Anti-Inflammatory and Analgesic Activities of Ethanol Extract of Stem-Bark of Hymenodictyon Pachyantha (Udelose) in Experimental Animals

ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF ETHANOL EXTRACT OF STEM-BARK OF HYMENODICTYON PACHYANTHA (UDELOSE) IN EXPERIMENTAL ANIMALS

AHAM, EMMANUEL CHIGOZIE (PG/M.Sc/16/81353)

DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA NSUKKA

NOVEMBER, 2018

TITLE

ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIES OF ETHANOL EXTRACT OF STEM-BARK OF HYMENODICTYON PACHYANTHA (UDELOSE) IN EXPERIMENTAL ANIMALS

A RESEARCH PROJECT REPORT SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE (M.Sc) DEGREE IN PHARMACOLOGICAL BIOCHEMISTRY, UNIVERSITY OF NIGERIA, NSUKKA

AHAM, EMMANUEL CHIGOZIE (PG/M.Sc/16/81353)

DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA NSUKKA

SUPERVISOR: PROF. O. F. C. NWODO

NOVEMBER, 2018

CERTIFICATION

Aham, Emmanuel Chigozie, a postgraduate student of the Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, with Registration Number; PG/M.Sc/16/81353, has satisfactorily completed the requirements for the award of degree of Master of Science (M.Sc) in Biochemistry (Pharmacological). The work embodied in this dissertation is original and has not been submitted in part or full for any other diploma or degree of this or any other university.

______PROF. O. F. C. NWODO PROF. F. C. CHILAKA (Supervisor) (Head of Department)

______External Examiner

DEDICATION

This research work is dedicated to Late Mr. Aham Joe, whose tenets I aim at upholding. May perpetual light continue to shine on you as you continue to rest in the bossom of the Almighty.

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ACKNOWLEDGEMENTS

My foremost gratitude goes to the Almighty God for sustaining me throughout this stage and making it possible for this research work to be successfully completed.

I would also like to express my heartfelt gratitude to my supervisor, Prof. O. F. C. Nwodo who despite his tight schedule supervised this work. It was a privilege having him as a supervisor. He went beyond being a supervisor, but also played the role of a father, counselor and advisor. His scholarly expertise and constructive criticism was useful in achieving this great feat. I am really grateful sir.

A sincere appreciation to the present administration of the Department of Biochemistry, University of Nigeria, Nsukka under the headship of Prof. F. C. Chilaka, whose words of encouragement and moral support kept me on the task that was set before me. Indeed, your administrative competence and skills have constituted a great blessing to my academic plight. With a deep sense of appreciation, I hold in esteem all my lecturers and staff particularly Prof. L. U. S. Ezeanyika, Prof. O. U. Njoku, Prof. I. N. E. Onwurah, Prof. H. A. Onwubiko, Prof. B. C. Nwanguma, Prof. S. O. O. Eze, Dr. P. E. Joshua, Dr. C. S. Ubani, Dr. O. C. Enechi, Dr. V. E. O. Ozougwu, Dr. A. L. Ezugwu, Dr. (Mrs.) C. A. Anosike, Dr. (Mrs.) O.U. Njoku, Dr. (Mrs.) F. N. Nworah, and Dr. (Mrs.) C. Nkwocha; not forgetting the assistance from Mr. I. U. Okagu, and Dr. E. Anaduaka including the entire staff of the Department of Biochemistry, University of Nigeria, Nsukka, for the knowledge they have bestowed in me; thereby making me a well ground Biochemist.

I wish to express my heartfelt gratitude to my dear mother, Mrs. Aham Assumpta. She played the motherly role in encouraging me. In a special way I appreciate the family of Prof. C.A. Igwe, Mr. Uchenna A. Ibekwe and Mr. Patrick Onyi’s family, for the financial and moral supports they accorded me; may God reward you bountifully. I would not also forget the likes of Nath Francis, Akubunwa Emmanuel, Egwim Frank, Anyanwu Justus and to my numerous friends for their tireless efforts in seeing to the completion of this work. To all my classmates, Mr. Osuagwu Evans and those who donated their cherished blood to be used in the course of this work. I am grateful. May God reward you all abundantly.

ABSTRACT

This study was aimed at investigating the anti-inflammatory and analgesic activities of ethanol extract of Hymenodictyon pachyantha stem-bark as well as the possible mechanisms of anti- inflammatory action of the plant extract. The plant material was extracted using 3.5 litres of absolute ethanol. The anti-inflammatory activity of the ethanol extract was evaluated by determining its effect on egg albumin-induced rat paw oedema, phospholipase A2 activity, calcium chloride-induced platelet aggregatory response and membrane stabilization activity. The effect of the extract on acetic acid-induced writhing responses was also investigated. The percentage yield of the ethanol extract was 3.32%. Phytochemical analyses of the extract revealed the presence of flavonoids (1359.268 ± 0.02 mg/100g), terpenoids (2154.695 ± 0.01 mg/100g), steroids (3.782 ± 0.05 mg/100g), saponins (0.405 ± 0.03 mg/100g), alkaloids (268.856 ± 0.12 mg/100g), tannins (1375.930 ± 0.08 mg/100g) and phenols (2900.169 ± 0.15 mg/100g). The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. The stem-bark extract at 100, 200 and 400 mg/kg body weight significantly (p < 0.05) and dose- dependently inhibited egg albumin-induced rat paw oedema in both early and late stages of inflammation when compared with the untreated control, sustained over a period of 0.5 to 5 hrs. The standard anti-inflammatory drug (indomethacin, 10 mg/kg b. w.) followed a similar trend. The extract also significantly (p < 0.05) inhibited phospholipase A2 activity in a dose-related manner when compared to the control, with a range of 0.1 to 0.5 ml; inhibiting the enzyme activity by 78.92 to 95.59%. The extract also significantly (p < 0.05) and concentration- dependently inhibited platelet aggregatory response when compared to the control. The extract significantly (p < 0.05) inhibited hypotonicity-induced red blood cell membrane lysis in a concentration- dependent manner, similar to the standard drug indomethacin. Ethanol extract of Hymenodictyon pachyantha stem-bark (100, 200 and 400 mg/kg b. w.) significantly (p < 0.05) reduced the number of writhings induced by 0.6% acetic acid solution in a dose dependent manner counted over a period of 20 mins. The results, therefore suggest that the mechanisms of the anti-inflammatory effect may be due to the stabilization of lysosomal membrane, by inhibiting phospholipase A2 and aggregation of platelets. Findings of this investigation provide empirical evidence for the use of Hymenodictyon pachyantha stem-bark extract in folkloric treatment of inflammatory disorders.

TABLE OF CONTENTS

Title Page i

Certification ii

Dedication iii

Acknowledgements iv

Abstract v

Table of Contents vi

List of Figures xii

List of Tables xiii

List of Abbreviations xiv

CHAPTER ONE: INTRODUCTION

1.0 Introduction 1

1.1 Description and uses of Hymenodictyon pachyantha 2

1.1.1 Taxonomic classification of Hymenodictyon pachyantha 3

1.1.2 Common names of Hymenodictyon pachyantha 3

1.1.3 Geographical distribution 3

1.2 Previous studies on Hymenodictyon pachyantha 3

1.3 Overview of inflammation 4

1.3.1 Inflammatory mediators 6

1.3.1.1 Cell derived mediators 6

1.3.1.1.1Vasoactive amines 6

1.3.1.1.2 Prostaglandins 7

1.3.1.1.3 Thromboxane A2 and prostacyclin 7

1.3.1.1.4 Cytokines 8

1.3.1.1.5 Leukotrienes 8

1.3.1.1.6 Chemokines 9

1.3.1.1.7 Nitric Oxide 9

1.3.1.1.8 Platelet-activating factor 10

1.3.1.2 Plasma-derived mediators 10

1.3.1.2.1 Complement system 11

1.3.1.2.2 Clotting or coagulation system 11

1.3.1.2.3 Fibrinolytic system 11

1.3.1.2.4 Kinin system 12

1.3.2 Cells of inflammation 13

1.3.2.1 Agranulocytes 13

1.3.2.1.1 Macrophages 13

1.3.2.1.2 Monocytes 14

1.3.2.1.3 Lymphocytes 14

1.3.2.1.3.1 T-cells and B-cells 15

1.3.2.1.3.2 Natural killer cells 15

1.3.2.2 Granulocytes 16

1.3.2.2.1 Mast cells 16

1.3.2.2.2 Neutrophils 17

1.3.2.2.3 Basophils 17

1.3.2.2.4 Eosinophils 17

1.3.2.3. Platelets 18

1.3.3 Classification of inflammation 18

1.3.3.1 Acute inflammation 18

1.3.3.1.1 Mechanism of acute inflammation 19 vii

1.3.3.1.2 Responses in acute inflammation 20

1.3.3.1.3 Vasodilatation 21

1.3.3.1.2.2 Increased vascular permeability 20

1.3.3.1.2.2 Neutrophil extravasation 22

1.3.4 Resolution of the acute inflammatory response 24

1.4 Chronic inflammation 26

1.4.1 Types of chronic inflammation 27

1.4.1.1 Non-specific proliferative 27

1.4.1.2 Granulomatous inflammation 27

1.4.2 Inflammatory response in chronic inflammation 28

1.5 Inflammatory disorders 28

1.6 Anti-Inflammatory agents 28

1.6.1 Corticosteroids 29

1.6.2 Non-steroidal anti-inflammatory drugs (NSAIDs) 30

1.7 Aim and objectives of the study 32

1.7.1 Aim of the study 32

1.7.2 Specific objectives of the study 32

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials 33

2.1.1 Plant materials 33

2.1.2 Animals 33

2.1.3 Bacterial organism 33

2.1.4 Blood samples 33

2.1.5 Drugs 33

2.1.6 Chemicals and reagents 34 viii

2.1.7 Equipment and instruments 34

2.2 Methods 34

2.2.1 Preparation of plant extract 34

2.2.2 Qualitative phytochemical analysis 35

2.2.2.1 Test for alkaloids 35

2.2.2.2 Test for flavonoids 35

2.2.2.3 Test for saponins 35

2.2.2.4 Test for tannins 35

2.2.2.5 Test for terpenoids and steroids 36

2.2.2.6 Test for phenols 36

2.2.3 Quantitative phytochemical analysis 36

2.2.3.1 Estimation of the concentration of flavonoids 37

2.2.3.2 Estimation of the concentration of terpenoids 37

2.2.3.3 Estimation of the concentration of steroids 37

2.2.3.4 Estimation of the concentration of saponins 37

2.2.3.5 Estimation of the concentration of alkaloids 37

2.2.3.6 Estimation of the concentration of tannins 38

2.2.3.7 Estimation of the concentration of phenols 38

2.2.4 Acute toxicity and lethal dose determination 38

2.2.5 In vivo anti-inflammatory studies 38

2.2.5.1 Determination of the effect of ethanol extract of Hymenodictyon pachyantha

stem- bark on egg albumin-induced rat paw oedema 38

2.2.6 In vitro anti-inflammatory studies 40

2.2.6.1 Determination of the effect of ethanol extract of Hymenodictyon pachyantha

stem- bark on phospholipase A2 activity 40 ix

2.2.6.2 Determination of membrane stabilization effect of ethanol extract of

Hymenodictyon pachyantha stem-bark on hypotonicity induced haemolysis of

red blood cells 41

2.2.6.3 Determination of the ethanol extract of Hymenodictyon pachyantha

stem- bark on platelet aggregation 42

2.2.7 Analgesic activity test 43

2.2.8 Statistical Analysis 43

CHAPTER THREE: RESULTS

3.1 Percentage yield of effect of ethanol extract of Hymenodictyon pachyantha

stem-bark 44

3.2 Qualitative phytochemical constituent of ethanol extract of Hymenodictyon

Pachyantha stem-bark. 44

3.3 Quantitative phytochemical composition of ethanol extract of Hymenodictyon

Pachyantha stem-bark 44

3.4 Result of the acute toxicity test of ethanol extract of Hymenodictyon pachyantha

stem-bark 44

3.5 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on egg

albumin-induced rat paw oedema 49

3.6 Effect of effect of ethanol extract of Hymenodictyon pachyantha

stem-bark on phospholipase A2 activity on phospholipase A2 activity 51

3.7 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on

hypotonicity-induced haemolysis of human red blood cells 51

3.8 Effect of the ethanol extract of Hymenodictyon pachyantha stem-bark on platelet

aggregation 54

3.9 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark and x

aspirin on acetic acid-induced nociceptive response in mice 54

CHAPTER FOUR: DISCUSSION

4.1. Discussion 57

4.2 Conclusion 62

4.3 Suggestion for further studies 62

REFERENCES 63

APPENDICES 77

LIST OF FIGURES Figure 1: Leaves and Stem-bark of Hymenodictyon pachyantha 4

Figure 2: The inflammatory response to injury 5

Figure 3: Generation of arachidonic acid metabolites and their roles in inflammation 10

Figure 4: Intercommunication between inflammatory cascade and the coagulation cascade 12

Figure 5: Overview of vascular changes in acute inflammation 21 Figure 6: The multistep process of leukocyte migration through the blood vessels 24

Figure 7: Chemical structures of prednisolone and deflazacort 29

Figure 8: Chemical structures of common non-steroidal anti-inflammatory drugs 31

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LIST OF TABLES Table1: Percentage yield of ethanol extract of Hymenodictyon pachyantha stem-bark 45

Table 2: Qualitative phytochemical constituents of ethanol extract of Hymenodictyon pachyantha stem-bark 46

Table 3: Quantitative phytochemistry of ethanol extract of Hymenodictyon pachyantha stem- bark 47

Table 4: Acute toxicity test of ethanol extract of Hymenodictyon pachyantha stem-bark 48

Table 5: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on egg albumin-induced rat paw oedema 50

Table 6: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on phospholipase A2 activity 52

Table7: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on hypotonicity- induced haemolysis of human red blood cells 53

Table 8: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on calcium chloride-induced platelet aggregation 55

Table 9: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark and aspirin on acetic acid induced nociceptive response in mice 56

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LIST OF ABBREVIATIONS

% Percentage

5-HT 5-hydroxytryptamine

ANOVA Analysis of variance

AP-1 Activator protein-1

APC Antigen processing cell

ATP Adenosine triphosphate

CAM Cell adhesion molecule cNOS Constitutive nitric oxide synthase

CNS Central nervous system

COX Cyclooxygenase

DAMPs Damage-associated molecular patterns

DMARDs Disease-modifying antirheumatic drugs

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid

GR Glucocorticoid receptor

HMWK High-molecular-weight kininogen

HRBCs Human red blood cells

IFN-γ Interferon gamma

IgG Immunoglobulin G

IKK Inhibitor of kappa β kinase

IL Interleukin iNOS Inducible nitric oxide synthase

IRF Interferon regulator factor

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Iκβ Inhibitor of kappa β

LOX Lipoxygenase

LTs Leukotrienes

LXs Lipoxins

MAC Membrane attack complex

MAC-1 Macrophage-1 antigen

MAPK Mitogen-activated protein kinase

MCP-1 Monocyte chemoattractant protein-1

MHC Major histocompatibility complex

MMP -1 Membrane mitochondrial potential -1

NO Nitric oxide

NOS Nitric oxide synthase

NSAIDs Non-steroidal anti-inflammatory drugs

PAF Platelet-activating factor

PAMPs Pathogen-associated molecular patterns

PDGF Platelet-derived growth factor

PECAM-1 Platelet/endothelial cell adhesion molecule-1

PGs Prostaglandins

PLA2 Phospholipase A2

PMNs Polymorphonuclear neutrophils

PRP Platelet-rich plasma

PRRs Pattern-recognition receptors

PSGL-1 P-selectin glycoprotein ligand-1

PUFAs Polyunsaturated fatty acids

RBC Red blood cell xv

ROS Reactive oxygen species

RNS Reactive nitrogen species

TF Tissue factor

TGF Transforming growth factor

TNF-α Tumour necrosis factor-alpha tPA Tissue plasminogen activator

TXs Thromboxanes uPA Urinary plasminogen activator

VCAM-1 Vascular-cell adhesion molecule-1

VEGF Vascular endothelial growth factor

WBC White blood cell

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CHAPTER ONE

INTRODUCTION

The diverse natural products from medicinal plant continues to be an accepted form of treatment in the orient, and plant drugs based on traditional practice represent a huge portion of the pharmaceutical products in modern western countries (Dhami, 2013). Concerns have been raised that modern pharmaceutical practice too often involves costly drugs that produce unacceptable side effects. Experience shows that natural substances can apparently address several modern health concerns with fewer side effects. The acceptance that natural is better, fear or distrust of physicians, disappointment with allopathic care, and cultural or religion influences, increases interest in the use of natural bio-resources to manage chronic diseases and reduce issues of side effects and prices of pharmacological therapies. Medicinal plants with anti-inflammatory activity are considerably employed in the traditional treatment of several disorders of inflammation (Iwueke et al., 2006). Inflammation is a biological reaction to a disrupted tissue homeostasis (Medzhitov, 2008). Inflammation is a complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells or irritants (Malaya et al., 2003). It can be acute or chronic (Ferrero-Miliani et al., 2007). Its purpose is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair and restoration of functions. Inflammation is normally closely regulated by the body. When inflammation is too little, it could lead to progressive tissue destruction by the harmful stimulus (e.g. bacteria) and compromise the survival of the organism. On the other hand in chronic inflammation, the inflammatory response is out of proportion resulting in damage to the body. Inflammation is critically implicated in the development of many complex diseases and disorders including autoimmune diseases, metabolic syndromes, neurodegenerative diseases, cancers, and cardiovascular disease, heart attacks, Alzheimer’s disease and cancer (Coussens and Werb, 2002; Libby et al., 2002).

