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OKONKWO A.C. (PG/M.PHARM/08/50349)

AN EVALUATION OF THE ANTI-INFLAMMATORY PROPERTIES OF EXTRACTS AND FRACTIONS OF AERIAL PARTS OF Phyllanthus niruri L.

A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARCEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA PHARMACOLOGY AND TOXICOLOGY

JUNE, 2011

Digitally Signed by Webmaster’s Name

Webmaster DN : CN = Webmaster’s name O= University of Nigeria, Nsukka OU = Innovation Centre

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AN EVALUATION OF THE ANTI-INFLAMMATORY PROPERTIES OF EXTRACTS AND FRACTIONS OF AERIAL PARTS OF Phyllanthus niruri L.

BY

OKONKWO A.C. (PG/M.PHARM/08/50349)

A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARCEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA, IN PATRTIAL FULFILMENT OF THE REQUIRMENT FOR THE AWARD OF MASTER OF PHARMACY (M.PHARM) DEGREE

PROF. C. O. OKOLI (SUPERVISIOR)

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA

JUNE, 2011 iii

CERTIFICATION

I, OKONKWO ANGELA CHIEMENAM, a postgraduate student in the Department of

Pharmacology and Toxicology and with registration number PG/M.PHARM/08/50349 have satisfactorily completed the requirements for course and research work for the degree of

Master of Pharmacy (M. Pharm.) in Pharmacology and Toxicology. The work embodied in this dissertation report is original and has not been submitted in part or in full for any other diploma or degree of this or any other university.

______

Prof. Charles O. Okoli Prof. Charles O. Okoli (Supervisor) (Head of Department)

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DEDICATION

This work is dedicated to God, for His abundant graces, and to my family. v

ACKNOWLEDGEMENT

I am grateful to God Almighty whose unfailing love and faithfulness saw me through to the completion of this work.

My profound gratitude goes to my supervisor, Prof. Charles O. Okoli who patiently and tirelessly supervised this work. Thank you very much for your encouragement and unflinching support. Your sacrifices are immeasurable.

I also wish to express my love and regards to my husband, Engr. Nwezza for his support and encouragement during the course of my work. You are simply wonderful. And I remain grateful to my parents, Mr. and Mrs. J. Okonkwo for their unalloyed support and contributions to the success of this work. I also wish to send warm regards to my siblings.

The assistance and warm company I enjoyed from my lecturers are worthy of note. I wish to thank Prof Akah, Pharm Ndu, Mrs. Mbaoji and all the staff of the Department of

Pharmacology and Toxicology for being part of this work. I also wish to thank specially Dr.

(Mrs) A.C. Ezike who helped me with some of the materials for the work. My thanks go to

Dr. S. Udegbunam of the Department of Veterinary Medicine for his professional assistance.

I also cherish the friendship I enjoyed from my fellow postgraduates students and friends.

And finally, I am greatly indebted to the various scholars and publishers whose works were consulted in the course of this study.

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TABLE OF CONTENTS

Title page ------i

Certification ------ii

Dedication ------iii

Acknowledgement ------iv

Table of Contents ------v

List of Tables ------ix

List of Figures ------x

Abstract ------xi

CHAPTER ONE: INTRODUCTION ------1

1.1 Inflammation ------1

1.1.1 Acute Inflammation ------2

1.1.2 Chronic inflammation ------3

1.2 The inflammatory response ------4

1.2.1 Macrophage and Neutrophil Responses during inflammation - 5

1.2.2 Resolution of Inflammation ------7

1.3 Mediators of inflammation ------8

1.3.1 Cell Derived Mediators ------9

1.3.1.1 Histamine ------9

1.3.1.2 Serotonin (5 -Hydroxytryptamine) - - - - - 10

1.3.1.3 Endotoxin ------10

1.3.1.4 Nitric Oxide ------10

1.3.1.5 Eicosanoids ------11

1.3.1.6 Platelet Activating Factor (PAF) ------12

1.3.1.7 Cytokines ------12

1.3.1.7.1 Cytokines involved in acute inflammation - - - - 12

1.3.1.7.1.1 Interleukin-1 ------12 vii

1.3.1.7.1.2 Tumor necrosis factor ------13

1.3.1.7.1.3 Interleukin-6 ------13

1.3.1.7.1.4 Interleukin-11 ------14

1.3.1.7.1.5 Interleukin-8/ chemokines ------14

1.3.1.7.1.6 Colony Stimulating factor ------14

1.3.1.7.2 Cytokines involved in chronic inflammation - - - - 15

1.3.1.7.2.1 Interleukin-2 ------15

1.3.1.7.2.2 Interleukin-3 ------15

1.3.1.7.2.3 Interleukin-4 ------15

1.3.1.7.2.4 Interleukin-5 ------16

1.3.1.7.2.5 Transforming growth factor-B - - - - - 16

1.3.1.7.2.6 Interferons ------16

1.3.2 Plasma Derived Mediators ------17

1.3.2.1 The Complement system ------17

1.3.2.2 The kinin system ------18

1.3.2.3 The coagulation system ------18

1.3.2.4 The fibrinolysis system ------19

1.4 Disorders of inflammation ------19

1.4.1 Rheumatoid arthritis ------19

1.4.2 Systemic Lupus Erythematosus - - - - - 20

1.4.3 Crohn’s disease (CD) ------21

1.4.4 Asthma ------21

1.5 Anti-inflammatory agents ------22

1.5.1 Non-steroidal anti-inflammatory drugs - - - - 22

1.5.2 Disease modifying anti-rheumatic drugs (DMARDs) - - - 24

1.5.3 Glucocorticoids drugs ------24

1.6 Anti-inflammatory medicinal plants - - - - - 25 viii

1.6.1 Botanical profile of Phyllanthus niruri - - - - 32

1.6.1.1 Taxonomy of plant ------32

1.6.1.2 Plant description ------33

1.6.1.3 Geographical distribution of plant - - - - - 33

1.6.1.4 Ethnomedicinal uses ------33

1.6.1.5 Literature Review of Phyllanthus niruri L. - - - - 34

1.6.1.6 Aim and scope of study ------36

CHAPTER TWO: MATERIALS AND METHODS - - - - 37

2.1 Materials ------37

2.1.1 Chemicals, solvents and reagents - - - - - 37

2.1.2 Animals ------37

2.1.3 Equipment ------37

2.2 Methods ------38

2.2.1 Collection and preparation of plant materials - - - - 38

2.2.2 Extraction of plant material ------38

2.2.3 Column chromatographic separation of the methanol extracts - 38

2.2.4 Phytochemical analysis of extracts and fractions - - - 39

2.2.5 Pharmacological tests ------43

2.2.5.1 Acute toxicity test ------43

2.2.5.2 Anti-inflammatory activity tests - - - - - 43

2.2.5.2.1 Studies on acute inflammation - - - - - 43

2.2.5.2.1.1 Topical edema induced by xylene in the mouse ear - - - 43

2.2.5.2.1.2 Carrageenaan induced pedal edema in rat - - - - 44

2.2.5.2.2 Studies on chronic inflammation - - - - - 45

2.2.5.2.2.1 Formaldehyde-induced arthritis in rats - - - - 45

2.2.5.2.2.2 Cotton-pellet granuloma test ------46

2.3 Statistical analysis ------46 ix

CHAPTER THREE: RESULTS ------47

3.1 Extraction and Fractionation ------47

3.2 Phytochemical constituents of extracts and fractions - - - 47

3.3 Acute toxicity studies ------47

3.4 Effect of extract and fractions on topical (acute) inflammation - 47

3.5 Effect of extract and fractions on systemic (acute) inflammation - 51

3.6 Effect of extract and fractions on formaldehyde induced arthritis - 51

3.7 Effect of extract and fractions on cotton pellet granuloma - - 51

CHAPTER FOUR: DISCUSSION AND CONCLUSION - - - 58

References ------61

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LIST OF TABLES

Tables Page

Some Medicinal Plants with Anti-inflammatory Ethnomedicinal uses 26

Phytochemical constituents of Extract and Fractions 48

Acute Toxicity (LD50) of Extract 49

Effect of Extract and Fractions on Acute Topical Edema of the Mouse Ear 50

Effect of Extract and Fractions on Acute Edema of the Rat Paw 52

Percent Inhibition of Edema caused by Carrageenan in Rats 53

Effect of Extract and Fractions on Formaldehyde Induced Arthritis in Rats 54

Percent Inhibition of Arthritis Induced by Formaldehyde in Rats 55

Global effect of Extract and Fractions on Formaldehyde Arthritis in Rats 56

Effect of Extract and Fractions on Cotton Pellet Granuloma 57

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LIST OF FIGURES

Figures Pages

Figure 1 Extraction and Fractionation Scheme 39

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ABSTRACT

The effects of extract and fractions of aerial parts of Phyllanthus niruri Linn (Euphorbiacaea) on acute and chronic inflammation were studied. The methanol extract (210 g; 10.5 % w/w) obtained by cold maceration for 48 h was fractionated in a silica gel column successively eluted with dichloromethane and methanol (100%) to yield the dichloromethane (31 g; 25.8 % w/w) and methanol ( 31.5 g; % w/w) fractions. The effect of the extract and fractions on acute inflammation was studied using topical edema induced by xylene in the mouse ear and carrageenan-induced pedal edema of the rat paw. Effect on chronic inflammation was evaluated using cotton pellet-induced granuloma test and formaldehyde induced arthritis in rats. The acute toxicity of the methanol extract was further studied in mice using the oral route.

The results showed that the extract and fractions significantly (P<0.05) inhibited the development of topical edema in the mouse ear and systemic edema of the rat paw. They also inhibited granuloma tissue growth and the global edematous response to formaldehyde. The dichloromethane fraction showed the greatest inhibitory effect in all the models. Acute toxicity test on the extract established an oral LD50 >5000 mg/kg in mice. Phytochemical analysis revealed the presence of alkaloids, carbohydrates, resins, saponins, tannins and terpenoids in the extract and fractions. The most active fraction, the dichloromethane fraction gave positive reactions for terpenes, sterols, resins, and alkaloids. These findings showed that the aerial parts of P. niruri possess anti-inflammatory activity in acute and chronic inflammation. This may be attributable to the alkaloids, resins, sterols and terpenes contained in the most active fraction. 1

CHAPTER ONE

INTRODUCTION

1.1 INFLAMMATION

Inflammation is defined as the fundamental pathologic process consisting of a dynamic complex of histological apparent cytological changes, cellular infiltration and mediator release that occurs in the affected blood vessels including adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent including the local reactions and resulting morphologic changes, the destruction or removal of the injurious material and the response that lead to repair and healing (Stedman, 2006).

Although in ancient times, inflammation was recognized as part of the healing process up to the end of the 19th century, it was viewed as an undesirable response harmful to the host.

However, beginning with the work of Metchnikoff and others in the 19th century, the contribution of inflammation to the body’s defensive and healing process was recognized

(Gallin, 1993).

The four basic symptoms of inflammation-redness, swelling, heat, and pain were described by

Celsius in the first century A.D. The cellular processes of inflammation fall into four distinct categories (Evans and Whicher, 1992):

(a) Changes in blood flow caused by changes in smooth muscle cell function

causing vasodilatation

(b) Alterations in vascular permeability engendered by cytoskeletal cells.

(c) Migration of phagocytic leukocytes to the site of inflammation.

