Screening for the effects of selected Zimbabwean plant extracts on enzymes and processes involved in pain and inflammation.

By Elaine Chirisa R055334H

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE MASTER OF PHILOSOPHY (SCIENCE) DEGREE IN BIOCHEMISTRY

Department of Biochemistry Faculty of Science University of Zimbabwe

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ABSTRACT Inflammation is a complex process that is mediated by signalling radicals and prostaglandins. Prostaglandins are produced by conversion of arachidonic acid by cyclooxygenase (COX) isoenzymes. Selective inhibition of the inducible cyclooxygenase isoform, COX-2, would probably relieve inflammation without adversely affecting physiological function. Chronic inflammation can be attenuated by the unregulated production and poor elimination of free radicals leading to oxidative stress. One of the major contributors of free radicals is the overproduction of nitric oxide (NO) during inflammation. Chronic inflammation and oxidative stress can create micro-environments that favour development of degenerative diseases such as cancer and rheumatoid arthritis. Herbal remedies are used in folk medicine to treat inflammatory ailments when conventional drugs are unavailable or inaccessible. The plant species used by herbalists to treat pain and signs of inflammation could be potential sources of novel anti- inflammatory agents. Zimbabwean plants that are used to treat pain have compounds that can inhibit enzymes and processes that are involved in inflammation. The objective of this study was to investigate the in vitro anti-inflammatory and antioxidant activities of eight selected Zimbabwean medicinal plant extracts. Amaranthus spinosus , Brachystegia boehmii , Cassia abbreviata , Combretum molle , Combretum platypetalum , Combretum zeyheri , Gymnosporia senegalensis and Parinari curatellifolia were tested for anti-inflammatory activities using the COX enzyme inhibitory activity. Of these, six plants were further tested for membrane stabilisation using the erythrocyte membrane stabilization assay, protein denaturation inhibition using the albumin denaturation inhibition assay and antioxidant activity using the diphenyl picryl hydrazine and tetramethoxy azobismethylene quinone assays. Eight plant extracts were tested for COX-1 and -2 enzyme inhibitory activities. Combretum zeyheri and Combretum molle showed greater COX-2 inhibitory activity of 42 and 68 % respectively while showing the least COX-1 inhibition with percentage inhibition. Combretum platypetalum inhibited COX-2, with a percentage inhibition of 85 %, COX-1 inhibition of 42 %, making it COX-2 selective. IC 50 s of the COX-2 selective C. platypetalum extract and indomethacin against COX-2 were determined to be 571 and 414 µg/ml respectively. The six plant extracts, B. boehmii , C. molle , C. platypetalum , C. zeyheri , G. senegalensis and P. curatellifolia that were active against COX isoforms were evaluated for membrane stabilisation, albumin denaturation inhibition and free radical scavenging activity. C. platypetalum , C. molle and B. boehmii were able to stabilize the erythrocyte membrane and inhibit the precipitation of bovine serum albumin in solution. P. curatellifolia extract showed potent antioxidant activity using both assays with the maximal free radical scavenging at 16 and 18 µg/ml respectively. C. zeyheri and Gymnosporia senegalensis extracts also showed antioxidant activity with values of 21 and 32 µg/ml respectively. P. curatellifolia and C. platypetalum extracts were further evaluated for their effect on NO production in RAW 264.7 cells. C. platypetalum ethanol extract and P. curatellifolia water extract inhibited NO production in RAW 264.7 murine macrophage cells. In conclusion, Zimbabwean plant species evaluated in the study showed anti-inflammatory activity and could be potential sources of novel and potent anti-inflammatory agents.

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DEDICATIONS I dedicate this thesis to my family and friends that have shared this exciting journey with me. Thank you for your unwavering belief, love and support.

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ACKNOWLEDGEMENTS I express my profound gratitude to my supervisor Professor Stanley Mukanganyama for his guidance, advice and constructive criticism. I am also grateful to my co-supervisor Dr Farisai Chidzwondo for her wisdom and advice as well as her keen interest in my work. I thank the entire Biomolecular Interactions Analyses Group for their support throughout the project. I acknowledge the financial support from the International Science Program (ISP), International Foundation for the Sciences (IFS), TWAS, DAAD German In-country Scholarship and University of Zimbabwe Research Board.