No matter the initiating stimulus, the classic inflammatory response is characterised by five clinical signs: calour (warmth), dolour (pain), rubour (redness), tumour (swelling) and functio laesa (loss of function) (Rigler, 1997). Cyclooxygenase (COX) is the key enzyme in the synthesis of prostaglandins, prostacyclins and thromboxanes which are involved in

inflammation, pain and platelet aggregation (Pilotto et al., 2010). The clinical symptoms such as fever, aches and pains associated with several diseases are directly or indirectly due to inflammatory disorders. Non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are classically used to alleviate inflammation. Long term uses of NSAIDs causes side effects including gastric ulceration and renal toxicity (Payne, 2000; Ezekwesili et al., 2011) since they concurrently inhibit both isoforms of cyclooxygenase (COX). The development of NSAIDs which are selective COX inhibitors still have side effects as reports have connected these drugs with an increased risk of heart attack and stroke (Salmon, 2006; Nelson and Cox, 2008). There is therefore, the need for potent anti-inflammatory drugs with fewer side effects from natural sources as alternatives to these drugs. This has necessited the research into plants used in folk medicine to ameliorate inflammation. An example of such plant is Hymenodictyon pachyantha which is used in Nigeria folk medicine for the management of inflammatory disorders.

1.1 Description and uses of Hymenodictyon pachyantha

Hymenodictyon pachyantha is very distinct from the other Hymenodictyon species because of its recurved calyx lobes, which are longer than the mature corollas, and its elongate ovaries. Hymenodictyon is a genus of flowering plants in the family Rubiaceae comprising of about 30 species (David, 2008). The generic name is derived from two Greek words, hymen, ‘membrane’, and diktyon, ‘net’. It refers to the wing that surrounds each seed. Molecular phylogenetic studies have shown that Hymenodictyon is paraphyletic over the Madagascan genus Paracorynanthe (Ulrika and Birgitt, 2010). In Hymenodictyon and Paracorynanthe, the stipules bear large deciduous glands called colleters. The corolla tube is narrow at the base, gradually widening toward the apex. The fruit is a woody capsule. The species belonging to this genus are having oppositely arranged serrated leaves, small, clustered flowers and many seeded capsules. Stipules are linear to lanceolate or ovate 15 mm long, apex acuminate and pubescent. Leaves are deciduous; petioles are 10-60 mm long, green-white-tinged, puberulous; secondary veins are seven to ten pairs per side. Fruit, a capsule, is ellipsoid, 2 to 2.5 cm long, growing on recurved, thick pedicels 5 to 12 mm long. Seeds are many, flat, winged all around the margin, about 1 cm long, including the wing. Bark is mostly furrowed and rough, 10-20 cm thick, exfoliating in irregularly shaped with soft scales. The wood of H. pachyantha is soft and has limited use, mostly for boxes. The wood is used as planks in

building houses and boats, making boxes, packing cases, pencils, toys model making and matches. In India, it is used as a cheaper grade of wood for making furniture, wrapper bobbins and wool boards. The bark obtained is useful for tanning purposes while leaves are useful for dyeing and as fodder for cattle while the remaining are only useful for timber wood (David, 2008). 1.1.1 Taxonomic classification of Hymenodictyon pachyantha Kingdom: Plantae Phylum: Angiosperms Class: Eudicots Order: Gentianales Family: Rubiaceae Sub-family: Cinchonoideae Genus: Hymenodictyon Species: pachyantha Source: (David, 2008)

1.1.2 Common names of Hymenodictyon pachyantha

Hymenodictyon pachyantha is commonly known in Igbo as Udeleose ( Enugu), (Jane and Edwin, 2011) and óbadaṇ (Farquhar) in Edo (Benin).

1.1.3 Geographical distribution

The genus comprises of trees and shrubs, distributed mostly in tropical and sub-tropical parts of Asia and Africa (Sylvain and Birgitta, 2006). H. pachyantha is mainly found in secondary forests at low altitudes, often about cliffs near the sea (Retief and Leistner, 2000). It is distributed in Nigeria (Benin, Enugu), Cameroon, and Ivory Coast.

1.2 Previous studies on Hymenodictyon pachyantha

Phytochemical studies carried out on species of the genus Hymenodictyon pachyantha has shown considerable number of important phytoconstituents. The chemical constituents previously reported to be found in this plant were coumarins (Parichat et al., 2009) and anthraquinones. The stem bark contains tannin, toxic alkaloid hymenodictine, a bitter

substance, aesculin (Sylvain and Birgitta, 2006), an apioglucoside of scopoletin, hymexelsin (Prashant and Vijay, 2011). Anthraquinones, rubiadin and its methyl ether, lucidin, damnacanthal, nordamnacanthal, 2-benzylzanthopurpurin and anthragallol, (Gurung, 2002) have also been isolated from roots. Hymenodictyoline obtained from H. pachyantha is one of the few alkaloids which do not contain oxygen (Razafimandimbison and Brermer, 2002). Studies have also reported acetylenic fatty acids, a new triglyceride, and 11 known compounds, among them, ursolic acid, oleaqnolic acid, uncarinic acid E, b-sitosterol (Parichat et al., 2009). The roots of H. pachyantha also reported to contain anthragallol, 6- methylalizarin, soranjidiol, morindone and triterpenes (Sultana et al., 2015); oleanolic acid; uncarinic acid E (3β-hydroxy-27-(E) (Nareeboon et al., 2009).

Figure 1: Leaves and Stem-bark of Hymenodictyon pachyantha Source: (Zahoui et al., 2010)

1.3 Overview of inflammation Inflammation is a pervasive phenomenon that operates during severe perturbations of homeostasis, such as infection, injury and exposure to contaminants (Ashley et al., 2012). Inflammation is the result of concerted participation of a large number of vasoactive, chemotactic and proliferative factors at different stages (Gobinda et al., 2011). Inflammatory response is brought about or mediated by inflammatory mediators such as chemokines, cytokines, cell adhesion molecules, extracellular matrix proteins (Simon et al., 2000; Nanyak et al., 2013) which when in excess are deleterious (Liu and Hong, 2002). It is triggered by infectious agents such as, viruses, fungi, bacteria and protozoa. It is also due to trauma,

physical and chemical agents, tissue necrosis and immune reactions. The mechanisms involved in the inflammatory process are common to all, regardless of the triggering factor (Ferrero-Miliani et al., 2007).

Figure 2: The inflammatory response to injury Source: (Marieb and Mitchell, 2007)

It involves a cascade of biochemical events comprising of the local vascular system and the immune system (Da Silveira e Sá et al., 2013). It also involves the production of factors that could cause damage to tissues when not properly regulated. Although inflammation is a defense mechanism, the complex events and mediators involved in inflammatory reaction can be induced, maintained and aggravated by many diseases (Anosike et al., 2012a), thus inflammation is critical for the development of many complex diseases and disorders including autoimmune diseases, metabolic syndrome, neurodegenerative diseases, cancers, and cardiovascular diseases. Inflammation comes in two types: chronic inflammation, which can be defined as a dysregulated form of inflammation, and acute inflammation, which can be defined as a regulated form.

1.3.1 Inflammatory mediators

Inflammatory mediators are substances triggered by inflammatory stimuli (Bhadrapura and Sudharshan, 2016). They are derived from inflammatory cells, or released as plasma proteins (Vishal et al., 2014). They also mediate inflammatory response by either acting as pyrogens, thereby triggering off the synthesis and release of prostaglandin to the insult or by causing the lysis of cells, especially the mast cells in the case of injury of infections. Most mediators bind to specific target receptors on the cells to elicit their effects. Exceptions are lysosomal enzymes that have a direct enzymatic effect and reactive oxygen species (ROS) that have a direct toxic effect. Mediators can stimulate target cells to release secondary mediators. The local lipid mediators constitute a new genus of anti-inflammatory and pro-resolving endogenous compounds that have proven to be very potent in treating a number of inflammation-associated models of human disease.

1.3.1.1 Cell derived mediators

These are mediators derived from stimulated inflammatory cells. They may be preformed and stored in the granules of these cells or may be synthesized de novo and secreted as needed. Circulating platelets, basophils, PMNs, endothelial cells monocyte and macrophages, tissue mast cells and the injured tissue itself are all potential cellular sources of vasoactive mediators. In general, these mediators are; (i) derived from metabolism of phospholipids and arachidonic acid (e.g., prostaglandins, thromboxanes, leukotrienes, lipoxins, platelet-activating factor PAF), (ii) preformed and stored in cytoplasmic granules (e.g., histamine, serotonin, lysosomal hydrolases), or

(iii)derived from altered production of normal regulators of vascular function (e.g., nitric oxide and neurokinins).

1.3.1.1.1 Vasoactive amines

Vasoactive amines include histamine and serotonin. The latter is also known as 5- hydroxytryptamine (5-HT). Histamine (β-Imidazolylethylamine) is a vasodilator, a constrictor of smooth muscle, and a potent stimulant of vascular permeability, respiratory mucus, and gastric acid secretion. It exerts its effects on a variety of cell types including

smooth muscle cells, neurons, glandular cells (endocrine and exocrine), blood cells, and cells of the immune system. In addition to its role in immediate hypersensitivity reactions, histamine can exert H2-receptor-mediated anti-inflammatory activity including inhibition of human neutrophil lysosomal enzyme release, inhibition of IgE-mediated histamine release from peripheral leukocytes, and activation of suppressor T-lymphocytes. Histamine is synthesized in the golgi apparatus of mast cells and basophils by decarboxylation of the amino acid histidine and is then stored in secretory granules in complexes with heparin, protein, or both. Histamine is released during antigen reaction with mast cell bound antibody molecules and during the inflammatory response to skin injury (Trautmann et al., 2000). It is also released from basophils and platelets. It causes the contraction of endothelial cells of venules leading to increased vascular permeability. Its effect is rapidly inactivated by histaminase. Serotonin is released from the platelets along with histamine and acts similarly to histamine (Stone et al., 2010).

1.3.1.1.2 Prostaglandins

Phospholipase A2 (PLA2), in response to any disturbance of the cell membrane activates the hydrolysis of phospholipids from the lipid bilayer into free fatty acids such as, arachidonic acid. Arachidonic acid plays an important role in many metabolic pathways and is useful when produced in moderation (George et al., 2014). When produced in excess, it acts as a substrate for COX (or prostaglandin H2 synthase) to release prostanoids, comprising of prostaglandins (PGs), thromboxanes (TXs) and prostacyclins (Ricciotti and FitzGerald,

2011). Examples of PGs are prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2).

Prostaglandin D2 is a major prostaglandin produced by mast cells. It is a bronchoconstrictor and also acts as a chemoattractant for leukocytes (Stone et al., 2010). Prostaglandin E2 which is more widely distributed causes pain, vasodilatation and increases vascular permeability. All cells are capable of synthesizing PGs, apart from non-nucleated erythrocytes.

1.3.1.1.3 Thromboxane A2 and prostacyclin

Thromboxanes and Prostacyclins are also produced through the action of COX on arachidonic acid. Thromboxane A2 (TxA2) is a potent platelet-aggregating agent and a vasoconstrictor. It is unstable and is rapidly converted to inactive Thromboxane B2 (TXB2).

Prostacyclin or prostaglandin I2 (PGI2) on the other hand is a vasodilator and a potent

inhibitor of platelet aggregation (Pilotto et al., 2010). It increases blood flow as well as blood vessel permeability by assisting in the release of NO from the endothelium (Vishal et al., 2014).

1.3.1.1.4 Cytokines

These are soluble immune signalling proteins of low molecular weight that modulate the differentiation, proliferation and function of immune cells, and coordinate inflammatory responses (Kidd and Urban, 2001). They are secreted primarily by activated tissue macrophages, lymphocytes and endothelial cells. Their main effects are to induce the acute phase reaction and to activate vascular endothelium, leukocytes, platelets and fibroblasts, thus, initiating the cascade of vascular, cellular and humoural events which together comprise the inflammatory response (Tan et al., 1999). Several cytokines play essential roles in orchestrating the inflammatory process, especially interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α) which are monokines, produced by monocytes and macrophages (Simopoulos, 2002). Lymphokines such as interferon‐gamma (IFN-γ) are produced by lymphocytes and they play an important role in the first line of defense against viral infections.

1.3.1.1.5 Leukotrienes

Leukotrienes (LTs), a family of lipid mediators, play a key role in the pathogenesis of inflammation. LTs are classified into two classes: LTB(4) and cysteinyl LTs (CysLTs). LTB(4) is one of the most potent chemoattractant mediators of inflammation. It exerts its actions through a seven transmembrane-spaning through G protein receptors, LTB4 R-1 and LTB4 R-2 CysLTs (LTC(4), LTD(4), and LTE(4)). Leukotrienes (LTs) like prostanoids, are released from excess arachidonic acid due to the activity of 5-lipoxygenase (5-LOX). The lipoxygenase (LOX) pathway is a parallel inflammatory pathway to the COX pathway (George et al., 2014). Leukotrienes are released from leukocytes and mast cells and are generally pro-inflammatory. Leukotriene B4 (LTB4) is a potent chemotactic agent for neutrophils. It also promotes their adhesion to vascular endothelial cells, their trans- endothelial migration and stimulates the synthesis of pro-inflammatory cytokines from macrophages and lymphocytes (Dalgleish and O’Byrne, 2002; Medzhitov, 2008).

Leukotriene B4 also promotes degranulation and the generation of ROS. Cysteinyl-containing

leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) cause intense vasoconstriction, bronchospasm and increased vascular permeability in venules. Leukotriene (LT) C4 and its products, LTD4 and LTE4, make up the biologic mixture previously known as the slow-reacting substance of anaphylaxis.