(d) Phagocytosis. 2

Early inflammatory changes in damaged tissues are known to involve the release of various biological active materials from polymorph nuclear leukocytes, lysosomal enzymes and others.

The vascular effects are primarily mediated by kinins, prostaglandins and vasoactive amines

(histamine) released by mast cells. Vasoactive amines cause increased vascular permeability leading to plasma exudation. The inflammatory process involves a complex interplay between blood cells, blood vessels and the cells of the involved tissue. The process can be seen as a coordinated response of a large number of cells to the initial stimulus (Singh et al., 2008).

The causes of inflammation are numerous and include pathogenic living micro-organisms, toxins from animal parasites (Cruise and Lewis, 1995). Other causes include physical injury, blunt or penetrating, chemical irritants, burns, trauma, ionising radiation, foreign bodies, including splinters, dirt, debris and immune reactions due to hypersensitivity (Green et al.,

1993). Inflammation can be classified as either acute or chronic. While acute inflammation has an immediate onset of action and lasts a few days, chronic inflammation is delayed and runs for several months to years.

1.1.1 Acute Inflammation

Acute inflammation is a short term process, usually appearing within a few minutes or hours and ceasing upon the removal of the injurious stimulus (Cotran et al., 1999). It is characterized by five cardinal signs: rubor (redness), calor (increased heat), tumour (swelling), dolor (pain), and functio laesa (loss of function) (Parakrama and Olive, 2005). The first four cardinal signs were discussed by Celsius in ancient Rome (30BC-38AD), while loss of function was added later by Galen (Porth, 2007).

Redness and heat are due to increased blood flow at body core temperature to the inflamed site; swelling is caused by accumulation of fluid, pain is due to release of chemicals that stimulate nerve endings. While loss of function has multiple causes (Parakrama and Olive, 2005). 3

These five signs appear when acute inflammation of internal organs may not result in the full set. Pain only occurs where the appropriate sensory nerve endings exist in the inflamed area e.g. acute inflammation of the lung (Pneumonia) does not cause pain unless the inflammation involves the parietal pleura, which does have pain-sensitive nerve endings (Parakrama and

Olive, 2005).

The process of acute inflammation is initiated by cells already present in all tissues mainly resident macrophages, dendritic cells, histiocytes, Kuppfer cells and mastiocytes. At the onset of an infection, burn or other injuries, these cells undergo activation and release of inflammatory mediators responsible for the cardinal signs of inflammation. Vasodilatation and its resulting increased blood flow cause the redness and increased heat. Increased permeability of the blood vessels results in an exudation of plasma proteins and fluid into the tissue, which manifests itself as swelling. Some of the released mediators such as bradykinin increase the sensitivity to pain. The mediator molecules also alter the blood vessels to permit the migration of leukocytes, mainly neutrophils, out of the blood vessels into the tissue. The neutrophils migrate along a chemotactic gradient caused by the local cells to reach the site of injury

(Cotran et al., 1999).

Generally, acute inflammation is a reversible process which requires constant stimulation to be sustained. Inflammatory mediators have short half lives and are quickly degraded in the tissue.

Hence, inflammation ceases once the stimulus has been removed (Cotran et al., 1999).

1.1.2 Chronic Inflammation

Chronic inflammation has a slow onset and persists for weeks or more. The symptoms are not as severe as with acute inflammation, but the condition is insidious and persistent. Chronic inflammation may be consequent on acute inflammation or exist by itself. An acute inflammation will become chronic if the immune system is unable to rid the body of the 4 offending foreign agent or if the agent is constantly able to re-enter the body. The main cells involved in chronic infection are macrophages and lymphocytes (Cotran et al., 1999). Chronic inflammation involves the release of a number of mediators that are not prominent in the acute response. One of the most important disorders involving these mediators is rheumatoid arthritis, in which chronic inflammation results in pain and destruction of bone and cartilage that can lead to severe disability that may shorten life (Katzung et al., 2004). Chronic inflammation is characterized by infiltration with mononuclear cells which include macrophages, lymphocytes and plasma cells. It is generally irreversible (Howarth et al., 1991).

Also, macrophage accumulation persists in chronic inflammation and they can at the same time induce considerable tissue damage (Gallin, 1993).

1.2 THE INFLAMMATORY RESPONSE

The inflammatory response is the combination of a number of overlapping reactions within the body. Although a host of these reactions occur simultaneously, certain number of events may be seen.

The initial response of the body to an infection or trauma is called the acute inflammatory response. This response is non-specific and is the first line of defense of the body against danger. It consists of a coordinated local and systemic mobilization of immune, endocrine and neurological mediators. In a healthy response the inflammatory response becomes activated, clears the pathogen (in the event of infection), begins a repair process and abates. However, inflammation itself can damage otherwise healthy cells which could then further stimulate inflammation. This runoff inflammation can lead to organ failure and death (Kumar et al.,

2004). The inflammatory response is characterized by a number of factors which include vasodilatation of local blood vessels with consequent excessive local blood flow, increased permeability of the capillaries allowing leakage of large quantities of fluid into the interstitial spaces, clotting of fluid in the interstitial space, because of the excessive amount of fibrinogen 5 and other proteins leaking from the capillaries, migration of large number of agranulocytes and monocytes into the tissues and swelling of the tissue cells (Guyton and Hall, 2001).

Innumerable insults (a mosquito bite, a splinter, a virus infection, a bruise) can trigger an inflammatory response and dispatch cells and chemicals to the site to repair the damage. The damaged cells release chemicals including histamine, bradykinin, and prostagladins which cause blood vessels to leak fluid into the tissues, causing swelling. The chemicals also attract white blood cells called phagocytes that engulf microorganisms and dead or damaged cells.

This process is called phagocytosis and is formed from a collection of dead phagocytes

(Guyton and Hall, 2001). The inflammatory response is the combination of a number of overlapping reactions within the body. Although a host of these reactions occur simultaneously, certain order of events may be seen.

1.2.1 Macrophage and Neutrophil Responses During Inflammation

Within minutes after inflammation begins, the macrophages already present in the tissue, 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 and become mobile, forming the first line of defense against infection during the first hour. Within this hour after inflammation, large numbers of neutrophils begin to invade the inflamed area.

This is caused by products from the inflamed tissues that initiate the following reactions:

a) They alter the inside surface of the capillary endothelium, causing neutrophils to

stick to the capillary walls in the inflamed area. This effect is called margination 6

b) They cause the intercellular attachments between the endothelial cells of the

capillaries and small venules to loosen, allowing openings large enough for

neutrophils to pass by diapedesis directly from the blood into the tissue spaces.

c) Other products of inflammation can cause chemotaxis of the neutrophils toward

the injured tissues (Guyton and Hall, 2001).

Thus, within several hours after tissue damage begins, the area becomes well supplied with neutrophils from a normal range of 4000 - 5000 to 15,000 - 25,000/ml, this is called neutrophila. It is caused by products of inflammation that enter the blood stream, transported to the bone marrow and act on the stored neutropils of the marrow thus mobilizing them into the circulating blood. Along with the invasion of neutrophils, monocytes from the blood enter the inflamed tissue and enlarge to become macrophages. The build-up of macrophages in the inflamed tissue area is much slower than neutrophils, requiring several days to become effective. After several days to several weeks, the macrophages finally come to dominate the phagocyte cells of the inflamed area because of greatly increased bone narrow production of new monocytes (Toratora and Grabowski, 1996). Eventually there is greatly increased production of both granulocytes and monocytes by the bone marrow. If the stimulus from the inflamed tissue continues, the bone marrow can continue to produce these cells in tremendous quantities. The combination of tumor necrosis factor ,interleukin-1 and colony stimulating factors produce a powerful feedback mechanism that begins with tissue inflammation and proceed to formation of large numbers of defensive white blood cells that help remove the cause of the inflammation (Guyton and Hall, 2001).

When these neutrophils and macrophages engulf large numbers of bacterial and necrotic tissue, essentially all the neutrophils and many, if not most, of the macrophage eventually die. After several days, a cavity is often excavated in the inflamed tissue that contains varying portions of 7 necrotic tissue, dead neutrophils, dead macrophages, and tissue fluid: Hence, the formation of pus (Guyton and Hall, 2001).

1.2.2 Resolution of Inflammation

The inflammatory response must be actively terminated when no longer needed to prevent unnecessary “by stander” damage to tissues (Cotran et al., 1999). Failure to do so results in chronic inflammation, and cellular destruction. Resolution of inflammation occurs by different mechanisms in different tissues. These mechanisms (Cotran et al., 1999; Eming et al., 2007) include:

(i) Short half life of mediators in vivo

(ii) Production and release of transforming growth factor (TGF) beta (TGF-β) from

macrophages (Werner et al., 2000)

(iii) Production and release of interleukin 10 (IL-10) (Sato et al., 1999)

(iv) Production of anti-inflammatory lipoxins (Serhan and savill, 2005)

(v) Down regulation of pro-inflammatory molecules, such as leukotrienes

(vi) Up regulation of anti-inflammatory molecules such as the interleukin 1 receptor

antagonist soluble tumor necrosis factor receptor (TNFR)

(vii) Apoptosis of pro-inflammatory cells

(viii) Desensitization of receptors

(ix) Increased survival of cells in regions of inflammation due to their interaction

with the extra–cellular matrix (ECM)

(x) Down regulation of receptor activity by high concentrations of ligands. 8

Acute inflammation normally resolves by mechanisms that have remained somewhat elusive.

Emerging evidence now suggests that an active, coordinated program of resolution is initiated in the first few hours after one inflammatory response begins. After entering tissues, granulocytes promote the switch of arachidonic acid-derived prostaglandins and leukotrienes to lipoxins, which initiate termination. Neutrophil recruitment thus ceases and programmed death by apoptosis is engaged. These events coincide with the biosynthesis, from omega-3 poly- saturated fatty acids, of resolvins and protectins, which critically shorten the period of neutrophil infiltration by initiating apoptosis. Consequently, apoptotic neutrophils undergo phagocytosis by macrophages, leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines such as transforming growth factor -β. The anti-inflammatory process ends with the departure of macrophages through the lymphatics (Serhan and Savill, 2005).

1.3 MEDIATORS OF INFLAMMATION

These are associated with the component events in inflammation. Model systems have provided insight into the temporal appearance and importance of mediators in the various processes of inflammation in humans. In one model, a derivative of endotoxin administered intravenously resulted in the accumulation of mediators in the peripheral tissues, accompanied by a characteristic change in body temperature and white blood cell count. The critical components of the inflammatory response- fever, neutrophil margination in the circulatory vessels, and then mobilization from the bone marrow were associated with readily detected changes in circulating levels of certain mediators of inflammation e.g TNFα, IL-8, and IL-6

(Gallin, 1993).

Chemical mediators of inflammation have various functions. Examples include histamine and bradykinin which increase vascular permeability. They can trigger complement activation, resulting in the formation of anaphylatoxins C3a and C5a which cause vasodilatation and 9 increased vascular permeability of small blood vessels, vasoconstriction caused by leukotrienes and thromboxane A2 , and cytokines (Gallin, 1993). Endogenous mediators of inflammation are produced from within the immune system itself, as well as other systems. For example, they can be derived from molecules that are normally present in the plasma in an inactive form, such as peptide fragments of some components of complement, coagulation and kinin systems

(Wikipedia, 2010). Platelets produce a group of acetyl-alkylglycerol ether analogs of phosphatidylcholine called platelet activating factors (PAFs). They contribute to inflammatory responses through a variety of mechanisms including (Gallin, 1993):

a) The release of vasoactive amines and other permeability factors,

b) The release of lysosomal enzymes,

c) The release of coagulation factors which lead to localized and generalized fibrin

deposition, and

d) The release of platelet aggregates of thrombi which result in the blockade of vessels

and capillaries. Leukocytes also produce PAFs in response to inflammatory

mediators (Gallin, 1993).