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TABLE OF CONTENTS

Abstract …………………………………………………………………………...... i Dedication ……………………………………………………………………...... ii Acknowledgements ………………………………………………………………………... iii Table of contents …………………………………………………………………...... iv List of tables ……………………………………………………………………………….. viii List of figures ……………………………………………………………………………… ix List of appendices …………………………………………………………………...... xi List of abbreviations ………………………………………………………………………. xii

CHAPTER ONE…………………………………………………………………………..1 Introduction and Literature Review………………………………………...... 1 1.1 Inflammation …………………………………………………………………………... 1 1.2 Inflammatory mediators………………………………………………………………... 4 1.2.1 Prostaglandins……………………………………………………………………. 7 1.2.2 Inducible nitric oxide synthase…………………………………………………... 10 1.3 Reactive oxygen species and oxidative stress ……………………………...... 12 1.4 Inhibition of inflammatory mediators…………………………………………...... 16 1.5 Rheumatoid arthritis……………………………………………………………...... 19 1.6 Current allopathic treatment for inflammation and oxidative stress……………...... 21 1.7 Ethnopharmacology……………………………………………………………...... 23 1.8 Treatment of pain using plants…………………………………………………...... 25 1.8.1 Combreteceae species in their use as pain remedies……………………………... 27 1.8.2 Parinari curatellifolia ……………………………………………………...... 30 1.8.3 Other species …………………………………………………………………….. 31 1.9 Bioassay model systems……………………………………………………………….. 32 1.9.1 Antioxidant assays……………………………………………………………….. 33 1.9.2 RAW 264.7 cell line……………………………………………………………... 35

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1.9.3 Menadione……………………………………………………………...... 36 1.10 Rationale of study ……………………………………………………...... 37 1.11 Hypothesis …………………………………………………………………………….38 1.12 Objectives ………………………………………………………………………...... 38 1.12.1 Main objective ………………………………………………………………38 1.12.2 Specific objectives ………………………………………………...... 39 1.13 Overview of study…………………………………………………………………….,.40

CHAPTER TWO………………………………………………………….………………42 Materials and Methods…………………………………………………………….……….. 42 2.1 Chemicals ……………………………………………………………………………… 42 2.2 Cell lines ………………………………………………………………………………. 42 2.3.1 Plant selection and collection and authentication …………………………………… 42 2.3.2 Plant extraction ……………………………………………………………………… 43 2.4.1 Qualitative analysis of phytoconstituents from Combretum platypetalum …………. 45 2.4.2 Isolation of phytoconstituents from Combretum platypetalum ……………………... 46 2.5 COX assays …………………………………………………………………………… 47 2.5.1 Cyclooxygenase enzymes inhibitor screening assay ………………………... 47 2.5.2 The effect of plant extracts on cyclooxygenase enzyme activity …………... 48

2.5.3 IC 50 determination of Combretum platypetalum ……………………………. 49 2.5.3 The effect of isolated phytoconstituents on COX-2 enzyme ……………………….. 50 2.6 Antioxidant Assays ……………………………………………………………...... 50 2.6.1 Diphenyl picryl hydrazyl (DPPH) free radical scavenging assay …………… 50 2.6.2 (TMAMQ) free radical scavenging assay …………………………………… 51 2.7 Protein anti-inflammatory Assays …………………………………………………….. 51 2.7.1 Albumin denaturation inhibition assay ……………………………………… 52 2.7.2 Sheep erythrocyte membrane stabilizing activity assay …………………….. 53 2.8 Nitric oxide production Assays ………………………………………………………... 54 2.8.1 RAW 264.7 cell culture ……………………………………………………... 54

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2.8.2 The effect of combining plant extracts and oxidative compounds on nitric oxide production in RAW murine macrophage ………………………………………….. 54 2.8.3 The effect of plant extracts on nitric oxide production in lipopolysaccharide activated RAW murine macrophage cell line ……………………………………. 55 2.8.4 Nitrite quantification assay…………………………………………………... 56 2.9 Statistical analysis……………………………………………………………………… 57