1.3.1.1.6 Chemokines

Chemokines such as IL-8, macrophage inflammatory protein-1α, macrophage chemoattractant protein-1, -2, and -3, and rantes function primarily as chemoattractants and activators (Kolaczkowska and Kubes, 2013). Chemokines are inducible molecules. They include, C-X-C (or α) chemokines and C-C (or β) chemokines. C-X-C chemokines are so called because they have an intervening amino acid between the first two of the four conserved cysteine residues at their amino terminal. C-C chemokines on the other hand, do not have an intervening amino acid between their first two amino-terminal cysteine residues (Tan et al., 1999). C-X-C chemokines are primarily neutrophils chemoattractants while C-C chemokines are primarily chemoattractants for monocytes and T-cells. Example of C-X-C chemokine is interleukin-8 (IL-8) while an example of C-C chemokine is monocyte chemoattractant protein-1 (MCP-1). Interleukin-8 and MCP-1 also induce degranulation and respiratory burst in neutrophils and monocytes respectively.

1.3.1.1.7 Nitric oxide

A free radical gas produced endogenously by a variety of mammalian cells including macrophages and endothelial cells, synthesized from arginine by nitric oxide synthase. Nitric oxide is one of the endothelium-dependent relaxing factors released by the vascular endothelium and mediates vasodilation. It also inhibits platelet aggregation, induces disaggregation of aggregated platelets, and inhibits platelet adhesion to the vascular endothelium. Nitric oxide activates cytosolic guanylate cyclase and thus elevates intracellular levels of cyclic GMP. Three major isoforms of NOS include two constitutively expressed forms, which are calcium-calmodulin dependent and are collectively known as constitutive nitric oxide synthase (cNOS). The third which is calcium-calmodulin independent, induced by cytokines and regulated in the gene by a variety of inflammatory mediators is inducible nitric oxide synthase (iNOS) (Kumar et al., 2013). Nitric oxide is toxic to bacteria and

directly inhibits viral replication. It also combines with ROS to yield peroxynitrate radicals which have potent antimicrobial activity (Chukwuka et al., 2011).

1.3.1.1.8 Platelet-activating factor

Platelet-activating factor (PAF) is an ether phospholipid which is released from most proinflammatory cells and platelets by the action of PLA2 (Sato et al., 2009). PAF- like molecules are also generated by activated vascular endothelium in a membrane-bound form. They are chemotactic for neutrophils, enhances the adhesion of platelets and leukocytes to endothelium, involved in vasoconstriction and bronchoconstriction and cause increased vascular permeability.

Figure 3: Generation of arachidonic acid metabolites and their roles in inflammation Source: (Priyadarshini et al., 2013)

1.3.1.2 Plasma-derived mediators

Plasma proteins circulate in an inactive form waiting to undergo proteolytic cleavage to become active. Mediator-producing systems in plasma include: complement, clotting, fibrinolytic and kinin systems.

1.3.1.2.1 Complement system

Complement is part of the humoral immune system. Its role is to generate biologically active products from the pathways of complement activation which include: classical, lectin, alternative, properdin and thrombin pathways (Neher et al., 2011). Peptides generated by complement activation play a critical role in the elimination of invading pathogens. They include, C3b and C4b which opsonise pathogens for phagocytosis, the anaphylatoxins C3a and C5a which acts as chemoattractants for leukocytes and the membrane attack complex (MAC or C5b-9) involved in the direct lyses of pathogens (Alexander et al., 2008; Griffiths et al., 2009). The generation of anaphylatoxins C3a and C5a further induces degranulation of mast cells, basophils and eosinophils. They also induce the expression of adhesion molecules on endothelial cells and cause smooth-muscle contraction (Klos et al., 2009).

1.3.1.2.2 Clotting or coagulation system

The mechanism of coagulation involves activation, adhesion and aggregation of platelets, along with the deposition and maturation of fibrin. Fibrin activation involves two pathways, the intrinsic and the extrinsic cascades. Following vascular injury, the extrinsic clotting cascade is triggered as activated endothelium expresses tissue factor (TF) on their luminal surfaces and expresses increased levels of plasminogen activator inhibitor-1 which inhibits fibrinolysis (Tan et al., 1999). In the presence of clot activating factors, prothrombin is converted to thrombin by prothrombinase. Thrombin in turn converts fibrinogen to fibrin, which is the major product involved in clot formation. The intrinsic cascade occurs with the engagement of Hageman factor (factor XII) which interacts with factors Va and VIIIa and becomes converted to its active form, factor XIIa. Factor XIIa in turn activates factor Xa which directly converts prothrombin to thrombin and the subsequent generation of fibrin

from fibrinogen (Ward, 2010). Fibrin is deposited on aggregated platelets to bring about thrombus formation.

1.3.1.2.3 Fibrinolytic system

The fibrinolytic system acts in opposition to the coagulation system, to counterbalance clotting. It is activated by urinary plasminogen activator (uPA) and tissue plasminogen activator (tPA) which converts plasminogen to plasmin. Plasmin directly interacts with fibrin to bring about the breakdown of fibrin clots as they are formed within the intravascular compartment (Ward, 2010).

1.3.1.2.4 Kinin System

Kinins are vasoactive peptides generated through the kinin-generating cascade. The most important product of this cascade is the plasma protein, bradykinin. Prekallikrein is converted to the protease, kallikrein by factor XIIa from the clotting cascade. Kallikrein interacts with high molecular weight kininogen (HMWK) to bring about the hydrolysis and release of bradykinin (Stankov, 2012). Bradykinin is a powerful vasopermeability agent. It also causes pain, vasodilatation and oedema, all contributing to inflammation.

Figure 4: Intercommunication between inflammatory cascade and the coagulation cascade.

Source: (Ward, 2010)

1.3.2 Cells associated with inflammation

The cellular component involves leukocytes, which normally reside in blood and must move into the inflamed tissue via extravasation to aid in inflammation. Some act as phagocytes, ingesting bacteria, viruses, and cellular debris. Others release enzymatic granules that damage pathogenic invaders. Leukocytes also release inflammatory mediators that develop and maintain the inflammatory response. In general, chronic inflammation is mediated by agranulocytes such as monocytes and lymphocytes whereas acute inflammation is mediated by granulocytes.

1.3.2.1 Agranulocytes

These leukocytes are characterised by the apparent absence of granules in their cytoplasm are referred to as agranulocytes. Among these categorization includes the macrophages, monocytes and lymphocytes.

1.3.2.1.1 Macrophages

Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the types of proteins specific to the surface of healthy body cells on its surface in a process called phagocytosis. They are found in essentially all tissues (Ovchinnikov, 2008) where they patrol for potential pathogens by amoeboid movement. They play a critical role in non-specific defense (innate immunity), and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. In humans, dysfunctional macrophages cause severe diseases such as chronic granulomatous disease that result in frequent infections. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called M1 macrophages, while those that decrease inflammation and encourage tissue repair are known as M2 macrophages (Mills, 2012). This difference is reflected in their metabolism, M1 macrophages have the unique ability to metabolize arginine to the killer molecule nitric oxide, whereas M2 macrophages have the unique ability to metabolize arginine to the repair molecule ornithine. Human macrophages are produced by the differentiation of monocytes in tissues. Macrophages are essential for wound healing. They replace polymorphonuclear neutrophils as the predominant cells in the wound two days after injury. The main role of macrophages is to phagocytize bacteria and damaged tissue, and they also debride damaged tissue by releasing proteases. Macrophages also secrete growth factors and other cytokines, especially during the third and fourth post-wounding days. These factors attract cells involved in the proliferation stage of healing to the area. Macrophages may also restrain the contraction phase (Newton et al., 2004). Macrophages are stimulated by the low oxygen content of their surroundings to produce factors that induce and speed angiogenesis (Greenhalgh, 1998) and they also stimulate cells that re-epithelialize the wound, create granulation tissue, and lay

down a new extracellular matrix (Stashak et al., 2004). By secreting these factors, macrophages contribute to pushing the wound healing process into the next phase.

1.3.2.1.2 Monocytes

Monocytes play multiple roles in immune function. Such roles include replenishing resident macrophages under normal states and in response to inflammation signals, monocytes can move quickly (approximately 8–12 h) to sites of infection in the tissues and differentiate into macrophages and dendritic cells to elicit an immune response. Half of them are stored in the spleen (Swirski et al., 2009). Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter. Microbial fragments that remain after such digestion can serve as antigens. The fragments can be incorporated into MHC molecules and then trafficked to the cell surface of monocytes (and macrophages and dendritic cells). This process is called antigen presentation and it leads to activation of T lymphocytes, which then mount a specific immune response against the antigen. Antimicrobial activity of monocytes includes oxygen-dependent mechanisms such as the respiratory burst, which through a complex series of reactions forms highly reactive hydroxyl radicals that damage host and microbial membranes.

1.3.2.1.3 Lymphocytes

A lymphocyte is one of the subtypes of white blood cell in a vertebrate's immune system. They consists of natural killer cells (NK cells) (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). They are the main type of cell found in lymph, which prompted the name lymphocyte. The three major types of lymphocyte are T cells, B cells and natural killer (NK) cells. Lymphocytes can be identified by their large nucleus.

1.3.2.1.3.1 T-cells and B-cells

T cells (thymus cells) and B cells (bone marrow- or bursa-derived cells) are involved in the acquired or antigen-specific immune response given that they are the only cells in the organism able to recognize and respond specifically to each antigenic epitope. The B Cells have the ability to transform into plasmocytes and are responsible for producing antibodies

(Abs). Thus, humoral immunity depends on the B Cells while cell immunity depends on the T Cells (Yang et al., 2010). B cells respond to pathogens by producing large quantities of antibodies which then neutralize foreign objects like bacteria and viruses. In response to pathogens some T cells, called T helper cells, produce cytokines that direct the immune response, while other T cells, called cytotoxic T cells, produce toxic granules that contain powerful enzymes which induce the death of pathogen-infected cells. Following activation, B cells and T cells leave a lasting legacy of the antigens they have encountered, in the form of memory cells. Throughout the lifetime of an animal, these memory cells will remember each specific pathogen encountered, and are able to mount a strong and rapid response if the pathogen is detected again.

1.3.2.1.3.2 Natural killer cells

Natural killer (NK) cells are part of the innate arm of the immune system. Their function is to eliminate aberrant cells, including virally infected and tumorigenic cells. For this purpose NK cells store cytotoxic proteins within secretory lysosomes, specialized exocytic organelles that are also known as lytic granules. NK cells are believed to be relatively short-lived, and at any one time there are likely more than 2 billion circulating in an adult (Blum and Pabst, 2007). NK cells distinguish infected cells and tumors from normal and uninfected cells by recognizing changes of a surface molecule called MHC (major histocompatibility complex) class I. NK cells are activated in response to a family of cytokines called interferons. Activated NK cells release cytotoxic (cell-killing) granules which then destroy the altered cells (Janeway et al., 2001). They were named natural killer cells because of the initial notion that they do not require prior activation in order to kill cells which are missing MHC class I.

1.3.2.2 Granulocytes

These WBCs are characterised by the presence of granules within their cytoplasm. Granulocytes comprise of mast cells, neutrophils, T-cells and B-cells, eosinophils and basophils.

1.3.2.2.1 Mast cells

Mast cells are found in mucosal and epithelial tissues, surrounding blood cells, smooth muscle, and hair follicles throughout the body and in all vascularized tissues except for the central nervous system and the retina (da Silva et al., 2014). They are also seen in tissues surrounding digestive tract, conjunctiva, nose and mouth (Prussin and Metcalfe, 2003). Mast cells are located at the junction point of the host and external environment at places of entry of antigen such as gastrointestinal tract, skin, respiratory epithelium (Metcalfe and Boyce, 2006; Jamur et al., 2005). The cytoplasm of the mast cell contains 50–200 large granules that store inflammatory mediators, including histamine, heparin, a variety of cytokines, chondroitin sulfate, and neutral proteases (da Silva et al., 2014). Mast cell is derived from the myeloid stem cell. It contains many granules rich in histamine and heparin. In order for mast cells to migrate to their target locations, the coordinated effects of integrins, adhesion molecules, chemokines, cytokines, and growth factors are necessary (Collington et al., 2011). They participate in wound healing, angiogenesis, defense against pathogens and blood-brain barrier function (da Silva et al., 2014; Polyzoidis et al., 2015). They are also known to play roles in allergy and anaphylaxis. Mast cells look very much like basophils but are different cells as they develop from different hematopoietic stem cells. Unlike basophils, which leave the bone marrow in the mature form, mast cells circulate in an immature form and only mature when they get to their target tissue site (Prussin and Metcalfe, 2003). Mast cells play important roles in the inflammatory process. They release histamine which dilates post- capillary venules, activates the endothelium, increases blood vessel permeability leading to local oedema and also depolarizes the nerve endings leading to pain or itching. In the skin, antigens, via IgE, activate mast cells in the deep layers of connective tissue. Mast cells release histamine as well as other vasoactive molecules, which cause urticaria (hives).

1.3.2.2.2 Neutrophils

Neutrophils are a type of phagocyte and are normally found in the bloodstream. During the acute phase of inflammation, particularly as a result of bacterial infection, environmental exposure (Jacobs et al., 2010) and some cancers (Waugh and Wilson, 2008; De Larco et al., 2004), neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation. They migrate through the blood vessels, then through interstitial

tissue, following chemical signals such as Interleukin-8 (IL-8), C5a, fMLP and Leukotriene B4 in a process called chemotaxis. They are the predominant cells in pus, accounting for its whitish/yellowish appearance. Neutrophils are recruited to the site of injury within minutes following trauma, and are the hallmark of acute inflammation (Cohen and Burns, 2002). However, due to some pathogens being indigestible, they can be less useful alone. With the eosinophil and the basophil, they form the class of polymorphonuclear cells.

1.3.2.2.3 Basophils

Basophils arise and mature in bone marrow (Voehringer, 2009). When activated, basophils degranulate to release histamine, proteoglycans (e.g. heparin and chondroitin), and proteolytic enzymes (e.g. elastase and lysophospholipase). They also secrete lipid mediators like leukotrienes (LTD-4), and several cytokines (Nakanishi, 2010). Histamine and proteoglycans are pre-stored in the cell's granules while the other secreted substances are newly generated. Each of these substances contributes to inflammation.

1.3.2.2.4 Eosinophils

Eosinophil granulocytes are white blood cells and one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Along with mast cells, they also control mechanisms associated with allergy and asthma. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood. These cells are eosinophilic or 'acid-loving' as shown by their affinity to coal tar dyes: Normally transparent, it is this affinity that causes them to appear brick-red after staining with eosin, a red dye, using the Romanowsky method. Eosinophils persist in the circulation for 8–12 h, and can survive in tissue for an additional 8–12 days in the absence of stimulation (Young et al., 2006).

1.3.2.3. Platelets

Platelets adhere to these sub-endothelial matrix proteins and become activated. Activated platelets lead to the formation of TXA2 which is a potent vasoconstrictor and stimulus of platelet aggregation. Thromboxanes are known toinduce blood vessel constriction and platelet aggregation by increasing intracellular calcium ion (Ca2+), which promote fusion of dense and alpha platelet granules with the platelet membrane, thus, releasing their contents.