The mediators involved in the inflammatory response are broadly classified into cell-derived and plasma-derived mediators.

1.3.1 Cell Derived Mediators

Cell derived mediators include histamine, serotonin, nitric oxide, prostaglandins, leukotrienes, platelet activating factor.

1.3.1.1 Histamine

Histamine is a biologically active amine found in many tissues. It exerts complex physiologic and pathologic effects through multiple receptor subtypes and is often released locally. Most 10 tissue histamine is sequestered and bound in granules (vessicles) in mast cells or basophils; the histamine content of many tissues is directly related to their mast cell content (Katzung et al.,

2004). It is the best known chemical mediator in acute inflammation. It causes vascular dilatation and the immediate transient phase of increased vascular permeability (Gaboury et al.,

1995). Histamine release from mast cells, basophil and eosinophil leukocytes, and platelets is stimulated by complement components C3a and C5a, and by lysosomal proteins released from neutrophils (Gaboury et al., 1995). Histamine causes vasodilatation of arterioles and increases vascular permeability of capillaries and venules (Cotran et al., 1999).

1.3.1.2 Serotonin (5 -hydroxytryptamine)

Serotonin is an indole ethylamine formed in biologic systems from the amino acid L- tryptophan. It is present in high concentration in mast cells and platelets. It also may play a role in mediating inflammation, but antagonists ameliorate only certain types of inflammatory responses. Serotonin causes vasodilation and increased vascular permeability (Katzung et al.,

2004).

1.3.1.3 Endotoxin

Bacterial products and toxins can act as exogenous mediators of inflammation i.e. endotoxin or

LPS (Lipopolysaccharide) of Gram-negative bacteria. Endotoxin can trigger complement activation, resulting in the formation of anaphylatoxins C3a and C5a, which cause vasodilation and increase vascular permeability. Endotoxin also activates the Hageman factor, leading to activation of the coagulation and fibrinolytic pathways as well as the kinin system. In addition, endotoxins elicit T cell proliferation, and have been described as super antigen for T cells

(Gallin, 1993).

1.3.1.4 Nitric oxide

Nitric oxide is formed from the amino acid arginine by nitric oxide synthase (NOS) present in endothelia and macrophages. Many of the long-lived actions of nitric oxide in vivo appear to be 11 caused by stable s-nitroso compounds (R-SNO). It has a role in both acute and chronic inflammation. The NOS-3 is involved in the vasodilation associated with acute inflammation.

Nitric oxide has a detrimental effect in chronic models of arthritis while dietary L-arginine supplementation is known to exacerbate arthritis. Protection is seen with NOS- 2 inhibitors

(Thomas and Ramwell, 2004).

Psoriasis lesions, airway epithelium in asthma, and inflammatory bowel lesions in humans all demonstrate elevated levels of nitric oxide and NOS-2. Synovial fluid from patients with arthritis contains increased oxidation products of nitric oxide, particularly peroxynitrite. Recent studies have shown that nitric oxide stimulates the synthesis of inflammatory prostaglandins by activating cyclooxygenase isoenzyme II (COX-2) (Thomas and Ramwell, 2004). Actions of nitric oxide include vasodilation, antiplatelet aggregation, and macrophage-induced cytotoxicity (Moody et al., 2001).

1.3.1.5 Eicosanoids

The families of prostaglandins, leukotrienes, and related compounds are called eicosanoids.

Arachidonic acid the most abundant precursor, IS derived from either dietary sources or from membrane lipids (Morrow and Roberts, 2001). Prostaglandins and leukotrienes are released by tissues in response to a host of mechanical, thermal, chemical, bacterial and other insults, and they contribute importantly to the genesis of the signs and symptoms of inflammation (Morrow and Roberts, 2001). Leukotriene (LK) B4 is a potent chemoattractant for polymononeuclear leukocytes and can promote exudation of plasma by mobilizing this source of additional inflammatory mediators. Although prostaglandins do not appear to have direct effect on vascular permeability, both PGE2 and PGI2 markedly enhance edema formation and leukocyte infiltration by promoting blood flow in the inflamed region. Moreover, they potentiate the pain-producing activity of bradykinin and other autacoids (Morrow and Roberts, 2001).

12

1.3.1.6 Platelet Activating Factor (PAF)

This is a newly defined class of biologically active lipids, which can produce effect at low concentrations (Morley et al., 1989). Like the eicosanoids, PAF is not stored in cells but synthesized in response to stimulation (Morrow and Roberts, 2001). It has actions on variety of different target cells (Morley et al., 1989). It is elaborated by leukocytes and mast cells and exerts pro-inflammatory effects. For example, intradermal injection of PAF duplicates many of the signs and symptoms of inflammation, including increased vascular permeability, hyperalgesia, edema and infiltration of neutrophils (Morrow and Roberts, 2001).

1.3.1.7 Cytokines

Inflammation is mediated by a variety of soluble factors including a group of secreted polypeptides known as cytokines. Inflammatory cytokines can be divided into two groups: those involved in acute inflammation and those responsible for chronic inflammation, most cytokines are multifunctional. They are pleiotropic molecules that elicit their effects locally or systemically in an autocrine or paracrine manner. They are major determinants of the make- up of the cellular infiltrate, the state of cellular activation, and the systemic responses to inflammation (Gallin et al., 1992).

1.3.1.7.1 Cytokines involved in acute inflammation

Several cytokines play key roles in mediating acute inflammatory reactions namely IL-I, TNF-

α, IL-6, IL-11, IL-8 and other chemokines, G-CSF, and GM-CSF. Of these, IL-I (α and β) and

TNF are extremely potent inflammatory molecules: they are the primary cytokines that mediate acute inflammation (Feghali et al., 1997).

1.3.1.7.1.1 Interleukin -1

The complement DNAs for IL-1α and β were cloned in 1984. They are encoded by two different genes, both located on human chromosome 2. Their main sources are mononuclear 13 phagocytes, fibroblasts, keratinocytes, and T and B lymphocytes. Both IL -1α and IL-I β can trigger fever by enhancing prostaglandin E2 (PGE2) synthesis by the vascular epithelium of the hypothalamus and can stimulate T-cell proliferation (Feghali et al., 1997).

In addition, IL-1 elicits the release of histamine from mast cells at the site of inflammation.

Histamine then triggers early vasodilatation and increase of vascular permeability (Dinarello,

1992).

1.3.1.7.1.2 Tumor Necrosis Factor

Tumor necrosis factors – (TNF) α and β are cytokines that bind to common receptors on the surface of target cells and exhibit several common biological activities. The TNFα and IL-1 share several pro-inflammatory properties. Like IL-1, TNFα can induce fever either directly via stimulation of PGE2 synthesis by the vascular epithelium of the hypothalamus or indirectly by inducing release of IL-1. The TNF is believed to be responsible for the metabolic alterations which result in the cachexia associated with chronic parasitic infections and some cancers

(Feghali et al., 1997).

1.3.1.7.1.3 Interleukin-6

Previous synonyms of interleukin-6 illustrate some of its biological activities. They include interferon B-2 (1FN-β2), hybridoma/plasmacytoma growth factor, hepatocyte-stimulating factor, B cell stimulation factor 2 (BSF-2) and B cell differentiation factor (BCDF) (IL-6 is produced by a variety of cells including mononuclear phagocytes, T cells and fibroblasts

(Feghali et al., 1997). Up regulation of IL-6 production has been observed in a variety of chronic inflammatory and autoimmune disorders such as thyroiditis, type 1 diabetes, rheumatoid arthritis (Hirano, 1992), systemic sclerosis (Feghali et al., 1997), mesangial proliferative glomerulonephritis and psoriasis, and neoplasms such as cardiac myxoma, renal cell carcinoma, multiple myeloma, lymphoma and leukaemia (Hirano, 1992). 14

1.3.1.7.1.4 Interleukin -11

It is a functional homologue of IL-6 and can replace IL-6 for the proliferation of certain plasmacytoma cell lines, and in the induction of acute phase protein secretion in the liver.

Additional IL-11 activities include stimulation of T cell-dependent B cell immunoglobulin secretion, increased platelet production, and induction of IL-6 expression by CD4 cells

(Feghali et al., 1997)

1.3.1.7.1.5 Interleukin 8/Chemokines

The IL-8 and other low molecular weight chemokines (e.g. platelet factor 4, macrophage inflammatory protein (MIP) -1α and β) belong to a chemotactic cytokine family and are responsible for the chemotactic migration and activation of neutrophils and other cell types

(such as monocytes, lymphocytes, basophils, and eosinophils) at sites of inflammation (Miller and Krangel, 1992). Chemokines have been implicated in inflammatory conditions from acute neutrophil-mediated conditions such as acute respiratory distress syndrome to allergic asthma, arthritis, psoriasis, and chronic inflammatory disorders. IL-8 was previously known as neutrophil chemotactic factor (NCF) and neutrophil activating protein (NAP-1) (Feghali et al.,

1997); its main inflammatory impact lies in its chemotactic effects on neutrophils and its ability to stimulate granulocyte activity. It can be detected in synovial fluid from patients with various inflammatory rheumatic diseases, and mucosal levels of IL-8 are elevated in patients with active ulcerative collitis (Mahida et al., 1992).

1.3.1.7.1.6 Colony Stimulating Factor

Colony stimulating factors (CSF) are named according to the target cell type whose colony formation in soft agar cultures of bone marrow they induce. Of the CSFs, granulocyte–CSF (G- 15

CSF) and granulocyte macrophage–CSF (GM-CSF) participate in acute inflammation (Nagata,

1994).

An example of the pathophysiologic role of GM-CSF is the airway inflammation accompanying asthma, where the implicated cytokines include IL3, IL-5, and GM-CSF which perpetuate eosinophil activation and survival (Arm and Lee, 1992).

1.3.1.7.2 Cytokines Involved In Chronic Inflammation

The cytokines known to mediate chronic inflammatory processes are: IL-2, IC-3, IL-4, IL-5,

IL-6, IL-7, IL-9, IL–10, IL–12 IL–13 and transforming growth factor, interferons, TNFα and β

(Feghali et al., 1997).

1.3.1.7.2.1 Interleukin-2

It is secreted mainly by activated T helper cells. It acts as a growth factor/activator for T cells.

It plays a critical role in regulating both cellular and humoral chronic inflammatory responses

(Feghali et al., 1997)

1.3.1.7.2.2 Interleukin -3

IL-3, also called multi-CSF, is produced by activated T cells and mast cells. It shares several biological activities with GM-CSF (Crossier and Clark, 1992)

1.3.1.7.2.3 Interleukin - 4

IL-4 is produced by CD4 (TH) cells, mast cells, and basophils. It also stimulates collagen and

IL-6 production (Feghali et al., 1997) by human dermal fibroblasts, and may thus play a role in the pathogenesis of fibrotic diseases such as systemic sclerosis. In rheumatoid arthritis, on the other hand, IL-4 appears to exhibit some anti-inflammatory properties by inhibiting the 16 production of several pro- inflammatory cytokines such as IL-1, IL-6, IL-8 and TNF-α, by synovial membranes of rheumatoid arthritis patients (Miossec, 1993).

