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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

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