CHAPTER THREE…………………………………….…………………………………59 Results ………………………………………………………………………………….….. 59 3.1 The effect of plant extracts on cyclooxygenase enzyme activity ………………….. 60 3.2 Fractionation of Combretum platypetalum ……………………………………………. 62 3.2.1 Isolation of phytoconstituents from Combretum platypetalum ……………… 63 3.2.2 The effect of isolated phytoconstituents on COX-2 enzyme ………….. 64 3.3 Free radical scavenging ……………………………………………………………….. 64 3.3.1 DPPH free radical scavenging activity of plant extracts ……………………. 64 3.3.2 TMAMQ free radical scavenging activity of plant extracts ………………… 65 3.4 The effect of plant extracts on inflammatory processes in vitro ………………... 68 3.4.1 The effect of plants on albumin denaturation ……………………...... 68 3.4.2 The effect of plant extracts on sheep erythrocyte membrane stability ……... 70 3.5 The effect of plant extracts on nitric oxide production induced by selected compounds in RAW 264.7 murine macrophage cells ………………………………………………... 72 3.5.1 Activation of nitrite oxide production by compounds……………………….. 73 3.5.2 The effect of combining menadione and plant extracts on nitric oxide production in RAW 264.7 cells………………………………………………………………...... 74

3.5.3 The effect of combining H 2O2 and plant extracts on nitric oxide production in RAW 264.7 cells…………………………………...... ………………….. 75

3.5.4 The effect of menadione and H2O2 combination with C. platypetalum flavonoids on nitric oxide production in RAW 264.7 cells ……...... 77

3.5.5 The effect of menadione and H 2O2 combination with C. platypetalum saponins on nitric oxide production in RAW 264.7 cells…………………………………… 78

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3.6 The effect of Combretum platypetalum and Parinari curatellifolia extracts on lipopolysaccharide activated RAW murine macrophage cells………………..……………79 3.6.1 The effect of Parinari curatellifolia water extract on lipopolysaccharide activated RAW murine macrophage at 24 hours...…………………………………………...79 3.6.2 The effect of Combretum platypetalum ethanol extract on lipopolysaccharide activated RAW murine macrophage cells at 24 hours …..…………………...……. 81 3.6.3 The effect of Combretum platypetalum ethanol extract on lipopolysaccharide activated RAW murine macrophage cells at 48hours…………………………….... 82

CHAPTER FOUR………………………………………………………………………….84 Discussion …………………………………………………………………………………. 84 4.1 The effect of plant extracts on cyclooxygenase enzyme activity…………………….... 84 4.2 The effect of plant extracts on free radical scavenging activity ………………………. 89 4.5 The effect of plants on albumin denaturation …………………………………………. 90 4.6 Membrane stabilizing activity of plant extracts ……………………………………….. 91 4.7 The effect of plant extracts on nitric oxide production in RAW cells ………………....92 4.9 The effect of Combretum platypetalum and Parinari curatellifolia extracts on RAW cells………………………………………………………………………………………,,,, 95 4.10 Potential of Zimbabwean plants as sources of anti-inflammatory agents ………...... 100 4.11 Conclusions ………………………………………………………………………. 102 4.12 Limitations ……………………………………………………………………...... 102 4.13 Suggested future work …….………………………………………………………... 103

CHAPTER FIVE References ………………………………………………………………………………...104

APPENDINCES ………………………………………………………………………….. 121

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LIST OF TABLES TABLE TITLE PAGE 1.1 Attributes of acute and chronic inflammation……………………... 2 1.2 Medicinal uses of Combretum species……………………………... 28 2.1 Zimbabwean plant samples collected and extracted and their uses in traditional medicine …………………………………………….. 44 2.2 Summary of pipetting into Enzyme Immunoassay Analysis plate… 50 3.1 Inhibitory activity of plant extracts on cyclooxygenase enzymes…. 60 3.2 Phytoconstituent analysis of C. platypetalum leaf extract…………. 63 3.3 Anti-COX-2 activity of C. platypetalum phytoconstituents……….. 63 3.4 Antioxidant activity of selected plant extracts using TMAMQ and DPPH free radical scavenging assays…………………………. 67 3.5 Percentage inhibition of bovine serum albumin by plant extracts..... 69 3.6 Percentage stabilisation of erythrocyte membrane by ethanol plant extracts……………………………………………………….. 71

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LIST OF FIGURES FIGURE TITLE PAGE 1.1 Nuclear factor kappa beta pathway that activates inflammatory gene expression…………………………………………………………………...5 1.2 Prostaglandin synthetic pathway ………………………………………...... 8