Adenosine diphosphate (ADP) which also mediates platelet aggregation is released. Activated platelets also trigger the coagulation cascade leading to the formation of thrombin. Thrombin and other platelet agonists such as, ADP, PAF and adrenaline bind to specific receptors on platelet membrane and stimulate aggregation in which more platelets are recruited and begin to adhere to each other. This forms haemostatic plug onto which fibrin may be deposited, thus, stabilizing the clot formed. The degranulation of platelets also releases P-selectin and histamine. Histamine increases vascular permeability while P-selectin facilitates leukocyte migration from blood to tissues (Zarbock et al., 2006).

1.3.3 Classification of inflammation

Inflammation could be classified as either acute or chronic, depending on the type and duration of the antigen challenge and is mediated by some chemical substances such as histamine, serotonin, slow reacting substances of anaphylaxis (SRS-A), prostaglandins and some plasma enzyme systems such as the complement system, the clotting system, the fibronolytic system and the kinin system.

1.3.3.1 Acute inflammation

In a classical acute inflammatory response, cellular events are temporally activated. Acute inflammation is a rapid response characterized by classical symptoms of redness, heat and oedema. It is defined as a series of tissue responses that occur within the first few hours following injury. It is initiated by resident tissue macrophages, mast cells and endothelial cells (Anosike et al., 2012b). Upon initial challenge, protein exudation increases and polymorphonuclear leukocytes (neutrophils) accumulate in inflamed tissue. Neutrophils are the most prominent cells in acute inflammation (Phillipson and Kubes, 2011; Sakic et al., 2011). Neutrophil infiltration follows a rapid response from sentinel cells prestationed in the tissues at the time of injury, including macrophages and mast cells (Makriyannis and Nikas, 2011). As primary defenders, neutrophils transmigrate into tissues in large numbers to neutralize pathogens and promote the clearance of cellular and other debris by phagocytosis. As the lesion matures, neutrophils accumulate in the local tissue and die via apoptosis (programmed cell death). The initial accumulation of neutrophils is followed by a second wave of cellular infiltration, of mononuclear phagocytes (monocytes). Differentiation of monocytes into macrophages promotes the removal of apoptotic neutrophils and debris by

nonphlogistic phagocytosis. Macrophages that have completed the elimination of apoptotic neutrophils are cleared from the inflamed tissue either by egression to the lymphatic system or by apoptosis, by a process termed efferocytosis. This temporal regulation of inflammation requires that this conserved cellular phenotype is highly regulated to clear the original insult (Savill et al., 2002). Outcomes of acute inflammation include, complete resolution, healing by connective tissue replacement, abscess formation or progression to chronic inflammation. Failure to resolve the inflammatory response, or continuous activation of the responses, become harmful to the tissue and consequently develop into the chronic lesion that we call inflammatory diseases. Diseases associated with uncontrolled acute inflammation are characterized by a lack of activation of resolution programs and by the inappropriate release and maintenance of high levels of toxic substances and pro-inflammatory mediators, which may result in damage to host tissues and prolong the inflammatory response (Bannenberg et al., 2005).

1.3.3.1.1 Mechanism of acute inflammation

Inflammation consists of a tightly regulated cascade that is orchestrated by cytokines. Recognition of antigen is the first step of the inflammatory cascade. This is achieved by innate immune system as it detects a broad range of molecular patterns called pathogen- associated molecular patterns (PAMPs) that are commonly found on pathogens but are foreign to the host, (Janeway and Medzhitov, 2002). Alarmins or damage-associated molecular patterns (DAMPs) which are endogenous molecules generated in response to a sterile injury, such as burn, hypoxia or chemical insult are also recognized by the innate immune system (Bianchi, 2007; Osterloh et al., 2009). Detecting these signals helps in minimizing inadvertent targeting of host cells and tissues. These damage signals are recognized by tissue-resident cells such as macrophages and mast cells, through multi-ligand pattern-recognition receptors (PRRs) expressed on their surfaces (McGhan and Jaroszewski, 2011). Examples of these receptors are transmembrane toll-like receptors (TLRs) and NOD- like receptors (NLRs). Transmembrane TLRs recognize and bind to PAMPs while intracellular NLRs bind DAMPs.

Ligation of PRRs leads to the activation of signal transduction pathways that regulate diverse transcriptional and post-transcriptional processes. For example, TLRs couple to the adaptor

protein, myeloid differentiation primary response gene 88 (MyD88), resulting in activation of nuclear factor kappa β (NF-κβ) and mitogen-activated protein kinase (MAPK) pathways. These in turn control the activities of multiple signal dependent transcription factors that include members of the NF-κβ, activator protein-1 (AP-1) and interferon regulator factor (IRF) families (Glass et al., 2010). Nuclear factor kappa β is found in virtually all cell types and remains in an inactive state bound to an inhibitor protein known as, inhibitor of kappa β (Iκβ). Upon transduction of the signal, NF-κβ is released from Iκβ through the phosphorylation of Iκβ by inhibitor of kappa β kinase (IKK). Active NF-κβ translocates to the nucleus, where transcription is upregulated through binding to target inflammatory genes (Ashley et al., 2012).

Transcription and translation of genes lead to the third stage of the inflammatory cascade, which is the inducible expression of pro-inflammatory cytokines, such as interleukin-1-beta (IL- 1β), interleukin-6 (IL-6), TNF-α, IFN-γ and chemokines. Several pro-inflammatory proteins such ascyclooxygenase-2 (COX-2) and iNOS are also expressed (Russo-Marie, 2004). NOD-like receptors on the other hand, signal the inflammasome, which activates caspase-1 to convert cytokines into active forms (Martinon et al., 2009). In conjunction with chemokines, these cytokines facilitate the recruitment of effector cells, such as blood-borne neutrophils and monocytes, to the inflammatory site by chemotaxis. Neutrophils then eliminate pathogens usingdegranulation, NETs and phagocytosis. Macrophages and dendritic cells also participate in phagocytosis of antigen.

1.3.3.1.2 Responses in acute inflammation

Acute inflammation involves vascular and cellular responses. The vascular responses are vasodilatation and increased vascular permeability, while the cellular component involves the emigration of leukocytes from the vascular to the extravascular compartment.

1.3.3.1.2.1 Vasodilatation

This involves changes in the vascular caliber leading to increased blood flow to tissues. It is caused by arteriolar dilation which sometimes occurs after transient vasoconstriction, resulting in heat and redness. It is also due to the opening of new capillaries. It occurs

primarily as a result of histamine and NO acting on the vascular smooth muscle (Ferrero- Miliani et al., 2007).

1.3.3.1.2.2 Increased vascular permeability

This involves the contraction of endothelial cells, leading to the escape of plasma protein-rich fluid known as exudates from the vascular compartment into the extravascular space. This process is called exudation. It leads to increased extravascular osmotic pressure leading to oedema (Stankov, 2012).

Figure 5: Overview of vascular changes in acute inflammation (Levison et al., 2008)

This vascular leakage of exudates can be chemically mediated or injury induced. Chemical mediation involves the retraction of endothelial cells by mediators of acute inflammation such as histamine, bradykinins, LTs and PGs released by mast cells and tissue resident macrophages. These mediators bind to their receptors on endothelial cells leading to vasodilatation, contraction of endothelial cells and widening of intercellular junctions (Da Silveira e Sá et al., 2013). Injury induced vascular leakage is an abnormal leakage caused by toxins and physical agents as they may cause necrosis of the vascular endothelium.

1.3.3.1.2.3 Neutrophil extravasation

This involves the movement of leukocytes from the circulation to the site of inflammation. All granulocytes and monocytes respond to chemotactic factors and move along a concentration gradient (Cicchetti et al., 2002). Like the leakage of fluids, the delivery of leukocytes to inflammatory sites is assisted by the release of vasodilators, such as histamine and PGs. Neutrophil extravasation involves, margination, tethering and rolling, stable arrest and adhesion, diapedesis and migration in interstitial tissues.

Margination: Normally, PMNs and other leukocytes are carried in the blood stream without making contact with endothelial surfaces. Margination involves the exit of free-flowing leukocytes from the central blood stream at sites of inflammation leading to leukocyte and endothelial cell interactions by close mechanical contact (Ley et al., 2007). This is made possible as stasis develops.

Tethering and rolling: Tethering is the repeated formation and breaking of transient adhesion bonds between the luminal surface of activated vascular endothelium and leukocytes under the prevailing hydrodynamic forces of fluid flow (Kolaczkowska and Kubes, 2013). The cell adhesion molecules (CAMs) for tethering and the initiation of rolling are the selectins. The detection of pathogens by PRRs increases the expression of CAMs such as P-selectin and E-selectin on the endothelial surface, leading to their activation. The inflammatory response is maintained by upregulating the production of E-selectin and keeping the expression of P-selectin due to the binding of cytokines released by activated macrophages and vascular endothelial cells to receptors on endothelial cells (Zarbock et al., 2006). Neutrophils not only undergo rolling on activated endothelium but also on other neutrophils already stably adherent to the endothelial surface. These neutrophil-neutrophil adhesive interactions are mediated by L-selectin binding to PSGL-1.

Stable arrest and adhesion: For rolling leukocytes to form more stable or tight adhesion bonds, and thus arrest on the endothelial surface despite the prevailing force of fluid flow, leukocyte integrins have to be activated (Phillipson and Kubes, 2011). The rolling of leukocytes facilitates their contact with chemokines generated by activated endothelium and lining its luminal part, thus inducing conformational changes of leukocytes surface integrins, thereby activating them. Neutrophils are known to constitutively express only β2 integrins

while monocytes and lymphocytes express both β1 and β2 integrins (Tan et al., 1999). β2 integrins include, neutrophil lymphocyte function associated antigen-1 (LFA-1 or CD11a/CD18) and macrophage-1 antigen (MAC-1 or CD11b). Endothelial cells also undergo activation by expressing on their luminar faces, CAMs for leukocytes. Examples of these molecules are immunoglobulin-like CAMs such as vascular-cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1). Stable arrest in neutrophils and monocytes can thus be mediated by β2 integrin-ICAM-1 adhesion. The ligation of integrins with their ligands activates signalling pathways inside the neutrophil, thus stabilizing adhesion and initiating cell motility (Cicchetti et al., 2002; Ley et al., 2007).

Diapedesis: This is also known as transmigration or emigration. The adhesion step of the recruitment cascade prepares leukocytes for transmigration, but this does not necessarily occur at the initial site of their arrest on the endothelium. Stably adherent leukocytes thus, flatten and spread on the luminal surface of the endothelium and crawl towards intercellular borders by extending their pseudopods without being swept away by fluid flow (Kolaczkowska and Kubes, 2013). The crawling of leukocytes is made possible through the coordinated formation and breaking of adhesion bonds. At the intercellular border, leukocytes extend pseudopods between endothelial cells and transmigrate into the extravascular tissue spaces. In order to leave the vasculature, leukocytes must first cross the endothelium, and then the basement membrane (Ley et al., 2007). Leukocytes migrate through regions of basement membrane with low expression of ECM components (Wang et al., 2006). As the leukocyte leaves the vessel, it expresses β1 integrins that help it bind to ECM proteins in the tissue. Transmigration thus requires integrins as well as different junctional proteins such as platelet/endothelial cell adhesion molecule-1 (PECAM-1 or CD31) expressed on both the endothelium and the leukocytes i.e., CD31-CD31 adhesive interactions (Kolaczkowska and Kubes, 2013). Chemoattractants generated at the infected site may promote diapedesis by inducing chemotaxis or by integrin activation (Tan et al., 1999).

Migration in interstitial tissues: Emigrated leukocytes initially follow a subluminal chemokine gradient that is deposited by endothelium. These chemokines however becomes inactive by binding to scavenger receptors found on the endothelium. Functionally active chemokines and other chemoattractants are thus secreted by tissue macrophages and non- vascular cells such as fibroblasts (Kolaczkowska and Kubes, 2013). Examples include,

soluble bacterial products (e.g. endotoxin and formylmethionyl peptides), C5a, LTB4 and IL- 8. These chemoattractants induce chemotaxis and activation of extravasated leukocytes, which are induced to carry out their respective effect or functions such as cytokine secretion, phagocytosis and degranulation (Cicchetti et al., 2002; Eming et al., 2007).

Figure 6: The multistep process of leukocyte migration through the blood vessels Source: (Ley et al., 2007)

1.3.4 Resolution of the acute inflammatory response

Resolution of the acute inflammatory response is an active biochemical process that is important to protect the host from overt tissue damage and amplification of the acute inflammatory response towards chronicity (Serhan, 2010). It is also critical for successful repair after tissue injury. After the first few hours of inflammation, a coordinated program of resolution is set into motion by tissue-resident and recruited macrophages. The activation of macrophages during the inflammatory process must be tightly regulated in order to avoid unrestrained inflammatory process through inappropriate release of cytokines such as TNF-α, IFN-γ and IL-1 (Sakic et al., 2011). Macrophages therefore liberate the anti-inflammatory cytokine, TGF-β which inhibits the production of TNF-α. Resolution of inflammation also

involves upregulating anti-inflammatory molecules, like IL-1 receptor antagonist or soluble TNF-α receptor (Eming et al., 2007). Cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-12 (IL-12) in very low concentrations are inducible, and act as powerful anti-inflammatory mediators by stabilizing Iκβ, which blocks NF-κβ activation (Shaikh, 2011). These regulatory cytokines therefore greatly diminish the production of pro- inflammatory mediators, downregulate the expression of chemokines and thus reduce the numbers of leukocytes accumulating in tissues (Iwalewa et al., 2007).

Lipoxins (LXs), produced from arachidonic acid through the 5-LOX pathway are antiinflammatory. During the onset of acute inflammation, tissue-resident and recruited macrophages in the inflamed tissue, including PMNs produce pro-inflammatory lipid mediators (e.g. PGE2 and LTB4). With time, a lipid mediator switch occurs, and these cells begin to produce lipoxin A4 (LXA4) which is a pro-resolving mediator (Serhan et al., 2008; Serhan, 2010). Lipoxin A4 blocks neutrophil recruitment, initiates non-phlogistic recruitment of monocytes and the phagocytosis of apoptotic neutrophils by macrophages (Kolaczkowska and Kubes, 2013). Closely related to LXA4 are lipid mediators synthesized from omega-3 polyunsaturated fatty acids (PUFAs) that stop neutrophil migration. They include resolvins, protectins and maresins. Resolvins and protectins are produced by neutrophils while maresins are produced by macrophages (Serhan, 2010). Leukocytes are cleared in tissues via lymphatics or by apoptosis. This is because apoptotic PMNs releases signals that attract monocytes (Soehnlein and Lindbom, 2010) which phagocytose the PMNs, cellular debris and red blood cells (RBCs) in the extravascular compartment (Canturk et al., 2001).

Apoptosis of neutrophils is promoted by resolvins and protectins. Some of the neutrophils do not die in the tissue but are reverse-transmigrated (re-enter the vasculature) because they are more resistant to apoptosis compared with other extravasated neutrophils. Reverse- transmigrating neutrohils could be a way of preserving neutrophils whenthey are not needed to fight infection (Kolaczkowska and Kubes, 2013). Increased vascular permeability is reversed due to closure of the open endothelial cell-cell junctions, ceasing the escape of exudates and leukocyte emigration from the blood compartment (Serhan and Savil, 2005). Exudates are also cleared by the uptake of the fluids together with inflammatory cells into the draining lymphatics (Ward, 2010) or they pinocytose into macrophages. They could also be cleared through the hydrolysis of kinins by kininases, thereby preventing their

vasopermeability effect. Thrombosis within vessels can be cleared by the activation of the fibrinolytic system.