1.3.1.7.2.4 Interleukin – 5

IL-5, also known as B cell growth factor II (BCGF II) and T cell replacing factor (TRF), is produced by CD4 T helper cells. IL-5 is involved in eosinophil differentiation and activation and stimulation of macroglobulin class switching to IGA. Other properties of IL–5 include increased activation of B cell proliferation, and enhancement of T cell cytotoxicity (Yokota et al., 1988).

1.3.1.7.2.5 Transforming Growth Factor – β

The transforming growth factor-β (TGF-β) family of cytokines includes three isoforms, TGF–

β1 β2, and β3 which are encoded by separate genes yet bind to the same high affinity receptor.

At a site of injury, TGF-β stored in platelets is released upon degranulation. TGF–β then attracts monocytes and other leukocytes to the site, thus participating in the initial step of chronic inflammation. It also inhibits collagenase production, and if expression is prolonged, it may result in progressive fibrosis analogous to unregulated tissue repair. Conditions in which a role for TGF- B has been suggested include mesangial proliferative glomerulonephritis and diabetic nephropathy in rats, pulmonary fibrosis, and systemic sclerosis (Feghali et al., 1997).

1.3.1.7.2.6 Interferons

The interferons are a group of cytokines originally identified by and named for their anti-viral activity. Type I interferons include IFN–α, a product of leukocytes, and IFN-β, a product of fibroblasts. Type II interferon, immune interferon or IFN-У is produced by activated T cells and NK cells and has been implicated in the pathogenesis of a variety of autoimmune and 17 chronic inflammatory conditions including models of systemic lupus erythematosus (Feghali et al.,1997).

1.3.2 Plasma Derived Mediators

This is also known as the plasma cascade systems and includes: the Complement, Kinin, coagulation system or clotting cascade and fibrinolytic systems.

1.3.2.1 The Complement System

‘Complement’ is a collective term that describes a system of about 20 proteins, many of which are enzyme precursors. The principal actors in this system are 11 proteins designated C1 through C9. All these are present normally among the plasma proteins in the blood as well as among the proteins that leak out of the capillaries into the tissue spaces. The enzyme precursors are normally inactive, but they can be activated mainly by the so-called Classic pathway (Guyton and Hall, 2001).

The Classic pathway is initiated by an antigen-antibody reaction. That is, when an antibody binds with an antigen, a specific reactive site on the “constant” portion of the antibody becomes uncovered, or “activated”, and this in turn binds directly with the C1 molecules of the

Complement system, setting into motion a “cascade’ of sequential reactions (Guyton and Hall,

2001). Also the endotoxins of gram negative bacteria activate Complement via the Alternative pathway. Product of the kinin, coagulation and fibrinolytic system can activate Complement

(Rang and Dale, 1999). Among the more important effects caused by the reaction sequence

(Guyton and Hall, 2001) are:

a) Opsonisation and phagocytosis: One of the products of the complement cascade,

C3b, strongly activates phagocytosis by both neutrophils and macrophages

causing these cells to engulf the bacteria to which the antigen-antibody

complexes are attached. This process is called opsonisation 18

b) Lysis: One of the most important of all the products of the complement cascade

is the lytic complex, which is a combination of multiple complement factors and

designated C5b6789

c) Agglutination

d) Neutralisation of viruses

e) Chemotaxis: Fragment C5a initiates chemotaxis of neutrophils and

macrophages

f) Activation of mast cells and basophils; Fragments C3a, C4a, and C5a activate

mast cells and basophils causing them to release histamine, heparin, and several

other substances into the local fluids

g) Inflammatory effects (Guyton and Hall, 2001).

1.3.2.2 The Kinin System

The Kinins are peptides of 9-11 amino acids; the most important vascular permeability factor is bradykinin. Kinins play an important role in the inflammatory process. Kallikreins and Kinins can produce redness, local heat, swelling, and pain, and the production of kinins is increased in inflammatory lesions produced by a variety of methods. They elicit pain by stimulating nociceptive afferents in the skin and viscera (Reid, 2004).

1.3.2.3 The Coagulation System

This system is responsible for the conversion of soluble fibrinogen into fibrin, a major component of the acute inflammatory exudates (Davie et al., 1991). Coagulation factor xii (the

Hageman factor), once activated by contact with extracellular materials such as basal lamina, and various proteolytic enzymes of bacterial origin, can activate the coagulation, kinin and fibrinolytic systems (Katzung et al., 2004).

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1.3.2.4 The Fibrinolysis System

The fibrinolysis system acts in opposition to the coagulation system, to counterbalance clotting and generate several other inflammatory mediators. Plasmin responsible for the lysis of fibrin into fibrin degradation products may have local effects on vascular permeability (Rang et al.,

1995).

1.4 DISORDERS OF INFLAMMATION

Abnormalities associated with inflammation comprise a large, officially unrelated group of disorders which underlie a vast variety of human diseases. The immune system is often involved with inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation

(Wikipedia, 2010).

Examples of disorders association with inflammation include: rheumatoid arthritis (RA), systemic lupus erythematosus, Crohn’s disease, ulcerative colitis, acne vulgaris, asthma, pelvic inflammatory disease, Psoriasis, etc.

1.4.1 Rheumatoid Arthritis

Although the pathogenesis of rheumatoid arthritis is largely unknown, it appears to be an autoimmune disease driven primarily by activated T cells giving rise to T cell-derived cytokines, such as IL-1 and TNF. Many of these cytokines, including IL-1 and TNF, have been found in the rheumatoid synovium (Morrow and Roberts, 2001).

There is stiffness of the diarthrodial joints including the shoulders, lips, knees, elbows and ankles. As rheumatoid arthritis is a systemic disease, patients also manifest non-specific early symptoms like malaise, fatigue, fever and anorexia (Ukwe, 2004).

It has been proposed that salicylate and certain other NSAIDs can directly inhibit the activation and function of neutrophils. Some NSAIDs can inhibit leukocyte adhesion by a mechanism 20 that seems to be independent of their ability to inhibit prostaglandins biosynthesis (Ukwe,

2004).

Immunosuppressive agents such as methotrexate and cyclophosphamide are indicated in patients with severe rheumatoid arthritis refractory to standard therapy or in patients with severe symptoms, who cannot tolerate the agents utilized in standard therapy (Ukwe, 2004).

Glucocorticoids are the most potent anti-inflammatory agents available. Steroids are used only as adjuvants to other treatment because of their long term toxicity. They are indicated for RA

Patients who continue to have active synovitis in many joints in spite of sufficient trials with

NSAIDs (Ukwe, 2004).

1.4.2 Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a multisystem autoimmune disease characterized by circulating auto antibodies and immune complexes (Moore and Lisak, 1995). The systemic effects of SLE are widespread, most commonly involving the joints and the skin with symmetric arthritis in both large and small joints, hematologic effects; anemia, leukopenia, or thrombocytopenia and constitutional symptoms- fever, fatigue and myalgia (Moore and Lisak,

1995).

Diagnosis of SLE is based on a combination of clinical and laboratory data. The criteria include: seizures or psychosis, antinuclear antibodies, renal dysfunction, oral ulcers and photosensitivity.

Treatment involves the use of non steroidal anti-inflammatory drugs (NSAIDs) for myalgias, fever and arthritis. If manifestations are severe or disabling, immunosuppression may be necessary. High dose glucocorticoids are commonly used with close monitoring (Seiden and

Krumholtz, 2002).

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1.4.3 Crohn’s Disease (CD)

Crohn’s disease (CD) is an immune-mediated disorder that affects primarily the terminal ileum but may also affect the small and large intestines in a segmental fashion (Elsehety and

Bertorini, 1997). CD presents as abdominal pain, diarrhoea, fever and generalized fatigue.

Frequent systemic complications of CD include arthritis, ankylosing spondylitis, erythema nodusum, and pyoderma gangrenosum (Elsehety and Bertorini, 1997). Neurologic and

Psychiatric complication have also been reported.

The principal medical therapy of CD is anti-inflammatory agents such as sulfasalazine or glucocorticoids. Surgical therapy is not curative; relapses are frequent (Farmer et. al., 1985).

1.4.4 Asthma

The recognition that asthmatic airway narrowing both at baseline and during disease exacerbations is due to inflammation is based on studies involving bronchial lavage and lung biopsies. Increased numbers of inflammatory cells including eosinophils, basophils, macrophages and lymphocytes, can be found in broncho alveolar lavage fluid from asthmatic patients (Undem and Lichtenstein, 2001).

Lung biopsies in asthmatic subjects have revealed airway thickness and an increased number of basophils and other inflammatory cells in lung tissues. Epidemiologic studies also show correlation between increasing immunoglobulin E (IgE) levels and prevalence of asthma

(Undem and Lichtenstein, 2001).

Acute asthmatic attacks manifest as dyspnea and coughing, which appear some minutes after exposure to the precipitating factor or allergen. A dry hacking cough may be a prominent symptom. Expectoration of mucus plug may reveal a large number of sharp edged crystals, called Charcot-Leyden crystals, which are related to disintegrating eosinophils. Treatment involves use of oral therapy with B-adrenergic receptor agonists, inhaled glucocorticoids, 22 systemic glucocorticoids, leukotriene-receptor antagonist, anticholinergic agents, methylxanthines and cromolyn (Gyang, 2004).

1.5 ANTI-INFLAMMATORY AGENTS

The treatment of patients with inflammation involves two primary goals: first, the relief of pain, which is often the presenting symptom and the major continuing complaint of the patient; and second, the slowing or – in theory – arrest of the tissue–damaging process (Wagner et al.,

2004).

Reduction of inflammation with nonsteroidal anti-inflammatory drugs (NSAIDs) often results in relief of pain for significant periods. Furthermore, most of the non-opioid analgesics (e.g. aspirin) also have anti-inflammatory effects so they are appropriate for the treatment of both acute and chronic inflammatory conditions (Wagner et al., 2004)

The glucocortcoids also have powerful anti-inflammatory effects and when first introduced were considered to be the ultimate answer to the treatment of inflammatory arthritis.

Unfortunately, the toxicity associated with chronic corticosteroid therapy limits their use except in the control of acute flare- ups of joint disease (Wagner et al., 2004). Another important group of agents are characterized as slow acting anti-rheumatic drugs (SAARDs) or disease – modifying anti-rheumatic drugs (DMARDs).

1.5.1 Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)

NSAIDs are a heterogeneous group of compounds, often chemically unrelated (although most of them are organic acids) which share certain therapeutic effects and side effects (Insel, 1996).

They also share the capacity to suppress the signs and symptoms of inflammation through the inhibition of prostaglandin synthesis. They also exert antipyretic and analgesic effects, but it is their anti-inflammatory properties that make them most useful in the management of disorders 23 in which pain is related to the intensity of the inflammatory process (Wagner et al., 2004).

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 and enolic acids.

(b) Selective cyclo-oxygenase (COX-2) inhibitors such as diaryl/substituted

furanones (rofecoxib), diaryl substituted pyrazoles (celecoxib) and sulfonamide.