1.3 The relationship between cyclooxygenase and prostaglandin E2 …………. 9 1.4 Endogenous and exogenous sources of free radicals in the body………….. 13 1.5 Reactive oxygen species and their role in the induction of disease ……….. 15 1.6 Proposed mechanisms of action of phytochemicals against inflammation... 26 1.7 Image of Combretum platypetalum ………………………………………... 29 1.8 Image of Parinari curatellifolia …………………………………………… 31 1.9 DPPH radical quenching…………………………………………………… 33 1.10 Tetramethoxyazobismethylene quinone TMAMQ radical ………………... 34 1.11 RAW 264.7 cells seen under a light microscope…………………………... 35 1.12 Menadione redox cycling…………………………………………………... 37 1.13 Flowchart of study………………………………………………………….41 2.1 COX catalysed reaction in COX activity assay……………………………. 47 2.2 Schematic diagram of the determination and quantification of nitrite ions by the Griess assay……………………………………………………………. 57 3.1 Dose response curves for Indomethacin and Combretum platypetalum …… 61 3.2 TMAMQ free radical scavenging activity of Combretum zeyheri , Brachystegia boehmii , Combretum molle and Parinari curatellifolia plant extracts...... 65 3.3 DPPH free radical scavenging activity of Combretum molle , Parinari curatellifolia , Combretum platypetalum and Combretum zeyheri ...... 66 3.4 Sodium nitrite standard curve……………………………………………… 73 3.5 Effect of selected compounds on nitrite concentration in RAW 264.7 cells. 74 3.6 Nitrite concentration in the presence of menadione and extracts of Combretum platypetalum , C. zeyheri , C. molle and P. curatellifolia extracts………...... 76 3.7 Nitrite concentration in RAW cells the presence of hydrogen peroxide and plant extracts……………………………………………………………………... 77

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3.8 Nitrite concentration in the presence of menadione and H 2O2 in combination with Combretum platypetalum flavonoids………………………………………. 78 3.9 Nitrite concentration in RAW cells in the presence of menadione and hydrogen peroxide in combination with Combretum platypetalum saponin 80 3.10 Nitrite concentration of LPS activated RAW cells combined with Parinari curatellifolia extract at 24 hours…………………………………………… 81 3.11 Nitrite concentration of LPS activated RAW cells combined with Combretum platypetalum extract at 24 hours…………………………………………… 82 3.12 Nitrite concentration of LPS activated RAW cells combined with Combretum platypetalum extract at 48 hours……………………………………...... 84 4.1 Proposed mechanism of action for Combretum platypetalum extract against COX-2……………………………………………………………………… 87 4.2 Proposed mechanism of action of Parinari curatellifolia extract ………… 97

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LIST OF APPENDICES APPENDIX TITLE PAGE 1 List of titles of publications arising from the study………………... 121 2 Raw data for COX activity assay…………………………………... 122 3 Raw data for Membrane stability assay……………………………..123 4 Raw data for albumin denaturation inhibition assay ……………….124 5 Raw data for TMAMQ free radical scavenging assay……………... 125 6 Raw data for nitric oxide production assays……………………….. 126 7 Standard curve for nitrite quantification…………………………….127

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LIST OF ABBREVIATIONS ABBREVIATION DEFINITION TNF-α Tumor necrosis factor NF Κβ Nuclear factor kappa beta IFN-γ Interferon gamma IΚβ Nuclear factor kappa beta inhibitor molecule IΚΚ Nuclear factor kappa beta inhibitor molecule kinase COX Cyclooxygenase COX-1 Cyclooxygenase-1 COX-2 Cyclooxygenase-2

PA 2 Phospholipase A 2

PGH 2 Prostaglandin H 2

PGD 2 Prostaglandin D 2

PGE 2 Prostaglandin E 2

PGF 2α Prostaglandin F 2α NO Nitric oxide RNS Reactive oxygen species eNOS endothelial nitric oxide synthase nNOS neuronal nitric oxide synthase iNOS Inducible nitric oxide synthase MAPK mitogen activated protein kinases cGMP Cyclic guanidine monophosphate ROS reactive oxygen species NADP nicotinamide adenosine diphosphate GSH glutathione NOO - peroxynitrite ions SOD superoxide dismutase DMARD disease modifying arthritic xii

NSAIDs non-steroidal anti-inflammatory drugs FDA Food and Drug Administration BHT butylated hydroxytoluene BHA butylated hydroxyl anisole DPPH Diphenyl picryl hydrazyl TMAMQ Tetramethoxyazobismethylene quinone HAT Hydrogen atom transfer SET single electron transfer RAW Rasche, Ralphe, Watson cell line