1.4 Chronic inflammation

Chronic inflammation involves active inflammation, characterized by simultaneous destruction and attempts at repair of the tissue (Eming et al., 2007; Ferrero-Miliani et al., 2007). Sustained inflammation resulting in tissue pathology implies the persistence of an inflammatory stimulus or a failure in normal resolution mechanisms in which the acute phase responses are sustained and disease progression ensues. Chronic inflammation is associated with many human diseases including, allergy, cancer, arthritis, diabetes, cardiovascular, neurological and autoimmune diseases (Glass et al., 2010). It is often due to infections with higher order organisms such as mycobacteria, fungi and metazoan parasites which are resistant to killing and clearing by the body. It is also due to immune complexes in autoimmune diseases and prolonged exposure to toxins. Chronic inflammation leads to a progressive shift in the type of cells present at the site of inflammation (Eming et al., 2007; Ferrero-Miliani et al., 2007).

It is associated with the infiltration of mononuclear cells such as lymphocytes, macrophages and plasma cells (Vishal et al., 2014) due to the persistent stimulus. Plasma cells produce antigen-specific antibodies in a bid to clear the persistent antigen. Apart from recruited macrophages, there is also local proliferation of macrophages at the inflammatory site leading to their accumulation. These macrophages are activated by endotoxin, ECM proteins and chemical mediators. Activated macrophages present processed antigen fragments on their surface and interact with T-lymphocytes leading to the activation of the lymphocytes (Dalgleish and O’Byrne, 2002). Monokines from activated macrophages also activate lymphocytes. Activated lymphocytes releases IFN-γ which activates additional macrophages, leading to persistence of the inflammatory response.

Activated macrophages releases proteases (e.g. metalloproteinases), cytokines, coagulation factors, lipid mediators, ROS and reactive nitrogen species (RNS) that may amplify the inflammatory response and injure surrounding tissues (Cunha et al., 2007). Endothelial cells and fibroblasts are also potential sources of ROS. In addition to direct damage of cell membranes and structural proteins of the ECM, ROS can selectively affect signalling

pathways leading to the activation of transcription factors that control the expression of pro- inflammatory cytokines and chemokines (Wenk et al., 2001) leading to further tissue necrosis. As a consequence of the highly proteolytic microenvironment, there is an unbalanced proteolytic activity, which overwhelms local tissue protective mechanisms (Eming et al., 2007). Mediators and growth factors crucial for repair thus, become targets of these proteases. Chronic inflammation is also associated with attempts at healing by tissue regeneration or with fibrosis (Kumar et al., 2013). Fibrosis involves fibrous connective tissue replacement of damaged tissue when regeneration cannot be accomplished. It involves angiogenesis, migration and proliferation of fibroblasts, and deposition of ECM especially collagen. This is followed by the appearance of granulation tissue and the subsequent formation of scar tissue. Activated macrophages lead to repair due to the production of growth factors (PDGF, bFGF, and TGF-β), fibrogenic cytokines, angiogenesis factors and remodelling collagenases (Eming et al., 2007).

1.4.1 Types of chronic inflammation

The two major types of chronic inflammation are nonspecific proliferative and Granulomatous inflammation.

1.4.1.1 Non-specific proliferative

This is characterized by the presence of non-specific granulation tissue formed by infiltration of mononuclear cells (lymphocytes, macrophages, plasma cells) and proliferation of fibroblasts, connective tissue, vessels and epithelial cells, for example, an inflammatory polyp-like nasal or cervical polyp and lung abscess.

1.4.1.2 Granulomatous inflammation

This is a specific type of chronic inflammation characterized by the presence of distinct nodular lesions or granulomas formed with an aggregation of activated macrophages or its derived cell called epithelioid cells usually surrounded by lymphocytes. The macrophages or epithelioid cells inside the granulomas often coalesce to form Langhans or giant cells such as foreign body, Aschoff, Reed-Sternberg and Tumor giant cells. There are two types: i) Granuloma formed due to a foreign body or T-cell mediated immune response is termed as foreign body granuloma, for example, silicosis

ii) Granulomas that are formed from chronic infection is termed as infectious granuloma, for example, tuberculosis and leprosy.

1.4.2 Inflammatory response in chronic inflammation

The primary goal of the inflammatory response is to detect and eliminate factors that interfere with homeostasis. A typical inflammatory response consists of four components: inflammatory inducers; the detecting sensors; downstream mediators; and the target tissues that are affected. The type and the degree of inflammatory response activated is dependent on the nature of the inflammatory trigger (bacterial, viral or parasitic) and its persistence (Medzhitov, 2008).

1.5 Inflammatory disorders

The association of inflammation with modern human diseases such as acne vulgaris, asthma, autoimmune diseases, coeliac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases (crohn’s disease), pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis and interstitial cystitis, obesity, cardiovascular disease, type 2 diabetes mellitus and cancer remains an unsolved mystery of current biology and medicine. The clinical symptoms associated with these disorders such as fever and pains are directly or indirectly due to inflammatory process. The immune system is often involved in inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation. Non-immune diseases with aetiological origins in inflammatory processes include cancer, atherosclerosis, and ischaemic heart disease (Cotran et al., 1999). A large variety of proteins are involved in inflammation, and any one of them is open to a genetic mutation which impairs the normal function and expression of that protein.

1.6 Anti-inflammatory agents

The use of anti- inflammatory agents for the symptomatic relief of infection dates back to the use of Aspirin to reduce fever (Mayer and Johnson, 2000). Early intervention with a selective or a combination of an appropriate agent at different time will reduce inflammation, preserve organ function and will lead to increased survival rate. There are various drugs that are used

in the treatment of inflammation. These are grouped into steroidal (corticosteroids) and the non-steroidal anti-inflammatory drugs.

1.6.1 Corticosteroids

Corticosteroids are a class of steroid hormones that are produced in the adrenal cortex of vertebrates, as well as the synthetic analogues of these hormones. They are involved in a range of physiological processes including immune response, stress response and regulation of inflammation amongst others. They are of two types; the glucocorticoids (GCs) and mineralocorticoids. The glucocorticoids such as cortisol are actually involved in the inhibition of inflammation (Joseph, 2008).

Figure 7: Chemical structures of prednisolone and deflazacort

Source: (Scremin et al., 2010)

They inhibit PLA2 activity which in turn reduces arachidonic acid release. This they do indirectly by causing the release of the inhibitory protein, lipocortin (Schimmer and Parker,

2001) which binds to the cell membrane, preventing PLA2 from coming in contact with arachidonic acid. By inhibiting PLA2 activity, corticosteroids neutralize the two main pathways of the arachidonic acid cascade (COX and LOX pathways) thereby preventing the formation of PGs, TXs and LTs which are inflammatory mediators. Some examples of corticosteroid are deflazacort and prednisolone (Dinarello, 2010; Czaja, 2012). Some side- effects associated with the use of corticosteroids which are often severe or irreversible

include, bone damage, skin thinning, immune suppression and propensity to infection, gastrointestinal ulcers and bleeding (Rainsford, 2007).

1.6.2 Non-steroidal anti-inflammatory drugs (NSAIDs)

NSAIDs are defined as a diverse group of drugs with the shared ability to relieve pain(analgesic), decrease inflammation (anti-inflammatory), decrease elevated body temperature(antipyretic) and decrease blood clotting (non selective NSAIDs only) by inhibition of platelet aggregation (Gorelick et al., 2003) without the immunosuppressive and metabolic side effects associated with corticosteroids. They are different from the steroids but have similar eicosanoid depressing, anti-inflammatory action (Buer, 2014). Traditional non- selective NSAIDs are used to prevent recurrent stroke and evidence is growing for the ability of selective NSAIDs to delay progression of Alzheimer’s disease. NSAIDs also exhibit uricosuric effects beneficial to management of gouty arthritis, increase plasma volume and cardiac output and work, stimulate respiration and respiratory alkalosis, and in large doses may cause hyperglycemia. Nonsteroidal agents are distinguished from true steroid agents, such as cortisone (cortisol), prednisone, triamcinolone, or methylprednisolone, and from the opiate-derived analgesics such as codeine, oxycodone, morphine sulfate, and meperidine (Aisen et al., 2003).

Examples of NSAIDs include aspirin, ibuprofen, naproxen, indomethacin, diclofenac etc. The classification of NSAIDs is applied to drugs that inhibit one or several steps in the metabolism of arachidonic acid. NSAIDs selectively inhibit cyclooxygenase (COX), but have little or no effect on lipoxygenase which generates leukotrienes.COX has two isoforms namely COX-1 and COX-2. COX-1 which is expressed virtually in all tissues of the body, catalyzes the formation of constitutive PG (PGE2, and PGI2), which mediates a wide range of normal physiologic effect. COX-1 activation also leads to the production of prostacyclin which when released by the vascular endothelium is anti-thrombogenic (Botting, 2006) and when released by the gastric mucosa is cytoprotective. It is also COX-1 in the platelet that leads to thromboxane A2 production, causing aggregation of the platelets to prevent inappropriate bleeding (Funk et al., 1991). COX-2 the second isoform is activated is activated in damaged and inflamed tissues and catalyzes the formation of inducible PG, including PGE2 which is linked with intensifying inflammatory response (Topper et al.,

1996). It is also involved in thermoregulation and the pain response to injury. COX-2 inhibition by NSAIDs is thought to be responsible for the analgesic, and anti-inflammatory actions of NSAID (Breden et al., 1995). Both COX-1 and COX-2 play a role in nociception (Ballou et al., 2000).

NSAIDs Inhibiting COX-1 and COX-2 have side effects especially during long therapy duration and at high doses. Some NSAIDs increase the risk of myocardial infarction and stroke (Kearney et al., 2006; Trelle et al., 2011), increased risk of erectile dysfunction (Shiri et al., 2006). Group of NSAIDs that interfere with the normal housekeeping function of COX-1 will result in a serious side effect such as peptic ulcer disease (Cristina et al., 2002) and bleeding, which causes between 10 and 25% morbidity and mortality (Shah et al., 2001). However, over expression of COX-2 in epithelial cells inhibits apoptosis and increases the invasiveness of tumor cells. This complication led to development of COX-2–specific inhibitors. Therefore, a drug that inhibits COX-2 at a lower concentration than that necessary to inhibit COX-1 might be considered safer (Fitzgerald, 2003). Selective COX-2 inhibitors such as Celecoxib (celebrex) and reecoxib (vioxx), prevents the synthesis of anti-thrombotic prostagalndins, prostacyclin by endothelial cells while leaving unopposed, the action of prothrombothic thrombaxane in platelets (Brunton et al., 2006).

Figure 8: Chemical structures of common non-steroidal anti-inflammatory drugs

Source: (Stolfi et al., 2013)

1.7 Aim and Objectives of the Study

1.7.1 Aim of the Study

The aim of this research was to investigate the anti-inflammatory and analgesic activities of the ethanol extract of stem-bark of Hymenodictyon pachyantha. 1.7.2 Specific objectives of the study

1) To determine the qualitative and quantitative phytochemical constituents of ethanol extracts of H. pachyantha stem-bark.

2) To determine the median lethal dose (LD50) of ethanol extract of H. pachyantha stem- bark. 3) To determine the effect of ethanol extract of H. pachyantha stem-bark on egg albumin-induced rat paw oedema.

4) To determine the effect of ethanol extract of H. pachyantha stem-bark on

phospholipase A2 activity. 5) To determine the effect of ethanol extract of H. pachyantha stem-bark on membrane stabilization. 6) To determine the effect of ethanol extract of H. pachyantha stem-bark on calcium

chloride (CaCl2)-induced platelet aggregatory response. 7) To determine the effect of ethanol extracts of H. pachyantha stem-bark on acetic acid- induced nociceptive response in mice.

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Plant material

Fresh stem-barks of Hymenodictyon pachyantha were collected, from Orba, Udenu Local Government Area of Enugu State, Nigeria. The stem-bark was authenticated by Mr. Alfred Ozioko of the Bioresources Development and Conservation Programme (BDCP) Research Centre, Nsukka, Enugu State.

2.1.2 Animals

Adult Wistar albino rats (40 males) weighing between 130 g and 240 g and mice weighing between 20 g and 40 g (38 males) were purchased from the animal house of Veterinary Medicine, University of Nigeria, Nsukka. The animals were acclimatized under standard laboratory condition in the animal house of the Department of Biochemistry for one week prior to the commencement of the experiment with a 12 h light and dark cycle and maintained on a regular feed (commercial chicken grower’s mash) and water ad libitum throughout the period of the experiment.

2.1.3 Bacterial organism

The fungal organism used was strains of Aspergillus niger. The organism was cultured and obtained from the Department of Microbiology, University of Nigeria, Nsukka.

2.1.4 Blood samples

The blood samples used for the membrane stabilization assay, platelet aggregation tests and phospholipase A2 activity were collected from apparently healthy individuals. They were free from drug treatments for at least 2 weeks before sample collection.

2.1.5 Drugs

Indomethacin (product of Yangzhou Pharmaceutical Co. Ltd.) was used as reference drug for paw oedema, membrane stabilization and anti-platelet aggregation assays. Prednisolone (product of Kunimed Pharmaceutical Ltd.) was used as the reference drug for the phospholipase A2 activity assay. Acetylsalicylic acid (Aspirin product of Juhel Pharmaceuticals Ltd.) was used as the reference drug for the analgesic activity test.

2.1.6 Chemicals and reagents

All chemicals used in this study were of analytical grade and products of May & Baker (England), BDH Chemicals limited (Poole, England), Sigma Chemical Company (U.S.A). Sterile distilled and deionised water was used in the preparation of the chemicals, reagents and drugs.

2.1.7 Equipment and instruments

Weighing balance (Vickas Ltd, England), mechanical grinder, Whatman® 1 filter paper, rotary evaporator (Sigma, India), water bath, oral intubation tube, UV-Vis spectrophotometer (Jenway 6305, China), centrifuge (Sigma-Aldrich, England), spatula, autoclave, electric blender (Kenwood, England), pH meter, mortar, pestle, measuring cylinder (Pyrex, England) and refrigerator (Thermocool, England).

2.2 Methods

2.2.1 Preparation of plant extract

Fresh Hymenodictyon pachyantha stem-bark was collected and washed. The sample was cut into pieces and air dried with regular turning to avoid decaying, until crispy. The dried stem- bark was pulverized into powdered form using a mechanical grinder. A known weight of the pulverized sample (1000 g) was macerated in 3.5 liters of absolute ethanol, vigorously shaken and allowed to stand for 72 h. The mixture was filtered using Whatman No.1 filter paper and the filtrate concentrated under reduced pressure using a rotary evaporator at 45oC to obtain the crude ethanol extract. The concentrated extract was stored in an air-tight container in a

refrigerator at 4oC until required. The percentage yield for the extract was calculated using the formula:

Percentage yield = Weight of crude extract × 100 Weight of dried pulverized sample

2.2.2 Qualitative phytochemical screening of ethanol extracts of H. pachyantha stem- bark

The qualitative phytochemical analyses were carried out using the method of Harbone (1998) and Trease and Evans (2002).

2.2.2.1 Test for alkaloids

A quantity of the sample (0.2 g) was boiled with 5 ml of 2% HCl on a steam bath. The mixture was filtered and 1 ml of the filtrate was treated with 2 drops of the Wagner’s reagents. A creamy white precipitate indicated the presence of alkaloids.

2.2.2.2 Test for flavonoids

A quantity of the sample (0.2 g) was heated with 10 ml ethyl acetate in boiling water for 3 min. The mixture was filtered, and the filtrate was used for the following tests: 4 ml of the filtrate was shaken with 1ml of 1% aluminium chloride solution and observed for light yellow colouration that indicated the presence of flavonoids.