The anti-inflammatory activity of the NSAIDs is mediated chiefly through inhibition of biosynthesis of prostaglandins. Various NSAIDs have additional possible mechanisms of action, including inhibition of chemotaxis, down regulation of interleukin–1 production, decreased production of free radicals and superoxide, and interference with calcium–mediated intracellular events. The NSAIDs decrease the sensitivity of vessels to bradykinin and histamine, affect lymphokine production from T lymphocytes, and reverse vasodilation. Most

NSAIDs act as non selective inhibitors of the enzyme cyclooxygenase (COX), inhibiting both the COX 1 and COX 2 isoenzymes which catalyse the formation of prostaglandins and thromboxane from arachidonic acid. Prostaglandins act as messenger molecules in the process of inflammation. This process of inflammation was elucidated by John Vane (1927-2004), who later received a Nobel price for his work. (Hinz et al., 2008). NSAIDs have antipyretic activity and can be used to treat fever. Fever is caused by elevated levels of prostaglandin E2 which alters the firing rate of neurons that control thermoregulation within the hypothalamus

(Nabulsi, 2009). They are all gastric irritants as well, though as a group the newer agents tend to cause less gastric irritation than aspirin. Nephrotoxicity has been observed for all of the drugs for which extensive experience has been reported, and hepatotoxicity can also occur with any NSAIDs (Wagner et al., 2004). 24

Although these drugs effectively inhibit inflammation, there is no evidence that in contrast to drugs such as methotrexate and gold – they alter the course of an arthritic disorder.

1.5.2 Disease-Modifying Anti-Rheumatic Drugs (DMARDS)

DMARDs as the name implies slows the progression of bone and cartilage destruction. The effects may take 6 weeks to 6 months to become evident. These therapies include methotrexate, azathioprine, penicillamine, hydroxychloroquine and chloroquine, organic gold compounds/sulfasalazine, leflumonide. Considerable controversy surrounds the long term efficacy of many of these therapies (Wagner et al., 2004).

1.5.3 Glucocorticoid Drugs

Glucocorticoids are adrenocortical hormones secreted by the adrenal cortex of the kidney under the direct control of the pituitary gland through the release of corticotropin

(adrenocorticotropic hormone, ACTH) (Chrousus, 2004). Most of their known effects are mediated by widely distributed glucocorticoid receptors (proteins) which are members of the super family of nuclear receptors (Chrousus, 2004).

Glucocorticoids dramatically reduce the manifestations of inflammation. This is due to their profound effects on the concentration, distribution, and function of peripheral leukocytes and to their suppressive effects on the inflammatory cytokines and chemokines and on other lipid and glucolipid mediators of inflammation (Schimmer and Parker, 1996).

Glucocorticoids inhibit the functions of tissue macrophages and other antigen–presenting cells.

The ability of these cells to respond to antigens and mitogens is reduced. In additions to their effects on leukocyte function, they influence the inflammatory response by reducing the prostaglandin, leukotriene, and platelet activating factor synthesis that results from activation of phospholipase A2. The anti-inflammatory and immunosuppressive effects of these agents are 25 widely used therapeutically but are also responsible for some of their most serious adverse effects (Chrousus, 2004).

1.6 ANTI-INFLAMMATORY MEDICINAL PLANTS

Natural products have long been recognized as an important source of therapeutically effective medicines (Cragg et al., 2003). Before the availability of synthetic drugs, man was completely dependent on medicinal plants for curing diseases. With the synthesis of aspirin in 19th century, a new era started in the history of anti-inflammatory and analgesic drugs (Vane, 1997).

Traditional medicine healers have used herbal formulation for anti-inflammatory action with considerable success (Chatterjee and Pal, 1984). In addition to medical drugs, some herbs may have anti-inflammatory qualities, including hyssop, ginger, turmeric, Arnica montana which contains helenalin, a sesquiterpene lactone, and willow bark, which contains salicylic acid, the active ingredient in aspirin (Julkunentuto and Tahvanainen, 1989).

Several herbs have been employed as anti-inflammatory agents (Table 1). Studies on their actions have been carried out at different times. A typical example of this is the ancient “stone breaker”, Phyllanthus niruri. It has been shown to reduce kidney and gall bladder stones, hence its title “stone breaker” (Taylor, 2003).

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Table 1: Some medicinal plants with anti-inflammatory ethno medicinal uses

Plant Family Plant parts used Reference

Abies pindrow Spach Pinaceae Leaf Chatterjee et al., 1991

Abutilon mauritianum Malvaceae Juice, leaf Iwu, 1986

Acalypha Ciliata Forsk Euphorbiaceae Leaf Ogungbamila et al., 1997

Acalypha indica Linn Euphorbiaceae Leaf Ogungbamila et al., 1997

Acalypha hispida Burm F. Euphorbiaceae Leaf Ogungbamila et al., 1997

Acalypha wilkesiana. Euphorbiaceae Leaf Ogungbamila et al., 1997

Acanthus montanus Acanthaceae Leaf Okoli et al., 2008

Achyranthes aspera Amaranthaceae Whole plant Iwu, 1986

Adansonia digitata Bombaceae Fruit, leaf Okafor et al., 1999

Aframomum melegueta Zingiberaceae Seeds Okoli et al., 2007

Agave rigida Amaryllidaceae Leaf Iwu, 1986

Acacia albida Minocaceae Exudates/root bark Iwu, 1986

Alstonia congensis Apocynaceae Bark Iwu, 1986

Amaranthus spinosus Amanthaceae Leaf Iwu, 1986

Anacardium occidentale L. Anacardiaceae Bark, leaf Iwu, 1986 27

Ananas comosus Bromoliaceae Unripe fruit Iwu, 1986

Anchomanes difformis Araceae Rhizome Akah et al., 1994

Anthocleista djalonensis Loganiaceae Stem bark Akah et al., 1994

Apium graveolens Linn Umbelliferae Seeds Atta et al., 1998

Artocarpus heterophyllus Moraceae Roots Iwu, 1986

Aspilia Africana Compositae Leaf Okoli et al., 2007

Azadirachta indica Melicaceae Root bark Ivanovska et al., 1997

Beta vulgaris Linn Chenopodiaceae Root Atta et al., 1998

Bridelia ferruginea Euphobiaceae Aerial Assi et al., 1991

Brillantaisia patula Acanthaceae Leaf Ogungbamila et al., 1997

Bryophyllum pinnatum Crassulaceae Aerial part/tissue Ogungbamila et al., 1997

Buccholzia coriacea Capparidacaea Leaf Aguwa et al., 2009

Butyrospermum parkii Sapotaceae Seed oil Akubue et al., 1983

Calotropis procera Asilepiudaceae Leaf Ogungbamila et al., 1997

Cassia fistula Caesalpiniaceae Pod Alam et al., 1990

Catharanthus roseus Apocynaceae Leaf Okafor et al., 1999

Chrysanthemum bovaele Compositae Leaf Iwu, 1986

Cinnamomum zeylanicum Lauraceae Barks Atta et al., 1998 28

Cissus spp Ampelidaceae Stem Okafor et al., 1999

Cirulus spp Cucurbitaceae Juice Atta et al., 1998

Cirus urantum Rutaceae Juice/leaf Atta et al., 1998

Conium maculatum L. Apiaceae Aerial Ivanovska et al., 1997

Conyza Canadensis Asteraceae Leaf Lengfeld et al., 1986

Coptis chinensis Franch Ranunculaceae Root Cueller et al., 2001

Costus afer Zingiberaceae Stem & leaf Singha, 1965

Crataeva nurvala Caparidaceae Stem bark McGaw et al., 1997

Crataeva religiosa Forst Caparidaceae Stem bark McGaw et al., 1997

Culcasia scandens Araceae Leaf Okoli et al., 2006

Curcuma longa Zingiberaceae Aerial parts Iwu, 1986

Cymbopogon citrates Graminae Leaf Iwu, 1986

Dalbergia sissoo Roxb Fabaceae Leaf Hajare et al, 2001

Dennettia tripetala Annonaceae Root, bark Okafor et al., 1999

Dianthus barbatus Caryophyllaceae Aerial Parts Cordell et al., 1977

Diodia scandens Rubiaceae Whole plant Akah et al., 1994

Dioscorea dumetorum Dioscoreaceae Tubers Iwu, 1986

Echinacea purpurea Compositae Root Evans W.C. 1996 29

Elephantopus scaber Compositae Leaf Iwu, 1986

Erythrococca anomala Euphorbiaceae Leaf Iwu, 1986

Erythrophleum chinensis Caesalpinaceae Bark Iwu, 1986

Eucalyptus camaldulentis Dehn Myrtaceae Leaf Atta et al., 1998

Euphorbia royleana Euphorbiaceae Stem/leaf latex Singha, 1965

Ficus elastica Moraceae Root bark Sackeyfio et al., 1986

Garcinia Kola Guttifera Fruit Iwu, 1986

Gentianella achalensis (Gilg) Gentianaceae Flowering aerial part Debenedetti et al., 1999

Geophilic spp Pleosporaceae Leaf Iwu, 1986

Glaucium flavum Crantz Papaveraceae Aerial Ivanovski et al., 1997

Hibiscus esculentus Malvaceae Fruits Iwu, 1986

Hyptis suaveolens. Labiatae Leaf Iwu, 1986

Icacina trichantha lcacinaceae Tuber Asuzu et al., 1999

Ipomoea digitata Convolvulaceae Leavf Iwu, 1986

Ipomoea fistula Convolvulaceae Shrubs Toursarkissian 1980

Isopyrum thalictroides L. Ranunculaceae Root Ivanovska et al., 1997

Jasminum officinale L. Oleaceae Flower Atta et al., 1998

Jatropha curcas Euphobiaceae Seed/Leaf Iwu, 1986 30

Kalanchoe crenata (Andr.) H. Crassulaceae Aerial parts Ogungbamila et al., 1997

Keay Lecysthidaceae Bark Ogungbamila et al., 1997

Kigelia Africana (Lam.) Bignoniaceae Leaf Ogungbamila et al., 1997

Lagerstroemia indica Lythraceae Leaf Iwu, 1986

Lantana camara Verbinaceae Leaf Iwu, 1986

Lactuca sativa L. Compositae Seed Atta et al., 1998

Lonchocarpus cyanescens Papilionaceae Root Iwu, 1986

Macrolobum spp Caesalpinia Leaf Iwu, 1986

Mangifera indica Anacardiaceae Bark/leaf Iwu, 1986

Manihot esculenta Euphobiaceae Bark/Leaf Iwu, 1986

Marrubrum Polygonum Lamiaceae Root bark Cuellur et al., 2001

Microdesmis puberula Euphobeaceae Root Okafor et al., 1999

Morinda lucida Benth Rubiaceae Leaf Akah et al., 1994

Moringa pterygosperm Moringaceae Root Singha, 1965

Mussaenda afzelii. Rubiaceae Bark Iwu, 1986

Myrtaceae serrata Lam Myriaceae Root bark Mcgaw et al., 1997

Newbouldia laevis. Bignoniaceae Leaf Azuine et al., 1996

Palisota hirsute K. Schum Commelinaceae Leaf Akah et al., 1994 31

Piper guineense Piperaceae Fruits Iwu, 1986

Plantago major Plantaginaceae Leaf Tlahuimedic, 1999

Plumbago zeylanica Plumbaginoeceae Leaf, roots Iwu, 1986

Schwenkia American Solanaceae Plant Iwu, 1986

Schumaniophyton magnificum Rubiaceae Root Iwu, 1986

Scutellaria baicalensis Georgi Lamiaceae Root bark Cuellar et al., 2001

Securidaca longipedunculata Polygalaceae Root Iwu, 1986

Secamone afzelii (Schult.) Asclepiadaceae Leaf Ogungbamila et al., 1997

Sinomenium acutum Menispermaceae Root/stem Yamasaki, 1976

Smilax Kraussiana Smilaceae Root Iwu, 1986

Solanum indicum Solanaceae Fruits Iwu, 1986

Strophantus hibiscus . Apocinaceae Leaf Akah et al., 1994

Syzygium cuinini L Myrtaceae Bark Chandra et al., 2001

Tanacetum vulgare Asteraceae Aerial Schinella et al., 1998

Terminalia spp Combretaceae Bark, Fruit Iwu, 1986

Tetrapleura tetraptera Mimosaceae Bark, Fruit Iwu, 1986

Talitha minus L. Ranunculaceae Aerial Ivanovska et al., 1997

Thryallis glauca Malpighiaceae Flowers Okafor et al., 1999 32

Turnera micrantha Ulmaceae Leaves Barbera et al., 1992

Turnera ulmifolia Turneraceae Aerial parts Souzabrito et al., 1998

Vitex doniana Verbanacaea Leaf Iwueke et al., 2006

Withania somnifera Solanaceae Leaves Iwu, 1986

Zanthoxylum americanum Rutaceae Leaf Oriowo et al., 1982

1.6.1 BOTANICAL PROFILE OF Phyllanthus niruri

1.6.1.1 Taxonomy of Plant

Kingdom: Plantae

Phylum: Spermatophyta

Division: Angiospermae

Class: Dicotyledonceae

Order: Malpighiales

Family: Euphorbiaceae

Genus: Phyllanthus

Specie: Phyllanthus ninuri L.