H2O2 Hydrogen peroxide ELISA Enzyme Linked Immunosorbent assay

IC 50 Inhibitory concentration BSA bovine serum albumin DMEM Dulbecco´s modified eagle medium LPS Lipopolysaccharide

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CHAPTER ONE

Introduction and Literature review

1.1 Inflammation Inflammation is a protective process that guards the body against harmful stimuli and hastens the recovery process (Krishnaraju et al., 2009). The stimulus may be in the form of a wound, a pathogenic bacterium or virus or an endogenous trigger. The process is characterized by redness, pain, swelling, immobility and heat (Hsu et al ., 2013). The mechanisms of inflammation work to direct the immune response to the site of injury by increasing the blood supply and altering the permeability of the blood vessels which accounts for some the above mentioned characteristics such as the swelling and heat (Park et al. , 2013). The inflammatory response is complex as it involves an array of reactions that include enzyme activation, innate immune cell activation and migration, release of reactive oxygen species and tissue repair that act to remove the cause and repair the damage of the injury (Vadivu and Lakshmi, 2011). The migrated immune cells are usually macrophage and T lymphocyte cells that are activated to release pro-inflammatory cytokines. The pro-inflammatory cytokines are released to amplify inflammatory activity at the site of injury in order to resolve the cause of injury (Hong et al. , 2009). Inflammation is part of the innate immune response which means that is it not specific for any particular cause of injury.

The non-specific nature of the response means that the reactions are similar despite the cause of injury which can be detrimental if the injury is persistent. Therefore, the inflammatory reactions must be actively regulated to prevent unnecessary damage to tissues (Heras and Hortelano,

2009). The improper regulation of the process for the resolution of injury can lead to a perpetual state of inflammation which can become chronic.

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The regulation of inflammation, therefore, categorises the process into two distinct types, acute and chronic inflammation as in Table 1.1. Acute inflammation is the therapeutic state that allows the body to heal after an injury (Spite and Serhan, 2010). The immune cells, enzymes, reactive oxygen species all congregate at the site of injury leading to the propagation of biochemical cascades that act to remove the pathogen or seal the wound. Acute inflammation is terminated by anti-inflammatory mechanisms that remove the pro-inflammatory components from the site when the injury has been resolved (Spite and Serhan, 2010). The pain stops, mobility is restored and the heat and swelling dissipate.

Table 1.1 Characteristics of acute and chronic inflammation

Process Acute inflammation Chronic inflammation Initiators Microbial surface antigen and Non digestable microorganisms fragments Non-degradable foreign matter Injured tissue and fragments Auto-immune reactions Macrophage mediators Histamine Cytokines from T-lymphocytes Prostaglandins, leukotrienes Prostaglandins Bradykinin Proteases Lysosomal contents Reactive oxygen species Complement molecules Complement Cell types Neutrophils Plasma cells Macrophages Macrophages, fibroblasts Time course Acute onset Prolonged onset Minutes to hours Weeks to years Outcome Resolution Resolution Abscess formation Tissue destruction Chronic inflammation Fibrosis Degenerative disease

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Chronic inflammation, on the other hand, may be due to failure of the inflammatory process to recover from an acute event. The nature of the irritant can also cause chronic inflammation from the onset (Wakefield and Kumar, 2001). Chronic inflammation has been found to destabilize chromosomal DNA leading to mutations that could be potentially carcinogenic (Kaczor, 2010). Chronic inflammation has been found to be one of the major contributors to the development of cancer as it provides conditions that can persist for years allowing progression of disease (Aggarwal et al. , 2008). The occurrence of chronic inflammation in inflammatory bowel disease and prostatitis was shown in clinical trials to increase the risk of development of colon and prostate cancers respectively. The increase in risk makes the inflammatory process an attractive therapeutic target for cancer prevention (Kaczor,

2010). Chronic inflammation creates a microenvironment that facilitates transformation, malignancy and metastasis of cells (Khansari et al. , 2009). The effects of chronic inflammation are thought to be due to the excessive amounts of free radicals and depletion of antioxidant mechanisms. Chronic inflammation accompanied by oxidative stress has been linked to various steps involved in tumorigenesis, such as cellular transformation, promotion, proliferation, invasion, angiogenesis, and metastasis (Lai et al. , 2011).