2.2.2.3 Test for saponins

A quantity of the sample (0.1 g) was boiled with 5 ml of distilled water for 5 min. The mixture was filtered while still hot. The filtrate was used for the following tests. (i) Emulsion test: A quantity of the filtrate (1 ml) was added to two drops of olive oil. The mixture was shaken and observed for the formation of emulsion. (ii) Frothing test: A quantity, 1 ml of the filtrate was diluted with 4ml of distilled water. The mixture was shaken vigorously and then observed on standing for a stable froth.

2.2.2.4 Test for tannins

A quantity of the sample (2 g) was boiled with 5 ml of 45% ethanol for 5 min. The mixture was cooled and then filtered and the filtrate was treated with the following solutions. (i) Lead sub acetate solution: To 1 ml of the filtrate, 3 drops of lead sub acetate solution was added. A gelatinous precipitate indicated the presence of tannins. (ii) Bromine water: To 1 ml of the filtrate was added 0.5 ml of bromine water and then observed for a pale brown precipitate. (iii) Ferric chloride solution: a quantity, 1 ml of the filtrate was diluted with distilled water and then 2 drops of ferric chloride solution was added. A transient greenish to black colour indicated the presence of tannins.

2.2.2.5 Test for terpenoids and steroids

A known quantity, 9 ml of ethanol was added to 1 g of the sample and 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 1h, and the waxy matter filtered off. The filtrate was extracted with 2.5 ml of chloroform using a separating funnel. To 0.5 ml of the chloroform extract in a test tube was carefully added 1ml of concentrated sulphuric acid to form a lower layer. A reddish-brown interface showed the presence of steroids. Another 0.5 ml aliquot of the chloroform extract was evaporated to dryness on a water bath and heated with 3 ml of concentrated sulphuric acid for 10 min on water. A grey colour indicated the presence of terpenoids.

2.2.2.6 Test for phenols

To 0.2 g of the extract was added 2 ml of distilled water. Then 0.5 ml Na2CO3 and 0.5 ml Folin Ciocalteau reagent was subsequently added. Formation of a blue-green colour indicated the presence of phenol.

2.2.3 Quantitative phytochemical analysis

The quantitative phytochemical analysis of ethanol stem-bark extract of Hymenodictyon pachyantha using standard conventional protocols as illustrated by Harborne (1998); Evans

and Trease (2002). The concentration of the various phytochemical constituents were calculated thus:

Concentration (mg/100 g) =

Where,

2.2.3.1 Estimation of the concentration of flavonoids

The extract (1 g) was macerated in 20 ml of ethylacetate and filtered. To 5 ml of the filtrate was added 5 ml of 28% dilute ammonia. The mixture was shaken, the upper layer collected and its absorbance measured at 490 nm.

2.2.3.2 Estimation of the concentration of terpenoids

The extract (1 g) was macerated in 20 ml of ethanol and filtered. To 1 ml of the filtrate was added 1 ml of 5% phosphomolybdic acid solution. Concentrated sulphuric acid (1 ml) was gradually added and mixed. The mixture was allowed to stand for 30 min followed by the addition of 2 ml of ethanol. The absorbance was read at 700 nm.

2.2.3.3 Estimation of the concentration of steroids

The extract (1 g) was macerated in 20 ml of ethanol and filtered. To 2 ml of the filtrate, 2 ml of colour reagent was added and the solution left to stand for 30 min. The absorbance was read at 550 nm.

2.2.3.4 Estimation of the concentration of saponins

The extract (1 g) was macerated in 20 ml of petroleum ether and decanted into a beaker. The extract was washed again with 10 ml of petroleum ether. The filtrates were combined and evaporated to dryness. The residue was dissolved with 6 ml of ethanol after which 2 ml of it was transferred into a test tube and 2 ml of colour reagent added. This was allowed to stand for 30 min and the absorbance measured at 550 nm.

2.2.3.5 Estimation of the concentration of alkaloids

The extract (1 g) was macerated in 20 ml of ethanol and 20% sulphuric acid (1:1) and filtered. To 1 ml of the filtrate was added 5 ml of 60% sulphuric acid. After 5 min, 5 ml of 0.5% formaldehyde in 60% sulphuric acid was added and thoroughly mixed. This was allowed to stand for 3 h after which the absorbance was measured at 565 nm.

2.2.3.6 Estimation of the concentration of tannins

The extract (1 g) was macerated in 20 ml of distilled water and filtered. To 5 ml of the filtrate, 0.3 ml of 0.1N ferric chloride in 0.1N hydrochloric acid and 0.3 ml of 0.0008 M potassium ferricyanide were added. The absorbance was read at 720 nm.

2.2.3.7 Estimation of the concentration of phenols

The extract (1 g) was macerated in 20 ml of 80% ethanol and filtered. To 5 ml of the filtrate was added 0.5 ml of Folin-Ciocalteu’s reagent and allowed to stand for 2 min. This was followed by the addition of 2 ml of 20% sodium carbonate. The absorbance was measured at 650 nm.

2.2.4 Acute toxicity and lethal dose determination

Investigation on acute toxicity of the extract with estimation of the median lethal dose (LD50) was carried out using the modified method of Lorke (1983). This study was done only in two phases and a total of eighteen (18) mice were used. Six (6) groups of three (3) mice each were administered orally, doses of ethanol extract (10, 100 and 1000 mg/kg body weight) respectively for the first phase and (1900, 2600 and 5000 mg/kg b.w of the extract) for second phase by oral intubation. The mice were then observed for 24 h for lethality, neurological and behavioral change (signs of toxicity). The LD50 of the plant was calculated using the formula below:

2.2.5 In vivo Anti-inflammatory Study

2.2.5.1 Determination of the effect of ethanol extract of Hymenodictyon pachyantha stem- bark on egg albumin-induced rat paw oedema

This was done according to the method of Winter et al. (1962). The increase in the right hind paw size of the rats induced by the sub-plantar injection of freshly prepared egg albumin was used as a measure of acute inflammation.

Principle: Egg albumin just like agar, releases mediators of acute inflammation responsible for causing oedema. The ability of the ethanol extract to inhibit this release of mediators is a measure of the anti-inflammatory effect of the extract.

Experimental design: A total of twenty (20) male Wistar albino rats were used for the study. They were divided into five (5) groups of four (4) rats each and treated as follows: Group 1: Received 5 ml/kg of 3% (v/ v) Tween 80 in distilled water (vehicle) Group 2: Received 10 mg/kg body weight of indomethacin (standard drug) Group 3: Received 100 mg/kg body weight of Hymenodictyon pachyantha stem-bark ethanol extract Group 4: Received 200 mg/kg body weight of Hymenodictyon pachyantha stem-bark ethanol extract

Group 5: Received 400 mg/kg body weight of Hymenodictyon pachyantha stem-bark ethanol extract

Procedure: Rats were fasted for 18 h before the experiment to ensure uniform hydration and minimize variability in oedematous response, after which the right hind paw size of the rats at time zero (before the induction of oedema) was measured using a vernier caliper. This was followed by intraperitoneal administration of test substances as outlined above. One hour after administration, acute inflammation was induced by injecting 0.1 ml of freshly prepared egg albumin into the subplantar of the right hind paw of rats. The increase in the right hind paw size of rats was subsequently measured at 0.5, 1, 2, 3, 4 and 5 h after egg albumin injection. The difference between the paw size of the injected paws at time zero and at different times after egg albumin injection was used to assess the formation of oedema. These

values were used in the calculation of the percentage inhibition of oedema for each dose of the extract and for indomethacin at the different time intervals using the relation below:

Paw oedema = (Vt-Vo) Vo = Paw oedema at time zero; Vt = Paw oedema at time t (0.5, 1, 2, 3, 4, 5 h)

2.2.6 In vitro anti-inflammatory studies

2.2.6.1 Determination of the effect of ethanol extracts of Hymenodictyon pachyantha stem-bark on phospholipase A2 activity

The effect of the extract on phospholipase A2 activity was determined using modifications of the methods of Vane (1971).

Principle: Phospholipase A2 activity was assayed using its action on erythrocyte membrane. It releases free fatty acids from the membrane phospholipids thereby causing leakage, allowing haemoglobin to flow into the medium in the process. The enzyme activity is thus directly related to the amount of haemoglobin in the medium. This was measured at 418 nm since haemoglobin absorbs maximally at this wavelength.

Enzyme preparation: Fungal enzyme preparation was obtained from Aspergillus niger strain culture. The nutrient broth was prepared by dissolving 15 g of sabouraud dextrose agar in 1000 ml of distilled water, homogenized in a water bath for 10min and dispensed into 250 ml conical flasks. The conical flasks were sealed with cotton wool and foil paper. The broth was then autoclaved at 121oC for 15 min. The broth was allowed to cool to room temperature and then the organisms in the Petri dishes were aseptically inoculated into the broth and incubated for 72 h at room temperature. The culture was transferred into test tubes containing 3 ml phosphate buffered saline and centrifuged at 3000 rpm for 10 min. The fungal cells settled at the bottom of the test tube while the supernatant was used as the crude enzyme preparation.

Substrate preparation: Fresh human blood samples were centrifuged at 3000 rpm for 10 min and the supernatant (plasma) discarded. The red cells were washed three times with equal volume of normal saline, measured and reconstituted as a 40% (v/v) suspension with phosphate buffered saline. This served as the substrate for phospholipase A2.

Assay procedure: CaCl2 (2 mM) (0.2 ml), human red blood cell (HRBC) (0.2 ml), 0.2 ml of the crude enzyme preparation and varying concentrations of normal saline, the extract and the reference drug were incubated in test-tubes for 1hr. The control contained the human red blood cell suspension, CaCl2 and free enzyme. The blanks were treated with 0.2 ml of boiled enzyme separately. The incubation reaction mixtures were centrifuged at a speed of 3000 rpm for 10 min. Samples of the supernatant (1.5 ml) were diluted with 10 ml of normal saline and the absorbance of the solutions read at 418 nm. Prednisolone, a known inhibitor of phospholipase A2, was used as the reference drug. The percentage maximum enzyme activity and percentage inhibition was calculated using the following relation:

2.2.6.2 Determination of membrane stabilization effect of ethanol extract of Hymenodictyon pachyantha stem-bark on hypotonicity induced haemolysis of red blood cells

The effect of the ethanol extract on haemolysis induced by distilled water was evaluated by incubating various concentrations of the extract with human red blood cells and distilled water. This assay was carried out by a modified method of (Sikder et al., 2012).

Principle: This test is based on the susceptibility of RBCs membrane to oxidative damage, made possible asa result of the preponderance of PUFAs in the membranes leading to haemolysis. Haemoglobin and other internal cell components are thus released into the surrounding fluids. Absorbance readings at 418 nm are a reflection of the amount of haemoglobin in the medium.

Procedure: The effect of the ethanol extract of Hymenodictyon pachyantha stem-bark on haemolysis induced by distilled water was evaluated by incubating 0.1 ml solution containing graded concentrations (mg/ml) of the extract with 0.1 ml of the sodium citrate treated blood, 1.8 ml of normal saline and 0.5 ml of distilled water in a test tube at 37oC in a water bath for 1 h. After the incubation, the test tubes were centrifuged at 300 rpm for 10 min. The absorbance of the supernatants collected was read at 418 nm. These experiments were done in triplicates and mean absorbance values taken. The effect of the standard anti-inflammatory drug, indomethacin was determined as a positive control. Changes in absorbance were used to assess the extent of haemolysis; hence membrane stabilization. Percentage inhibition of haemolysis by the extract was calculated thus:

Where OD1 = Absorbance of control hypotonic solution and OD2 = Absorbance of test sample

2.2.6.3 Determination of the effect of ethanol extract of Hymenodictyon pachyantha stem-bark on platelet aggregation

This was achieved following a modification of the method of Born and Cross (1963).

Principle: The aggregation of platelets leads to increase transmittance, therefore less absorbance of light. CaCl2-induced platelet aggregation is thus shown by reduced absorbance at 520 nm. Any substance that has anti-aggregatory effect would thus lead to increased absorption by the medium.

Preparation of platelet-rich plasma (PRP): Blood samples were taken from healthy volunteers. Fresh blood samples (5 ml) were drawn intravenously using 5 ml plastic syringe into plastic tubes containing 1% EDTA as an anticoagulant. The tubes were centrifuged at 3000 rpm for 10 min and the supernatant was collected, diluted twice with normal saline and used as the platelet rich plasma (PRP) (Nwodo, 1981).

Procedure: An aliquot of PRP (0.2 ml) was put into each of a set of three test tubes containing 1 ml each of varying concentrations of extract (0.1, 0.2 and 0.4 mg/ml) in 3% tween 80 (dissolved in normal saline). Also, another set of two test tubes contained an aliquot (0.2 ml) of PRP and 1 ml each of 0.2 and 0.4 mg/ml indomethacin in 3% tween 80. The contents of the respective tubes were made up to 2.2 ml with the vehicle. A control tube contained 2 ml of the vehicle and 0.2 ml of PRP. The tubes were allowed to incubate before the induction of aggregation by the addition of 0.4 ml of 1.47% calcium chloride (CaCl2) solution. The tests were performed in triplicates. Changes in the absorbance of the solutions were taken at intervals of 30 sec for 120 sec at 520 nm. The blanks contained the extract or indomethacin without PRP.

2.2.7 Analgesic activity test

To determine the analgesic activity of ethanol extract, the writhing test was carried out according to the method described by Koster et al. (1959).

Principle: Nociception, initiated by pain receptors, is known as the neural process of encoding and processing noxious stimuli. It is the afferent activity produced in the peripheral and central nervous system by stimuli that have the potential to damage tissue.

Procedure: Vehicle (5 mg/kg), aspirin (200 mg/kg), three scalar amounts (100, 200 and 400 mg/kg) of the extract were respectively administered (p. o.) to 5 groups of 4 animals each. Thirty minutes afterwards, 0.6% acetic (10 mg/kg) was intraperitoneally injected into each animal. The number of writhings and stretchings was counted over a 20 min period.

2.2.8 Statistical analysis

The data obtained were expressed as Mean ± SD. Significant differences of the mean values were established by one-way and two-way ANOVA and the acceptance level of significance was p< 0.05 for all the results. This was done using the Statistical product and service solutions (SPSS) version 22.0.

CHAPTER THREE

RESULTS

3.1 Percentage yield of ethanol extract of Hymenodictyon pachyantha stem-bark

The percentage yield of ethanol extract of the Hymenodictyon pachyantha stem-bark is presented in Table 1. The dried crude sample (1,000 g) of Hymenodictyon pachyantha stem- bark powder was extracted with absolute ethanol. After extraction, the yield was calculated and gave 33.20 g, representing 3.32 % of the starting material.

3.2 Qualitative phytochemical constituent of ethanol extract of Hymenodictyon pachyantha stem-bark

Table 2 shows the results of the qualitative phytochemical composition of ethanol extract of Hymenodictyon pachyantha stem-bark. The result revealed the presence of all the phytochemicals that were screened for in varying proportions, which include: flavonoids, terpenoids, steroids, saponins, alkaloids, tannins and phenols. Flavonoids, phenols, terponoids and tannins were found to be present in very high concentrations in the ethanol extract while alkaloids were found to be moderately present. Steroids and saponins were only slightly present in the ethanol extract.

3.3 Quantitative phytochemical composition of ethanol extract of Hymenodictyon pachyantha stem-bark

Table 3 shows the results of the quantitative phytochemical composition of ethanol extract of Hymenodictyon pachyantha stem-bark. Phenol was the most abundant while saponins were the least. The results obtained correlated with the qualitative results obtained above.