Common Names: English – Black cat-nip, Child Pick-a-back, stone breaker, seed- under leaf. Igbo – Buchi oro Yoruba – Ehin Olobe Efik -Oyomo – ke – iso- anan ke – eden Hausa – Geeron-tsuntsaye ( Burkill, 1985)

33

1.6.1.2 Plant Description

The Phyllanthus niruri L is distributed through the tropical and sub-tropical regions of both hermispheres. It grows 30 – 60 cm high, quite glabrous, stem often branched at the base.

Leaves: They are numerous, subsessile, stipules are present, very acute.

Flowers: are yellowish, very numerous and axillary; male flowers are one to three in number, while female flowers are solitary in nature .Capsules measure 2.5 mm in diameter, smooth and scarcely lobed. The bark is smooth and light green (Taylor, 2003).

Fruits: are capsule-like, very small, globose, smooth, longitudinally ribbed on the back.

Capsules measure 2.5 mm in diameter (Taylor, 2003).

Seeds are contained in tiny smooth capsules. Seed to seed cycle occurs in two or four weeks

(Taylor, 2003).

1.6.1.3 Geographical Distribution of Plant

Phyllanthus niruri is a widespread tropical plant and is indigenous to the rain forest of the

Amazon and other tropical areas throughout the world, including the Bahamas, Southern India, and China. It is common in the coastal areas, prevalent in other wet rain forests, growing and spreading freely (Taylor, 2003).

1.6.1.4 Ethnomedicinal Uses

Phyllanthus niruri has been named for its effective use by generations of Amazonian indigenous people in eliminating gallstones and kidney stones. It has a long history in global medicine systems in every tropical country where it grows and generally, it is employed for similar conditions worldwide. A standard herb infusion or weak decoction is prepared as the traditional remedy for menstrual disorder, genitor-urinary disorders in India. Other properties/ actions documented by traditional use include carminative, detoxifier, diaphoretic, laxative and menstrual stimulant (Taylor, 2003). In Thailand, hot water extract of commercial sample of the entire plant is administered orally as an antipyretic (Mokkhasmit et al., 1971), hot water extract 34 of dried aerial parts as a diuretic and hot water extract of dried entire plant as an anti inflammatory agent (Wasuwat, 1967). It is taken orally to increase appetite in the Virgin

Islands (Oakes and Morris, 1958). Hot water extract of fresh entire plant is administered mainly for gonorrhea in Tanzania (Khan et al., 1978). Orally, the decoction of dried entire plant is used for coughs in infants of the Philippines (Velazco, 1980).

In Haiti decoction of the dried leaves is taken orally for indigestion while hot water extract of dried entire plant is administered orally as a spasmolytic (Werninger et al., 1980).

Hot water extract of leaf and stem is taken orally for fevers in Puerto-rico (Loustalot and

Pagan, 1949). In India, the fresh plant is taken orally for genito-urinary disorders (Sahu, 1984), and the fruit used externally for tubercular ulcers, scabies and ringworm (Chauhan et al.,

1997). Also the hot water extract of the dried plant is administered orally for diabetes; and in

Ayurvedic medicine is employed for asthma. In Papua-New Guinea, fresh leaf or fresh root juice are taken orally for veneral diseases (Holdsworth et al., 1989). Decoction of the dried leaf when taken orally is used to treat diarrhea (Holdsworth and Balun, 1992).

1.6.1.5 Literature Review of Phyllanthus niruri

In clinical research over the years, Phyllanthus niruri has demonstrated antihepatotoxic, analgesic, hypertensive, antispasmodic, diuretic, antibacterial, antimutagenic, anti viral and hypoglycaemic activities.

Since the mid-1960’s P. niruri has been the subject of much phyotochemical research to determine the active constituents and their pharmacological activities.

The first notable area of study has validated its longstanding traditional use for kidney stones.

In 1990, the Paulistic school of medicine in Sao Paulo, Brazil, conducted studies with humans and rats with kidney stones. They were given a simple tea of P. niruri for 1-3 months and it was reported that the tea promoted the elimination of stones (Taylor, 2003). 35

They also reported a significant increase in diuresis and sodium and creatinine excretion.

(Taylor 2003). Evaluation of the antidiabetic potentials of Phyllanthus niruri showed that the extract reduced fasting blood sugar in a dose-related manner and suppressed the post prandial rise in blood glucose after a heavy glucose meal in normoglycaemic rats. Thus the extract of aerial parts of P. niruri has great potentials as antidiabetic remedy (Okoli et al., 2010).

The plant’s traditional use for hypotension has been explored by research as well. The hypotensive effects were first reported in a study in 1952 using dogs (in which a diuretic effect was noted also) (Kitisin, 1952). The hypotensive effects were attributed to a specific phytochemical called geraniin (an ellagitannin phytochemical) in a 1988 study (Taylor, 2003).

Another area of research focused on the pain relieving and/or antinociceptive effects performed at a Brazilian university. The first three studies (published in 1994-1995) reported strong and dose-dependent analgesic effects in mice administered water and/or alcohol extracts of the plant (orally, intraperitoneally) against six different laboratory-induced nociception

(pain) models (Taylor, 2003)

In 1996, they isolated and tested the hypotensive phytochemical geraniin and reported that it was seven times more potent as an analgesic than aspirin or acetaminophen (Miguel, 1996)

The antihepatotoxic (liver-protecting) activity of P.niruri has been established with clinical research. These effects have been attributed to (at least) two novel lignans named phyllanthin and hypophyllanthin. The researchers who reported the cholesterol-lowering effects also reported that it protected rats from liver damage induced by alcohol, and normalized a fatty liver (Umerani, 1985). 36

Two human studies reported its antihepatotoxic actions in children with hepatitis and jaundice.

Indian researchers reported that it was effective in the treatment of jaundice in children,

(Bhumyamalaki et al., 1983).

Anti-mutagenic properties of P. niruri have been reported in several animal studies (as well as within cell cultures). Its extracts have stopped or inhibited cells (including liver cells) from mutating in the presence of chemical substances known to create cellular mutations. One of these studies indicates that it inhibited several enzyme processes peculiar to cancer cells replication and growth rather than a direct cytotic ability to kill the cancer cell. This cellular- protective quality was evidenced in other research which indicated that it protected against chemically-induced bone marrow chromosome damage in mice, as well as against radiation- induced chromosome damage in mice (Taylor, 2003).

1.6.1.6 Aims And Scope of Study

The ethnomedicinal uses of this plant suggest it may play a role in ameliorating inflammation component of diseases. This research was therefore aimed at (i) assessing the effect of extract of aerial parts of Phyllanthus niruri in models of acute and chronic inflammation and (ii) identifying the phytochemical constituents responsible for the anti-inflammatory activity. 37

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Chemicals, Solvents, Reagents, Drugs and Sources.

(i) Extraction, fractionation and chromatography- methanol, dichloromethane, Silica gel

(70-230 mesh)

(ii) Phytochemistry tests: Ferric chloride, iodide solution, ethanol, dilute ammonia solution,

H2SO4, potassium mercuric iodide solution, Fehlings solution, Millon’s reagent, picric

acid solutions

(iii) Pharmacological studies: Tween 80, carrageenan, xylene, indomethacin (anthracid by

Strides Vital) acetyl salicylic acid (Emzor), distilled water, cotton pellets, chloroform.

2.1.2 Animals

Adult swiss albino mice (15-25 g) and rats (110 -210 g) of either sex were obtained from the

Laboratory Animal Facility of the Department of Pharmacology and Toxicology, University of

Nigeria, Nsukka. The animals were housed in stainless steel cages and plastic cages under standard conditions and fed freely with standard pellet diet and clean drinking water.

2.1.3 Equipment

Rotary evaporator (Buchi Rotavapor-A Orme Scientific Ltd. Middleton, Manchester, England), oven, cork borer, spatula, separating funnel, stop watch, fractionating column, filter paper, electronic balance (B.Bran LP 202 Scientific Instrument, England), test tubes, autoclave (IH-

150 Gallenkamp, England), glass column.

38

2.2 METHODS

2.2.1 Collection And Preparation of Plant Materials

Fresh leaves of were collected from Orba in Udenu L G A of Enugu state precisely in October,

2008 and authenticated by Mr. A.O Ozioko of the International Centre for Ethnomedicine and

Drug Development (Inter CEDD) Nsukka, Enugu State, Nigeria. The leaves were cleaned and the aerial parts dried under the sun for 2 weeks and pulverized to coarse powder using a blender.

2.2.2 Extraction of Plant Materials

About 2 kg of the powdered leaves was extracted with 30 L of methanol by cold maceration for

48 h. The mixture was filtered and the plant material washed several times with fresh portions of methanol. The filtrate was concentrated in a rotary evaporator under reduced pressure and freeze dried to yield 210 g (10.5% w/w) of methanol extract.

2.2.3 Column Chromatographic Separation of The Methanol Extract

The methanol extract was separated on a silica gel glass column successively eluted with dichlomethane (100%) and methanol (100%). The silica gel (70-230 mesh) was packed in the column by dry packing method. About 120 g of the extract was mixed with sufficient quantity of dry silica gel and loaded on top of the already packed silica gel column. The column was then successively eluted with dichloromethane and methanol to obtain two fractions. The fractions were concentrated using a rotary evaporator to obtain 30 g (25.8 % w/w) of dichloromethane and 31.5 g (26.25 % w/w) of methanol fraction.

