway in the anti- oedematogenic action of
Pfaffia glomerata (Spreng) Pedersen in mice. J. Pharm. Pharmacol. 58, 667–675.
Tilley SL, Coffman TM, Koller BH (2001). Mixed messages: Modulation of
90
inflammation and immune responses by prostaglandins and thromboxanes. J.
Clin. Invest. 108:15-23.
Tjolsan A, Berge O, Hunskaar S Rosland JH, Hole K (1992). The Formalin test: An
evaluation of the method. Pain. 51:5-14.
Toda A, Yokomizo T , Shimizu T (2002). Leukotriene B4 receptors. Prostaglandins
Other Lipid Mediat., 68-69:575-585.
Tubaro A, Dri P, Delbello G, Zilli C, Della LR (1985). The croton oil ear test revisited.
Agents Actions, 17:347-349.
Umukoro S, Ashorobi RB (2006). Evaluation of anti-inflammatory and membrane
stabilizing properties of aqueous leaf extract of Momordica charantia in rats. Afr J
Biomed Res. 9:119-124
Vane, JR(1971). Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-
like Drugs. Nat. New Biol. 231, 232-235.
Vane JR and Bolting RM (1995). New insights into the mode of action of anti-
inflammatory drugs. Inflammation Res. 44: 1-10.
Viana CFG, Aragao AGM Jr., Ribeiro RA Magalhaes JFG Vale MR (1998). Effects of
Ageratum conyzoides in nociception and inflammatory response induced by Zymosan.
Fitoterapia 69, 349–354.
Viana GSB, Bandeira MAM, Matos FJA (2003). Analgesic and anti-inflammatory
effects of chalcones isolated from Myracrodruon urundeuva Allemao.
Phytomedicine. 10, 189–195.
Vimala R, Nagarajan S, Alam M, Susan T, Joy S (1997). Anti-inflammatory and antipyretic
91
activity of Michelia champaca Linn, (white variety), Ixora brachiata Roxb. and
Rhynchosia cana (Willd.) D.C. flower extract. Indian J Exp Biol. 35:1310-14.
Wagner JC, Roth AR (2000). Neutrophil migration mechanisms, with an emphasis on
the pulmonary vasculature 1. Pharmacol., 52:349-374.
Walker WS, Yen SE (1984). In Mononuclear Phagocyte Biology, ed. Volkman, A.
(Dekker, New York), pp. 207-222.
Weischenfeldt J, Porse B (2008). Bone Marrow-Derived Macrophages (BMM): Isolation and
Applications. Cold Spring Harb Protoc. 10:1101.
Weissmann G, Smolen JE, Korchak HM (1980). Release of inflammatory mediators
from stimulated neutrophils. New Engl. J Med. 303:24-27.
Wheeler AP, Bernard GR (1999). Treating patients with severe sepsis. N. Engl. J. Med.
340:207–214.
White M (1999). Mediators of inflammation and inflammatory process. J Allergy clin.
Immunol. 103: 5378-5381.
Williams CA, Hoult JR, Harborne JB, Greenham J, Eagles J (1995).A biologically
active lipophilic flavonol from Tanacetum parthenium. Phytochemistry 38:267–
270.
Winter CA, Risley EA, Nuss GW (1962). Carrageenin-induced edema in the hind paw of
the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med. 3:544-547.
Woode E, Poku RA, Ainooson GK, Boakye-Gyasi E, Abotsi WKM, Mensah TL (2009). An
evaluation of the anti-inflammatory, antipyretic and antinociceptive effects of
Ficus exasperata (Vahl) leaf extract. J Pharmaco Toxicol 4:138-51
92
Yesilada E, Ustun O. , Sezik E, Takishi E, Ono Y, Honda G (1997). Inhibitory effects of
Turkish folk remedies on inflammatory cytokines: interleukin-1, interleukin- 1ß and
tumor necrosis factor. J Ethnopharmacol. 58, 59-73.