3.4 Result of the acute toxicity test of ethanol extract of Hymenodictyon pachyantha stem-bark

There was neither death nor any sign of toxicity in the groups of mice administered 10, 100 and 1000 mg/kg body weight of ethanol extracts of Hymenodictyon pachyantha stem-bark for phase 1 as shown in Table 4. Similarly, no death or any sign of toxicity was recorded in the groups administered, 1900, 2600 and 5000 mg/kg body weight of the ethanol extracts of Hymenodictyon pachyantha stem-bark for phase 2.

Table 1: Percentage yield of ethanol extract of Hymenodictyon pachyantha stem-bark Weight of crude sample (g) Weight of extract (g) Percentage yield (%) 1000 33.20 3.32

Table 2: Qualitative phytochemical constituents of ethanol extract of Hymenodictyon pachyantha stem-bark Phytochemical constituents Relative abundance Flavonoids +++ Terpenoids +++ Steroids + Saponins + Alkaloids ++ Tannins +++ Phenols +++ Key + = Present ++ = Moderately present +++ = Highly present

Table 3: Quantitative phytochemistry of ethanol extract of Hymenodictyon pachyantha stem- bark Phytochemical constituents Concentration (mg/100 g) Flavonoids 1359.27 ± 0.02 Terpenoids 2154. 70 ± 0.01 Steroids 3.78 ± 0.05 Saponins 0.41 ± 0.03 Alkaloids 268.86 ± 0.12 Tannins 1375.93 ± 0.08 Phenols 2900.17 ± 0.15

Results are expressed in mean ± standard deviation (n = 3)

Table 4: Acute toxicity test of ethanol extract of Hymenodictyon pachyantha stem-bark

Treatment Dosage of extract (mg/kg) Mortality rate

PHASE 1

Group 1 10 0/3

Group 2 100 0/3

Group 3 1000 0/3

PHASE 2

Group 1 1900 0/3

Group 2 2600 0/3

Group 3 5000 0/3 n=3

3.5 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on egg albumin- induced rat paw oedema

Table 5 shows the effect of ethanol extract of Hymenodictyon pachyantha stem-bark on egg albumin-induced paw oedema in rats. It shows the mean paw oedema and percentage inhibition of egg albumin-induced oedema in the rat paw which was sustained over a period of 5 h. A significant (p < 0.05) reduction in the mean paw oedema was observed for all the treatment groups from 0.5 h to 5 h when compared to the control. There were no significant (p > 0.05) reductions in the mean paw oedema of rats in the control group at the different time intervals. The paw size of animals treated with increasing doses of the extract and indomethacin significantly decreased with time. At 1 h, the groups treated with 200 and 400 mg/kg body weight of the extract inhibited rat paw oedema by (54.24%) while the group treated with 100 mg/kg body weight inhibited rat paw oedema by (27.27%). At 5 h, the inhibition of rat paw oedema for the groups treated with 100, 200 and 400 mg/kg body weight of the extract were (57.14%), (67.92%) and (79.25%) respectively. The percentage oedema inhibition for rats treated with indomethacin 10 mg/kg after 0.5 h, 1 h, 2 h, 3 h, 4 h and 5 h was (49.18%) , (52.54%), (62.07%), (66.67%), (71.43%) and (75.47%) respectively. The percentage oedema inhibition of the group treated with 400 mg/kg of extract was significantly (p < 0.05) higher than that of indomethacin at 5 h period. The inhibitory effect of the extract was dose dependent with the group treated with 400 mg/kg showing the highest oedema inhibition over a 5 h period.

Table 5: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on egg albumin- induced rat paw oedema

Treatment Concentration Mean paw oedema (cm) and percentage inhibition of oedema

(mg/kg) 0.5h 1.0h 2.0h 3.0h 4.0h 5.0h Control (3% Tween 80) 5 ml/kg 0.61±0.02cE 0.59±0.01cD 0.58±0.01dCD 0.57±0.05dBC 0.56±0.01dB 0.53±0.01 eA

Indomethacin 10 0.31±0.01aF 0.28±0.02aE 0.22±0.03aD 0.19±0.01aC 0.16±0.02aB 0.13±0.01bA (49.18%) (52.54%) (62.07%) (66.67%) (71.43%) (75.47%)

Extract 100 0.35±0.01bF 0.31±0.01 bE 0.28±0.04cD 0.25±0.01cC 0.22±0.01 cB 0.19±0.03 dA (12.20%) (27.27%) (36.95%) (44.68%) (50.00%) (57.14%)

200 0.31±0.02aF 0.27±0.02 aE 0.25±0.01 bD 0.22±0.03bC 0. 20±0.04bB 0.17±0.02 cA (49.18%) (54.24%) (56.90%) (61.40%) (64.29%) (67.92%)

400 0.30±0.01a 0.27±0.05a 0.21±0.01 a 0.18±0.03a 0.15±0.04 a 0.11±0.01 a (50.82%) (54.24%) (63.80%) (68.42%) (73.21%) (79.25%) Results expressed as Mean ± SD (n = 4); Mean values having different lowercase letters as superscripts are considered significant (p < 0.05) down the column. Mean values having different uppercase letters as superscripts are considered significant (p < 0.05) across the row. ( ) Represents percentage inhibition of paw oedema calculated relative to control.

3.6 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on phospholipase A2 activity

Table 6 shows the effect of ethanol extract of Hymenodictyon pachyantha stem-bark on phospholipase A2 activity. The ethanol extract of Hymenodictyon pachyantha stem-bark significantly (p < 0.05) inhibited the activity of PLA2 in a dose-dependent manner when compared to the control. This is shown by the reduced absorbances of the supernatant solution. There was a decrease in the absorbance of the sample with increasing concentration of the extract hence a decrease in enzyme activity. The percentage inhibition for extracts at 0.1, 0.2, 0.3, 0.4 and 0.5 ml were 78.92%, 89.71 %, 91.67%, 93.63%, 95.59% respectively. Prednisolone, a standard anti-inflammatory drug followed a similar trend with the enzyme activity decreasing with increasing doses of prednisolone. The absorbance of extracts 0.1, 0.2, 0.3, 0.4 and 0.5 ml was found to be significantly (p < 0.05) lower when compared with control, the same was also observed with increasing concentrations of the standard drug (prednisolone) when compared to the control.

3.7 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on hypotonicity- induced haemolysis of human red blood cells

The result in Table 8 shows that, the ethanol extract of Hymenodictyon pachyantha stem-bark like indomethacin significantly (p < 0.05) protected the human erythrocyte membrane against lyses induced by hypotonic solution compared to the control. Observed, was also a significant (p < 0.05) inhibition with the lowest concentrations of the extract (0.2 mg/ml) compared to indomethacin. The highest percentage inhibition (92.68) of haemolysis here was obtained at 1.0 mg/ml of the extract.

Table 6: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on phospholipase A2 activity Treatment Concentration Absorbance % Maximum % (mg/ml) (418 nm) enzyme activity Inhibition Control -- 0.21±0.02f 100 --

Extract 0.1 0.04±0.01e 21.08 78.92 0.2 0.02±0.05d 10.29 89.71 0.3 0.02±0.01c 8.33 91.67 0.4 0.01±0.13b 6.37 93.63 0.5 0.01±0.01a 4.41 95.59

Predisolone 0.2 0.07±0.01a 41.67 58.33 Results expressed as Mean±SD (n=3); Mean values having different lowercase letters as superscripts are considered significant (p< 0.05) down the column

Table 7: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on hypotonicity- induced haemolysis of human red blood cells Treatment Concentration Hypotonic Test solution % Inhibition of (mg/ml) solution (ODI) (OD2) haemolysis Control -- 1.61±0.02g

Extract 0.2 0.40±0.11a 23.99%

0.4 0.69±0.05c 42.96%

0.6 0.89±0.02d 54.99%

Indomethacin 0.8 1.26±0.01e 78.18%

f 1.0 1.50±0.07 92.68%

0.2 0.48±0.02b 29.88%

Results are expressed in means ± standard deviation; n = 3, Absorbance = 418 nm Mean values having different letters as superscripts from top to bottom of the column are considered significant (p < 0.05)

3.8 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on calcium chloride induced platelet aggregation

Table 8 shows the effect of ethanol extract of Hymenodictyon pachyantha stem-bark on

CaCl2-induced platelet aggregatory response. The extract, like indomethacin, significantly (p < 0.05) inhibited platelet aggregatory response. The different concentrations of the extract inhibited CaCl2-induced platelet aggregation in a concentration-dependent manner. The maximum platelet aggregatory activity was attained at the 120 sec. The inhibition of platelet by the extract was similar to that of indomethacin. For example, 0.1 mg/ml of the extract gave a percentage inhibition of 67.14, 66.82, 60.44 and 66.18% at the different time intervals 30, 60, 90 and 120 sec. As the concentrations of the extract increases from 0.1 to 0.6 mg/ml, it inhibited the capacity of the CaCl2 which induce aggregation of human platelets leading to platelet aggregation decreases as shown below. For instance, at 120 sec 0.1, 0.2, 0.4 and 0.6 mg/ml of extract inhibited platelet aggregation as follows; 66.18, 61.35, 57.97 and 95.17% respectively. At the concentrations of 0.4 mg/ml and 0.6 mg/ml, there was no significant (p > 0.05) inhibition at the time intervals 30 sec and 60 sec.

3.9 Effect of ethanol extract of Hymenodictyon pachyantha stem-bark and aspirin on acetic acid-induced nociceptive response in mice

Table 9, shows that at the highest dose of (400 mg/kg) the plant extract caused significant (p < 0.05) suppression of nociceptive response (71.43%) in the mice when compared to the control. The analgesic assay of the ethanol extract exhibited significant (p < 0.05) potent analgesic activity compared to the aspirin. This showed that the extract possessed analgesic property. The number of writhings in all doses of the extract (100, 200, 400 mg/kg) and Aspirin (200 mg/kg) were significantly (p < 0.05) lower than the number of writhings in the control group. The analgesic effect of the extract was dose dependent. The number of writhings in the highest dose of the extract (400 mg/kg) was non-significantly lower when compared with standard drug (Aspirin 200 mg/kg). The % analgesic activity of extracts 100, 200, 400 and Apirin 200 mg/kg were 44.64, 58.04, 71.43 and 70.54% respectively. This indicates that the highest dose of the extract had similar analgesic properties when compared with the standard drug and can serve the same function.

57 57

Table 8: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark on calcium chloride-induced platelet aggregation Treatment Concentration ∆ Absorbance (520 nm) (mg/ml) 30 sec 60 sec 90 sec 120 sec

Control - 0.21±0.01fA 0.21±0.01fB 0.23±0.02fC 0.21±0.02fA

Extract 0.1 0.14±0.01eC 0.14±0.02eBC 0.14±0.02dB 0.14±0.05dA (67.14%) (66.82%) (60.44%) (66.18%)

0.2 0.14±0.02dC 0.13±0.01dBC 0.13±0.01cB 0.13±0.02cA (64.76%) (63.03%) (58.67%) (61.35%)

0.4 0.13±0.02cB 0.13±0.01cB 0.12±0.11bA 0.12±0.01bA (60.00%) (59.24%) (53.78%) (57.97%)

0.6 0.12±0.02bA 0.12±0.01bA 0.20±0.02eC 0.20±0.15eC (58.10%) (57.35%) (87.11%) (95.17%)

Indomethacin 0.2 0.13±0.01bcAB 0.13±0.02cB 0.12±0.02bA 0.12±0.01bA (60.48%) (59.72%) (53.78%) (58.45%) n = 3, Results expressed as Mean ± SD Mean values having different lowercase letters as superscripts are considered significant (p < 0.05) down the column. Mean values having different uppercase letters as superscripts are considered significant (p < 0.05) across the row. ( ) = % Inhibition of Platelet aggregation.

Table 9: Effect of ethanol extract of Hymenodictyon pachyantha stem-bark and aspirin on acetic acid-induced nociceptive

response in mice

Treatment Dosage (mg/kg) No of writhings (Count/20mins) % inhibition

Control (3% Tween 80) 0.1 ml/10 g 28.00±1.63d

Extract 100 15.50±2.08c 44.64

200 11.75±0.96b 58.04

400 8.00±0.82a 71.43

Aspirin 200 8.25±1.26a 70.54

Results expressed as Mean ± SD (n = 4); Mean values having different lowercase letters as superscripts are considered significant (p < 0.05) down the column

CHAPTER FOUR DISCUSSION

The present study was carried out to investigate the anti-inflammatory effect and mechanisms of action of ethanol extract of Hymenodictyon pachyantha stem-bark and have been established using in vivo (egg albumin-induced rat paw oedema) and in vitro (inhibition of phospholipase A2, membrane stabilization and platelet aggregation) approaches, as well as its effect on acetic acid-induced nociceptive response in experimental animals.

Results from the qualitative and the quantitative phytochemical analyses of the extract revealed that Hymenodictyon pachyantha has flavonoids, terpenoids, phenols, steroids, saponins, alkaloids and tannins in varying proportions. Some of these constituents are believed to be responsible for the anti-inflammatory activities of some plants (Garg et al., 2010; Sakat et al., 2010). For instance, flavonoids have been found to possess anti- inflammatory properties in various studies (Amarlal et al., 2009; Lopez-Lazaro, 2009; Shailasree et al., 2012). Terpenoids, steroids, saponins, tannins and alkaloids have been shown to exhibit anti-inflammatory actions (Gepdireman et al., 2005; Souza et al., 2007; Das et al., 2010). In addition, the anticoagulant and antiplatelet aggregatory activity of phenolic compounds and flavonoids have been reported (Imran et al., 2012). Secondary metabolites like flavonoids and alkaloids have been associated with analgesic and other properties (Afsar et al., 2015; Kumar et al., 2013; Fan et al., 2014). Flavonoids are the main constituents that have capacity to interfere with eicosanoids biosynthesis pathways (Robak and Gryglewski, 1996) and are suggested to suppress the release of arachidonic acid through inhibition of neutrophils degranulation (Tordera et al., 1994). Both of these actions result in suppression of inflammatory mediators like prostaglandins and lipoxygenase end products responsible for inflammation, pain, and fever.

Acute toxicity is defined as the unwanted effect that occurs either immediately or at a short time interval after a single or multiple administration of such substance within 24 h. The unwanted (or adverse) effect is any effect that produces functional impairments in organs and biochemical lesions, which could alter the functioning of the organism in general or individual organs (Akhila et al., 2007). It has been observed that an overdose of Hymenodictyon pachyantha extract is usually non-fatal; the victims tend to suffer self-

limiting gastrointestinal disturbances (Anokbonggo et al., 1990). The administration of the extract was by oral route to mimic route used by the traditional healers in treating their patients was thus used in the test animals. This would make any findings in the experimental animals easily translatable to what would be expected in the human subjects. On the basis of this fact, the acute toxicity study of ethanol extract of Hymenodictyon pachyantha stem-bark was carried out. The acute toxicity studies of oral doses of ethanol extract of Hymenodictyon pachyantha stem-bark in mice revealed that it has a high safety profile, as the extract was tolerated by the animals up to 5000 mg/kg. On administration of the extract, no immediate behavioural changes were observed. The mice moved about and fed normally. After twenty minutes, piloerection was noticed and the animals became restless, some trying to escape through the holes in the cages. The animals did not vomit, neither was there ptosis.