39

Plant material

Extract with methanol

Methanol extract

Preliminary pharmacological screening test

Fractionation of methanol extract

Dichloromethane Fraction Methanol Fraction

Figure 1: Extraction and Fractionation Scheme

2.2.4 Phytochemical Analysis of Extracts And Fractions

The extracts were screened for the presence of carbohydrates using Molisch test; reducing sugar using fehlings test, alkaloids using Dragendoff’s, Mayer’s, Wagner’s and Hager’s reagents; glycosides using Borntrager’s test; saponins using frothing and emulsion tests; tannins using ferric chloride. Phytochemical analysis of the extracts was done using the methods of Harborne (1984)

1. Test for Saponins

About 20 ml of water was added to 0.2 ml each of the plant extracts in 100 ml beaker 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: 40

About 5 ml of the filtrated was diluted with 20 ml of water and shaken vigorously and observed on standing.

2. Test for Tannins

Ferric chloride test:

About 1.0 ml of each was 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 color of the resulting precipitate observed.

3. Test for Carbohydrates

Iodine Test:

To 0.5 ml of each was mixed with a drop of iodine solution. A blue-black colour indicates the presences of starch

4. Tests for Reducing Sugar

Fehling’s test:

About 1.0 ml of each was shaken vigorously with 5 ml of distilled water and filtered the filtrate was used in the Fehling’s test as follows

To 1.0 ml portion of the filtrate was added equal volumes of Fehling’s solution A and B and boiled on a water bath for few minutes A brick- red precipitate indicates the presence of reducing sugar.

5. Test for Resins

Precipitation test: 41

About 5 ml of each of the extract 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.

6. Test for Oils

General tests:

About 0.1 ml of each was dropped on filter paper and observed translucency of the filter paper indicates the presence of oil

7. Test for Glycosides

Modified Borntrager’s test:

To 2 ml of each filtrate was added 5 ml of dilute sulphuric acid and ferric chloride solution, 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. 5 ml dilute ammonia solution 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.

8. Test for Flavonoids

Ammonium test:

About 10 ml of ethyl acetate 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: 42

About 4 ml of each filtrate was shaken with 1 ml of dilute ammonium solution. The sugars were allowed to separate and the yellow colour in the ammonical layer indicated the presence of flavonoids.

9. Test for Alkaloids (General Test)

About 20 ml of 5% sulphuric acid in 50% ethanol was added to 1 ml of the plant extract and heated on a boiling water bath for 10 min, cooled and filtered. The filtrate (2ml) was treated with a few drops of Meyer’s reagent, Dragendorff’s reagent, Wagner’s reagent and picric acid solution (1%) respectively.

The remaining filtrate in 100 ml separating funnel was made alkaline with dilute ammonia solution, the aqueous alkaline solution was separated and extracted with two 5 ml portions of dilute sulphuric acid The extract was tested with a few drops of Meyer’s Wagner’s and

Dragendorff’s reagent. Alkaloids give milky precipitate with one drop of Meyer’s regent, reddish-brown precipitate with few drops of Wagner’s reagent, yellow precipitate with few drop of picric acid solution and brick red precipitate with few drops of Dragendorff’s reagent.

10. Test for Steroids and Terpenoids

About 9 ml of ethanol was added to 1 ml of the plant extract, refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath and 5 ml of hot water was 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 separating funnel. To 0.5 ml of the chloroform extract in a test tube was carefully added 1 ml 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 3 ml of 43 concentrated sulphuric acid for 10 min in a water bath. A grey color indicates the presence of terpenoids.

2.2.5 Pharmacological Tests

2.2.5.1 Acute Toxicity (LD50) Test

The LD50 of the methanol extract (ME) was studied in mice using the method described by

Lorke (1983) using the oral route. The test was divided into two stages.

Stage one: Determination of the toxic range of the extracts. The mice were divided into 3 groups (n=3). Each group received a dose (10, 100 or 1000 mg/kg) of the extract (ME) suspended in 3% v/v Tween 80. The doses were administered orally and the treated animals monitored for 24 h for number of deaths.

Stage two: Determination of lethality. The doses used in this 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 a fresh batch of animals at one dose per animal (n=1). The treated animals were observed for number of deaths for 24 h.

2.2.5.2 Anti Inflammatory Activity Tests

2.2.5.2.1 Studies on Acute Inflammation

2.2.5.2.1.1 Topical Edema Induced By Xylene In The Mouse Ear

The effects of the methanol extract (ME), methanol fraction (MF) and Dichloromethane fraction (DMF) on acute topical inflammation were evaluated by a modification of the methods of (Tubaro et al., 1985) and (Atta and Alkohafi, 1998).

Adult albino mice (15 -25 g) of either sex were divided into 10 groups (n==10). Each group of animals received 5 mg/ear of the extract or fractions in 3% v/v Tween 80 applied on the outer (anterior) surface of the right ear. Topical inflammation was immediately induced on the 44 inner (posterior) surface of the same ear by application of xylene (0.05 ml). Control animals received either vehicle (3% v/v Tween 80) or Indomethacin (5 mg/ear). Two hours after induction or inflammation, mice were sacrificed by overdose of ether and both ears removed.

Circular discs of both the right (treated) and left (untreated) ears were punched using a cork borer (4 mm) and weighed. Edematous response was quantified as the weight difference between the two earplugs. The anti-inflammatory activity was evaluated as percent edema – inhibition or reduction in the treated animals relative to control animals (Tubaro et al., 1985;

Asuzu et al., 1999).

Reduction of edema (%) = 100 [1 – (Rt – Lt/ Rc – Lc)]

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 ear plug of control animals.

2.2.5.2.2.1.2 Carrageenan-Induced Pedal Edema In Rat

The rat paw edema method of Winter et al (1962) was used. Increase in the right hind paw volume (Bani et al., 2000) induced by the sub plantar injection of carrageenan was used as a measure of acute inflammation.

Adult albino rats (110-250 g) of both sexes were divided into 8 groups of 5 animals. Each group received one of two dose levels 200 or 400 mg/kg of the extract or fraction in 3% (v/v)

Tween 80 administered orally. Control animals received either indomethacin (100 mg/kg) or equivalent volume of 3% v/v Tween 80. Inflammation was induced 1h later by injection of 0.1 ml of 0.1% carrageenan into the sub plantar of the right hind paw of rats. The volume of the 45 paw was measured by water displacement before and at 0.5, 1, 2, 3, 4 h after carrageenan injection. Edema formation was assessed in terms of the difference in the zero time paw volume of the injected paw and its volume at the different times after carrageenan injection.

For each dose of extract the level of edema was calculated using the relation:

Inhibition of edema (%) =100 (1-[(a-x)/ (b-y)])

Where, a = mean paw volume of treated animals after carrageenan injection. x = mean paw volume of treated animals before carrageenan injection. b = mean paw volume of control animals after carrageenan injection. y = mean paw volume of control animals before carrageenan injection.

2.2.5.2.2 Studies On Chronic Inflammation

2.2.5.2.2.1 Formaldehyde – Induced Arthritis In Rats

Adult albino rats of both sexes were divided into 8 groups of 5 animals each. Each group received a drug treatment suspended in 3% v/v Tween 80. Control animals received either acetylsalicylic acid (100 mg/kg) or equivalent volume of vehicle. On day 1 of the experiment, one hour after extract administration, arthritis was induced by injecting 0.1 ml of 2% v/v formaldehyde solution into the sub- plantar of the left hind paw of rats. Formaldehyde injection was repeated on day 3 while drug (extract or vehicle) administration was continued from 1 to day 10.

Daily changes in the inflamed paws were evaluated by measuring the volume of water displaced by the paw once daily. Mean increase in the paw volume of each group over 10 days was calculated using the relation;

Inhibition of edema (%) = 100 [1–[(a – x) / (b-y)] 46

Where, a = mean paw volume of treated animals after formaldehyde injection. x = mean paw volume of treated animals before formaldehyde injection b = mean paw volume of control animals after formaldehyde injection y = mean paw volume of control animal before formaldehyde injection.

The global edematous response to formaldehyde arthritis was quantified using the area under the curve of the arthritis event.

2.2.5.2.2.2 Cotton Pellet Granuloma Test

Autoclaved cotton pellets (30 mg) were implanted subcutaneously one on each side of previously depilated back of the rats using the method of Penn and Ashford (1963) with some modifications. The animals were anaesthesized with chloroform. Extract and fraction were administered for 8 consecutive days starting from day one of pellet implantation. Control animals received an equivalent volume of vehicle (3% v/v Tween 80) or Aspirin (100 mg/kg).

On day 8, the animals were sacrificed by overdose of chloroform anesthesia and the pellets carefully dissected out freed from attachment to the surrounding tissues, and dried overnight in a hot air oven at 60oC to a constant weight. The weights of the dry pellets were measured.

2.3 Statistical Analysis

Data obtained were analyzed using ANOVA and subjected to Dunnett’s and Fischer LSD post hoc test. Differences between means were accepted significant at P < 0.05. Results are presented as Mean ± SEM. The area under the curve (AUC) was determined using WinNonlin

(Standard Edition Version 2.1.0.0). 47

CHAPTER THREE

RESULTS

3.1 EXTRACTION AND FRACTIONATION

About 2 kg of the aerial parts yielded 210 g of methanol extract ( M.E; 10.5% w/w). Column separation of extract (150 g) yielded 31 g ( DCMF; 25.8% w/w) and 31.5g ( ME; 26.25% w/w)

3.2 PHYTOCHEMICAL CONSTITUENTS OF EXTRACTS AND FRACTIONS

Phytochemical tests on the dichloromethane fraction gave positive reactions to terpenes, sterols, resins, and alkaloids, while the methanol fraction tested positive to carbohydrates, reducing sugars, saponins, and tannins. The extract tested positive to saponins, tannins, carbohydrates, terpenes, sterols, resins and alkaloids (Table 2).

3.3 ACUTE TOXICITY STUDIES

In the acute toxicity and mortality (LD50) test for the methanol extract no death was recorded in two stages of the test using oral routes. The LD50 was greater than 5000 mg/kg (Table 3).

3.4 EFFECT OF EXTRACT AND FRACTIONS ON TOPICAL (ACUTE

INFLAMMATION)

The extract and fractions significantly (P<0.05) inhibited the xylene-induced ear edema in mice. The dichloromethane fraction showed the greatest inhibition followed by the methanol fraction and methanol extract (Table 4).

48

Table 2: Phytochemical constituents of extract and fractions

Constituent Relative presence

Methanol extract Dichloromethane Methanol fraction

fraction

Alkaloids + + +

Anthraquinone - - - derivatives

Carbohydrates + - +

Flavonoids - - -

Reducing sugar + - +

Resins + + -

Saponins ++ - + +

Sterols ++ + -

Tannins ++ - + +

Terpenes + ++ -

Key: - Absent + Present + + Moderately Present + + + Abundantly Present

49

Table 3: Acute toxicity (LD50) of the extract

DOSE (mg/kg) MORTALITY

STAGE ONE 10 0/3

100 0/3

1000 0/3

STAGE TWO 1600 0/1

2900 0/1

5000 0/1

LD50 >5000 mg/kg 50

Table 4: Effect of Extract and Fractions on Acute Topical Edema of the Mouse Ear

Treatment Dose Edema Inhibition

(mg/ear) (g) (%)

M.E 5 0.004 ± 0.0003 33.33

DCMF 5 0.002 ± 0.0004* 66.66

MF 5 0.004 ± 0.0002 33.33

INDOMETHACIN 5 0.002 ± 0.0004* 66.66

CONTROL - 0.006 ± 0.0003

n=10; *P<0.05 compared to control. Inhibition (%) was calculated relative to the control

51

3.5 EFFECT OF EXTRACT AND FRACTIONS ON SYSTEMIC (ACUTE)

INFLAMMATION OF THE RAT PAW

The extract and fractions significantly (P<0.05) suppressed paw edema to varying degrees. The dichloromethane fraction showed a dose-dependent inhibition of paw edema. At a dose level of

400 mg/kg, it was comparable to that of indomethacine (100 mg/kg). The methanol fraction also evoked a dose related inhibition of paw edema. The potency of the extract and fractions decreased in the order – DCMF > MF > ME (Tables 5 and 6).

3.6 EFFECT OF EXTRACT AND FRACTIONS ON FORMALDEHYDE INDUCED

ARTHRITIS

The methanol extract and fractions caused a non dose dependent but significant inhibition of formaldehyde-induced arthritis. The inhibition exhibited by the fractions progressed increasingly from day 1 to almost complete inhibition on day 10. The global edematous response to formaldehyde arthritis quantified as area under the curve revealed the order of magnitude of potency as dichloromethane fraction > methanol fraction > methanol extract

(Tables 7, 8 and 9).

3.7 EFFECT OF EXTRACT AND FRACTIONS ON COTTON PELLET INDUCED

GRANULOMA

The methanol extract and fractions inhibited the development of cotton pellet granuloma to varying degrees. The magnitude of inhibition is of the order DCMF >MF > ME .The dichloromethane fraction at the two dose levels inhibited granuloma growth better than acetylsalicylic acid (100 mg/kg) (Table 10).

52

Table 5: Effects of Extract and Fractions on Acute Edema of the Rat Paw

Treatment Dose Paw edema (ml)

(mg/kg) 0.5 h 1 h 2 h 3 h 4 h

ME 200 0.19±0.015 0.34±0.015 0.33±0.015 0.32±0.010 0.29±0.007*

400 0.16±0.014 0.26±0.012 0.24±0.014 0.28±0.012 0.25±0.011*

DCMF 200 0.14±0.013 0.17±0.011 0.16±0.018 0.14±0.003 0.13±0.010

400 0.12±0.009 0.12±0.010* 0.10±0.009* 0.10±0.009 0.10±0.005*

MF 200 0.19±0.014 0.20±0.012 0.20±0.021 0.23±0.016 0.29±0.012

400 0.18±.0014 0.12±0.007 0.20±0.021 0.22±0.012 0.19±0.012

INDOMETHACIN 100 0.15±.011* 0.13±0.012 0.11±0.008 0.09±0.006* 0.06±0.007*

CONTROL 0.22±0.016 0.43±0.013 0.42±0.026 0.45±0.028 0.5±0.020

n=5; *P<0.05 compared to control (ANOVA; Dunnett’s test) Values of edema (ml) shown are

Mean ± SEM

53

Table 6: Percent Inhibition of Edema Induced By Carragenan in Rats

Dose Inhibition (%)

Treatment (mg/kg) 0.5 h 1 h 2 h 3 h 4 h

ME 200 13.63 20.93 21.42 28.89 42.00

400 27.27 39.53 42.85 37.77 50.00

DCMF 200 36.36 60.47 61.90 68.88 74.00

400 45.45 72.09 76.19 77.77 80.00

MF 200 13.63 53.45 52.38 48.88 42.00

400 15.18 51.16 32.38 51.11 62.00

INDOMETHACIN 100 31.81 69.76 73.80 80.00 88.00

n=5; Inhibition (%) was calculated relative to the control

54

Table 7: Effect of extract and fractions on formaldehyde induced arthritis in rats

Edema (ml) Mean ± SEM

Treatment Dose Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 m/kg M.E 200 0.29±0.012 0.28±0.014 0.29±0.010 0.45±0.010 0.32±0.008 0.23±0.010 0.25±0.011 0.18±0.020 0.17±0.021 0.15±0.200 400 0.28±0.010 0.25±0.184 0.22±0.025 0.39±0.025 0.35±0.010 0.25±0.009 0.24±0.013 0.19±0.007 0.15±0.014 0.12±0.019 DCMF 200 0.21±0.009 0.19±0.008 0.15±0.008 0.28±0.013 0.18±0.020 0.15±0.018 0.10±0.028 0.09±0.015 0.09±0.005 0.08±0.005 400 0.18±0.008 0.15±0.013 0.09±0.006 0.31±0.018 0.16±0.015 0.13±0.007 0.09±0.005 0.08±0.005 0.07±0.007 0.06±0.007 M.F 200 0.26±0.026 0.22±0.150 0.19±0.005 0.40±0.010 0.28±0.006 0.23±0.006 0.20±0.014 0.15±0.011 0.12±0.011 0.10±0.010 400 0.24±0.007 0.19±0.011 0.17±0.011 0.37±0.017 0.24±0.011 0.20±0.015 0.15±0.014 0.12±0.008 0.09±0.006 0.08±0.007 INDO 100 0.16±0.007 0.10±0.014 0.09±0.070 0.15±0.008 0.12±0.004 0.08±0.004 0.07±0.004 0.07±0.003 0.04±0.010 0.03±0.008 CONTROL 0.35±0.140 0.40±0.130 0.38±0.014 0.53±0.009 0.48±0.024 0.42±0.013 0.39±0.015 0.36±0.010 0.32±0.018 0.29±0.022

n = 5; P<0.05 compared to control. Values of arthritis (ml) shown are Mean ± SEM.

Key:

ME = Methanol extract MF = Methanol fraction DCMF = Dichloromethane fraction INDO = Indomethacine

55

Table 8: Percent inhibition of arthritis induced by formaldehyde in rats

Treatment Dose Inhibition (%)

(mg/kg) Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10

M.E. 200 17.14 30.00 23.68 15.09 33.33 45.23 35.89 50.00 46.87 48.27

400 20.00 37.50 42.10 26.41 27.08 40.47 38.46 47.22 53.12 58.62

DCMF 200 40.00 52.50 48.27 47.16 62.50 64.28 74.35 75.00 71.87 72.41

400 48.57 47.50 76.31 41.50 66.66 69.04 76.92 77.77 78.12 79.31

M.F 200 25.71 45.00 50.00 24.52 41.66 45.23 48.71 58.33 62.50 65.51

400 31.42 52.50 55.26 30.18 50.00 52.38 61.53 66.66 71.87 72.41

INDO 100 54.29 75.00 76.31 71.69 75.00 80.95 82.05 80.55 88.88 89.65

Percent inhibition of arthritis induced by formaldehyde in rats (n = 5). Inhibition was

calculated relative to control

Key:

ME = Methanol extract

MF = Methanol fraction

DCMF = Dichloromethane fraction

INDO = Indomethacine 56

Table 9: Global effect of extract and fractions on formaldehyde arthritis in rats

Treatment Dose (mg/kg) AUC (ml/day) Inhibition (%)

ME 200 2.54 32.84

400 2.38 36.95

DCMF 200 1.48 60.79

400 1.29 65.82

MF 200 2.10 44.37

400 1.81 52.05

INDOMETHACIN 100 0.90 76.29

CONTROL 3.78 -

Values of AUC shown are mean ± SEM: compared to control (n = 5) (ANOVA: LSD post hoc) AUC = Area under the curve.

57

Table 10: Effect of Extract and Fractions on Cotton Pellet Induced Granuloma

Treatment Dose Granuloma Tissue Inhibition

Weight (g) (mg/kg) (%)

ME 200 0.069 ± O.003 26.60

400 0.054 ± 0.003 42.55

DCMF 200 0.038± 0.006 59.57

400 0.023 ± 0.003* 75.53

MF 200 0.057± 0.004 39.36

400 0.053 ± 0.003* 43.61

ASA 100 0.043 ± 0.003* 54.26

CONTROL 0.094 ± 0.005 n=5; *P<0.05 compared to control. Inhibition (%) was calculated relative to the control.

58

CHAPTER FOUR

DISCUSSION AND CONCLUSION

The anti-inflammatory properties of the extract and fractions of aerial parts of Phyllantus niruri were evaluated. Extraction of the powdered aerial parts afforded the methanol extract which on solvent guided fractionation yielded the dichloromethane and methanol fractions. The effects of the extract and these fractions were evaluated on acute and chronic inflammation.

Assessment of effects on topical acute inflammation revealed potent suppression of xylene- induced edema of the mouse ear by the extract and fractions. Xylene, upon topical application causes instant irritation of the mouse ear, which leads to fluid accumulation and edema characteristic of the acute inflammatory response. Suppression of this response is a likely indication of antiphlogistic effect (Atta and Alkohafi, 1998) and suggests that the extract and fractions may possess anti-inflammatory activity in acute inflammation.

Consistent with this is their effect on systemic acute edema of the rat paw. In the systemic acute edema of the rat paw, the extract and fractions also inhibited the development of carrageenan–induced edema. Carrageenan edema is known to be mediated by pro- inflammatory mediators such as histamine, 5-HT, bradykinin and prostaglandins. Thus, the inhibitory effect suggests they may exact true anti-inflammatory effects in acute inflammation possibly by interfering with the pro-inflammatory effects of these mediators.

Studies on chronic inflammation of the formaldehyde type also revealed potent inhibition of the global edematous response to formaldehyde-induced arthritis by the extract and fractions.

The formaldehyde arthritis model has been used to evaluate the effect of anti-inflammatory agents on chronic inflammation where articular changes induced by formaldehyde injection 59 mimic those that occur in rheumatoid arthritis (Akah et al., 2007). Inhibition of formaldehyde induced arthritis is an indication of anti-inflammatory activity in chronic inflammation.

Further evaluation of the effect of the extract and fractions in chronic inflammation using the cotton pellet granuloma model showed pronounced inhibition of granuloma tissue growth.

Granuloma of chronic inflammation manifests as accumulation of modified macrophages arranged in small clusters or nodular collections or surrounded by a curf of lymphocytes

(Whaley and Burt, 1996). Thus, inhibition of granuloma formation suggests a modulation of leukocyte migration which also reduces the magnitude of the inflammatory response. The inhibitory effect of the extract and fractions in both models clearly suggests they may be effective in suppressing chronic inflammation. In all the models used in this study, the extract and fractions exhibited effective inhibitory effect in both acute and chronic inflammation.

Preliminary assessment of toxicity using acute toxicity test on the methanol extract revealed an oral LD50 >5000 mg/kg in mice. This high LD50 value suggest that the aerial part could be generally regarded as safe (Lorke, 1983) and possesses a remote risk of acute intoxication.

Biological activity guided technique was employed in the study to identify the anti- inflammatory constituents of the plant. A comparison of the magnitude of inhibition of inflammation in all the models revealed the order of potency dichloromethane fraction>methanol fraction> methanol extract. The dichloromethane fraction consistently exhibited the greatest anti-inflammatory potency in all the models which strongly suggests that it may contain more of the anti-inflammatory constituents of the plant. Phytochemical analysis of this fraction revealed abundant presence of terpenes, sterols, resins and alkaloids. These constituents have been reported to account for the anti-inflammatory activity of many plants

(Singh et al., 1997; Akihisa et al., 1997; Park et al., 2001). Further separation of the 60 dichloromethane fraction is likely to reveal any of them as the anti-inflammatory constituent of aerial parts of P. niruri.

In conclusion, the results of this study have shown that aerial parts of this plant contain constituents capable of suppressing acute and chronic inflammation. This justifies the traditional use of aerial parts of P. niruri to manage inflammatory disorders. The anti- inflammatory activity may be attributable to the terpenes, sterols, resins and alkaloids constituents.

61

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