The anti-inflammatory assay of the ethanol extract both displayed significant (p<0.05) inhibition of inflammation compared to the control which suggested that the phytoconstituents present in the plants may have inhibited the inflammation through their suppressive action on prostaglandin. Inflammation arrive when the immune system try to remove something that may turn out to be harmful. The anti-inflammatory activity of the ethanol extract of Hymenodictyon pachyantha stem-bark was confirmed by measuring its ability to reduce local oedema induced in the rat paw by injection of an irritant or phlogistic agent (Omkar et al., 2007). Egg albumin-induced oedema model has been used in anti- inflammatory studies. Subcutaneous injection of egg albumin into the rat paw produces oedema resulting from plasma protein-rich fluid exudation along with neutrophil extravasation. The increase in the paw volume of the rats after the injection of egg albumin correlates with the work of Ekwueme et al. (2011) that egg albumin induces paw oedema in rats. The feasible mechanism of egg albumin mediated inflammation is tri-phasic. The first phase (0 h – 2 h) is predominantly non-phagocytic and mainly mediated by histamine and serotonin, the second phase (2 h- 3 h) is mediated by kinin and the last phase (3 h- 5 h) is due to the liberation of PGs (Suba et al., 2005) which produces oedema dependent on neutrophils mobilization. The ethanol extract at 100, 200 and 400 mg/kg body weight showed a good anti-inflammatory activity as it significantly (p < 0.05) inhibited the increase in paw volume from 0.5 h to 5 h. This shows that the extract inhibited all the phases of the inflammatory response just as it was demonstrated by Silva et al. (2015) with Annona vepretorum species. The inhibition of the early phase of oedema exhibited by the ethanol extract in this study

suggests that it blocks the release of histamine and serotonin. The suppression of oedema in the second and third phase of inflammation suggests that the anti-inflammatory activity of the extract may also be due to the suppression of kinin and prostaglandin formation induced by egg albumin within this period. Since these mediators cause oedema by increasing vasodilatation and vascular permeability at the site of injury, the extract therefore reduces vascular permeability and fluid exudation, probably by preventing the contraction of endothelial cells, thus, suppressing oedema.

Consistent with this finding is the inhibitory effect of the ethanol extract on phospholipase A2 activity an acyl-hydrolase that cleaves fatty acid from membrane phospholipids in position two of the phospholipids, hydrolyzing the bond between the second fatty acid tail and the glycerol molecule and calcium chloride-induced platelet aggregation. Arachidonic acid released from these phospholipids is acted upon by COX and LOX which lead to the de novo synthesis of lipid mediators (George et al., 2014). The action of COX on arachidonic acid produces mediators such as PGE2, PGD2, PGI2 and TXA2, while the action of 5-LOX on arachidonic acid releases leukotrienes such as LTB4. The ethanol extract of Hymenodictyon pachyantha stem-bark from 0.1 to 0.5 ml exhibited a significant (p < 0.05) and concentration- dependent inhibition of PLA2 activity. This inhibition of PLA2 by the extract implies that it was able to suppress the release of free fatty acids from RBC membrane phospholipids and the consequent deprivation of COX and LOX substrates for the synthesis of inflammatory mediators, hence limiting their effects such as vasodilatation, vascular permeability, chemotaxis and pain, thus preventing inflammation. This inhibition of PLA2 also shows that the extract has potentials for preventing atherosclerosis and cancer as PLA2 has been implicated in their aetiology (Sato et al., 2009). The mechanism of inhibition of PLA2 by the extract could be like that of corticosteroids which induce lipocortin synthesis (Schimmer and Parker, 2001). This effect could be attributed to the presence of flavonoids in the extract as many studies have shown that flavonoids inhibit PLA2 (Wang et al., 2006; Kim et al., 2008; Messina et al., 2009). Tannins have also been found to inhibit PLA2 activity by Barbosa et al. (2012) as it was observed that the sub-fraction of Spiranthera odoratissima leaves which contained only tannins inhibited PLA2 activity.

Also in this study, the extract was found to significantly (p<0.05) inhibit lysis of erythrocytes compared to the control. The highest concentration of the ethanol extract displayed the most

inhibition of hemolysis of RBC. Therefore it may be assumed that the plant extracts may contain certain phytoconstituents that may be responsible for its membrane stabilization activity. Hossain et al. (2014) reported similar outcome in the study of membrane stabilization activity of different extracts from Spilanthes paniculata leaves. Membrane stabilization is the process through which the analgesic compounds work and is the possible assay to study the possible anti-inflammatory effect of any compound (Hossain et al., 2014).

The hypotonic solution-induced human erythrocytes lysis test is the effective model to examine membrane stabilization. Hypotonicity- induced haemolysis of red blood cells occurs due to water uptake by the cells and leads to the release of haemoglobin which absorbs maximally at 418 nm. Hence, the reduced optical density at 418 nm obtained for the various coconut test samples is a reflection of the stabilization of the red cell membrane caused by the extract. During inflammation, there are lyses of lysosomes which release their component enzymes which produce a variety of disorders. Since human red blood cell (RBC) membranes are similar to lysososmal membrane (Mounnissamy et al., 2007; Mohammedi and Atik, 2014) the stabilization of erythrocyte membranes by the extract thus, implies that it may as well stabilize lysosomal membranes and was therefore used as a method to study the mechanism of action of anti-inflammatory agents. During inflammation, lysosomal enzymes and hydrolytic components are released from the phagocytes to the extracellular space results in damages of the surrounding organelles and tissues and also guide a variety of disorders. Nonsteroidal anti-inflammatory drugs act either by inhibiting these lysosomal enzymes or through stabilization of lysosomal membranes. Furthermore, exposure of RBC to noxious substances such as hypotonic medium, results in the lysis of the membranes due to oxidation and the lysis of hemoglobin (Ferreira et al., 2004). So retaining the red blood cell membrane integrity by suppressing hypotonicity induced membrane lysis was taken as a possible mechanism of anti-inflammatory activity.

Platelets have been identified as crucial mediators of inflammation in a variety of animal models, including sepsis (Wang et al., 2005), acute kidney injury, dermal inflammation (Zarbock et al., 2006), vascular injury (Katoh, 2009), and myocardial infarction. Mechanism of platelet Aggregation involves platelet-to-platelet adhesion and is necessary for effective hemostasis following the initial adhesion of platelets to the site of injury. Following adhesion, platelets are activated by a number of agonists such as adenosine diphosphate (ADP) and

collagen present at the sites of vascular injury. These agonists activate platelets by binding to specific receptors on the platelet surface and the eventual formation of blood clot through the deposition of fibrin from the coagulation cascade.

This study demonstrated that the ethanol extract produced a concentration and time- dependent inhibition of CaCl2-induced platelet aggregatory response compared to control.

Inhibition of CaCl2-induced platelet aggregatory response could probably be due the extract’s ability to inhibit PLA2 and COX which are required for the synthesis of TXA2. The inhibition of TXA2 is imperative because it induces platelet aggregation by increasing intracellular Ca2+ which promotes fusion of platelet granules with the membrane, thus, releasing its contents such as ADP which also promotes the aggregation of platelets. The inhibition of platelet aggregation by the extract could also imply decreased vascular permeability and leukocyte extravasation which is mediated respectively by histamine and P- selectin normally released from the platelet granules (Zarbock et al., 2006). Since blood platelets participate in pathological thrombosis, leading to such conditions as myocardial infarction, stroke, embolism and peripheral vascular thrombosis (Golino et al., 2005; Fabre and Gurney, 2010), inhibition of platelet aggregation by the extract is thus indicative of its possible role as an antithrombotic agent and could be useful in the management of the above named disorders. This demonstrated effect of the extract could be due to the presence of phenolic compounds and flavonoids since the anticoagulant and antiplatelet aggregatory activity of these substances have been reported (Hommam et al., 2000; Imram et al., 2012). The beneficial effects of antioxidants on the inhibition of platelet activation and aggregation have also been reported (Sobotková et al., 2009). This confirms the involvement of flavonoids in the inhibition of platelet aggregation by the extract, since flavonoids are antioxidants (Messina et al., 2009). This effect could also be attributed to the presence of tannins, as the anticoagulant or anti-platelet aggregation activity of tannins have also been demonstrated (Mekhfi et al., 2006; Tognolini et al., 2006; Kee et al., 2008; Kim et al., 2008).

The ethanol extract evaluated for analgesic effect showed promising activity against acetic acid induced visceral pain. Pain is associated with the pathophysiology of various clinical consequences such as arthritis, muscular pain, cancer and vascular diseases (Akhtar et al., 2013). The characteristic of pain activity generated by intraperitoneal injection of acetic acid is presented with contraction of abdominal muscle followed by extension of hind limbs and

elongation of body part and such constriction is thought to be mediated by local peritoneal receptor (Bentley et al., 1983). Acetic acid induced writhing response is a suitable method for assessing peripheral analgesic effects as it is sensitive for various classes of analgesic drugs. Pain perception in acetic acid induced writhing assay is revealed by producing localized inflammatory response due to release of free arachidonic acid from tissue phospholipids by the help of COX, which in turn produces prostaglandin specifically PGE2 and PGF2α.

The level of lipoxygenase produced may also increase in peritoneal fluids and cause inflammation and pain by increasing capillary permeability. Thus the substance inhibiting the writhing will have analgesic effect preferably by inhibition of prostaglandin synthesis (Khan et al., 2010). NSAIDs relieve the pain response peripherally by inhibiting production of prostaglandins, thromboxane, and other inflammatory mediators by acting on cyclooxygenase enzymes. Ethanol extract of stem-bark at 100, 200 and 400 mg/kg significantly (p < 0.05) reduced the number of writhe induced by a 0.6% acetic acid solution in a dose dependent manner over a period of 20 min similar to that of the standard drug, aspirin. Central nervous system depressants have been shown to reduce the number of writhing in acetic acid pain models. The inhibition of acetic acid writhing by the ethanol extracts suggests that it has a depressant effect on the central nervous system.

4.2 Conclusion

The results revealed that the extract had significant (p < 0.05) anti-inflammatory activity when compared with the untreated control. The standard drug (indomethacin) showed similar effect. The results also infer that the extract inhibited acetic acid-induced nociceptive response through peritoneal receptors mediated cyclooxygenase inhibition. The results suggest that the mechanisms of this anti-inflammatory effect may be by inhibiting phospholipase A2 activity and platelet aggregation which are responsible for inflammation. The investigation provides empirical evidence for the use of Hymenodictyon pachyantha stem-bark extract in folkloric treatment of inflammatory disorder.

4.3 Suggestions for Further Studies a) Further investigation should be carried out to explore the precise mechanism of action of the extract.

b) The biologically active components present in the extract should be further investigated and elucidated structurally.

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APPENDICES

PREPARATION OF CHEMICALS AND REAGENTS

❖ 1% aluminium choride solution: A known quantity of ferric chloride (1 g) was dissolved and made up to 100 ml with distilled water.

❖ 1.47% CaCl2: This was prepared by dissolving 1.47 g of CaCl2 in 100 ml of distilled water and used for the anti-platelet aggregatory activity.

❖ 2mM CaCl2: This was prepared by dissolving 0.022 g of CaCl2 in 100 ml of distilled

water and used in the phospholipase A2 assay. ❖ Colour reagent: An iron stock solution was prepared by dissolving 5 g of ferric chloride in 200 ml of phosphoric acid, after which 4 ml of the stock was diluted with 24 ml of sulphuric acid. ❖ 28% dilute ammonia: A known volume, 28 ml of ammonia was mixed with distilled water and made up to 100 ml. ❖ 1% EDTA solution: A 1% solution of EDTA was prepared by dissolving 1g of EDTA powder in 100 ml of distilled water. ❖ 45% (v/v) ethanol: A known volume, 45 ml of ethanol was mixed with 55 ml of distilled water. ❖ 80% (v/v) ethanol: A known volume, 80 ml of ethanol was mixed with 20 ml of distilled water. ❖ 1% ferric chloride solution: A known quantity of ferric chloride (1 g) was dissolved and made up to 100 ml with distilled water. ❖ 0.1 N ferric chloride in 0.1 N hydrochloric acid: A known volume, 0.9 ml of hydrochloric acid and 2.7 g of ferric chloride were dissolved in 100 ml of distilled water. ❖ 5% (w/v) ferric chloride: A known quantity, 5 g of ferric chloride was dissolved in 100 ml of distilled water. ❖ 0.5% formaldehyde in 60% sulphuric acid: A known volume, 60 ml of concentrated sulphuric acid was diluted with distilled water and made up to 100 ml, after which 0.5 ml of formaldehyde was added. ❖ Folin-Ciocalteu’s reagent: Sodium tungstate (10 g), sodium molybdate (2.5 g) and lithium sulphate (15 g) were added to 10 ml of hydrochloric acid. To this was added 5 ml

of phosphoric acid. The solution was thereafter made up to 100 ml with distilled water. This was kept in the dark after which it was filtered and the filtrate diluted (1:10 dilution) with distilled water. ❖ 2% hydrochloric acid: A known quantity of hydrochloric acid (2 g) was dissolved in 100 ml of distilled water. ❖ Lead sub acetate solution: 15% lead acetate (i.e. 15.0 g of lead acetate in 100 ml of distilled water) was mixed with 20 ml of absolute ethanol and made up to 100 ml with distilled water. ❖ Normal saline: A known quantity (0.90 g) of sodium chloride was dissolved in 100 ml of distilled water. ❖ Picric acid: A known quantity, 1 g of picric acid was dissolved in 100 ml of distilled water. ❖ 5% phosphomolybdic acid: To a quantity, 35 g of molybdic acid was added sodium tungstate (5 g) and sodium hydroxide (20 g). The mixture was dissolved in 325 ml of distilled water in a volumetric flask after which 125 ml of concentrated phosphoric acid was added. The contents of the flask were boiled for 20-40 min, cooled and the volume diluted to 350 ml mark with distilled water. ❖ 0.0008M potassium ferricyanide: A known quantity, 0.02632 g of potassium ferricyanide was dissolved in 100 ml of distilled water. ❖ Phosphate buffered saline: This solution was prepared by dissolving 3.90 g of

NaH2PO4.2H2O and 4.45 g of Na2HPO4.2H2O in 250 ml of distilled water. 40.5 ml

aliquot of Na2HPO4.2H2O was mixed with 9.5 ml of NaH2PO4.2H2O solution and made up to 100 ml with distilled water. Then 0.9 g of NaCl was added to the buffer to form phosphate buffered saline. ❖ Sabouraud dextrose agar: Prepared by dissolving 15.0 g of sabouraud dextrose agar in 1000 ml of distilled water. The mixture was homogenized in a water bath at 100oC for 10 min; 5 ml was dispensed into petri-dishes and autoclaved at 121oC for 15 min. It was used for the first culture of Aspergillus niger. ❖ SDA broth: This was prepared by dissolving 15 g of sabouraud dextrose agar in 1000 ml of distilled water. It was used as the media for the submerged culture of Aspergillus niger

for the preparation of the crude phospholipase A2 enzyme.

❖ 20% (w/v) sodium carbonate: A known quantity (20 g) of sodium carbonate was dissolved in 100 ml of distilled water. ❖ Stock solution: A known quantity, 0.05 g (50 mg) of the extract or drug was dissolved in 50 ml of 3% tween 80 (suspended in distilled water or normal saline) to give a stock solution of 1 mg/ml. ❖ 20% sulphuric acid: A known volume, 20 ml of concentrated sulphuric acid was diluted with distilled water and made up to 100 ml. ❖ 60% sulphuric acid: A known volume, 60 ml of concentrated sulphuric acid was diluted with distilled water and made up to 100 ml. ❖ 0.02 M Tris-HCl buffer (pH 7.6): A known quantity, 2.482 g of tris (hydroxyl methyl amino methane) was dissolved in distilled water and made up to 1000 ml. The pH adjusted by adding 1 M HCl. ❖ 3% (v/v) tween 80: Tween 80 (3 ml) was dissolved in 97 ml of distilled water or normal saline. ❖ Wagner’s reagent: A known quantities, of iodine crystals (2.0 g) and of potassium iodide (3.0 g) were dissolved in minimum amount of water and then made up to 100 ml with distilled water.