Therefore, the treatment of chronic inflammatory conditions can become a possible target for disease chemoprevention (Kaur et al. , 2010). The therapeutic targets for resolving inflammation are numerous as the process is multifaceted. Inflammatory mediators, free radical activity and oxidative stress have been found to be attractive anti-inflammatory targets. The role of the components must be understood in order to effectively investigate inflammatory mediators as drug targets.

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1.2 Inflammatory mediators

Inflammation is mediated by chemokines that include tumour necrosis factor (TNF)-α, nuclear factor kappa beta (NF Κβ ), interferon (IFN)-γ, nitric oxide and interleukins (Hong et al. , 2009).

The mediators are from different sources and influence different reactions within the inflammatory process. Most inflammatory mediators are newly synthesized in the injured tissues or by the migrated immune cells during an inflammatory event (Wakefield and Kumar, 2001).

Pathogenic microorganisms such as bacteria activate NF-Κβ through receptors found on macrophages via several signalling pathways. The activated macrophages can release TNF-α which is responsible for the up-regulation of the production of other inflammatory mediators that include prostaglandins and nitric oxide. The activation of NF Κβ appears to play a pivotal role in the regulation of inducible enzymes as it has binding sites on the promoter regions of the genes as shown in Figure 1.1 (Franceschelli et al. , 2011). The transactivation of the gene is through the liberation of NF Κβ from its inhibitor molecule (Ikb-α). The Ikb masks the nuclear locator signal that allows the nuclear factor to move into nucleus and promotes gene expression (Posadas et al .,

2003). The removal of I Κβ is facilitated by IK kinase (IKK) that can be activated by TNF receptor or by the presence of large quantities of free radicals in the cell (Franceschelli et al .,

2011). The presence of large quantities of free radicals create conditions that allow the liberation of NF Κβ from its cytosolic inhibitor Ikb as well (Chen, 2005). IΚβ undergoes phosphorylation by IKK which is followed by ubiquination. The ubiquination tags the inhibitor molecule for degradation by proteasomes. NF Κβ is then able to move into the nucleus to promote transcription of COX-2 and iNOS genes.

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TNF

TNF receptor

ADP ATP IKK-P IKK

ubiquitination of IKB NFKB-IKB-PP NFKB-IKB-PP

IKB degradation by proteasome

NFKB nuclear localisation signal unmasked

NUCLEUS inflammatory mediators NFKB

Figure 1.1 Nuclear factor kappa beta pathway that activates inflammatory gene expression. The tumour necrosis factor (TNF) attaches to a receptor on the cell surface membrane leading to the activation of the IK kinase (IKK) that phosphorylates the nuclear factor kappa beta inhibitor molecule (Ikb) of Ikb-nuclear factor kappa beta (NFΚβ ) complex. Ubiquination follows the phosphorylation releasing NF Κβ to enter the nucleus and activate gene expression of inflammatory proteins. (Adapted from www.stat.rice.edu )

Inducible enzyme systems, cytokines and their reaction products whose expressions are influenced by NF Κβ are implicated in chronic inflammatory disease when they are improperly

5 regulated (Zhao et al. , 2009). The inflammatory response triggers the induction of enzymes that catalyse the production of important mediators. Cyclooxgenase-2 and inducible nitric oxide synthase are two examples of enzymes whose expressions are increased during inflammation.

Cyclooxygenase (COX) enzymes are membrane bound glycoproteins found in the endoplasmic reticulum (Gacche et al. , 2012). Arachidonic acid is converted by cyclooxygenase enzymes to prostaglandin H 2, the precursor molecule for all the eicosanoid molecules that include prostaglandins and thromboxanes (Bai and Zhu, 2008). There are two main isoforms of

COX enzymes that differ mostly in their pattern of expression. The isoforms share about 60% of their overall sequence and the maximum homology in the catalytically-important regions. The major differences between the isoforms are in substrate and inhibitor selectivity, their intracellular locations and the circumstances under which they are expressed. The substrate and inhibitor selectivities are found in the regions where the sequence differences between the two isoforms are located. COX-1 is constitutively expressed, that is, the enzyme is found under normal physiological conditions, throughout most of the tissues of the body. Whereas, COX-2 expression is induced by pro-inflammatory conditions during an innate immune response, although it is constitutively expressed in the brain and kidneys (Mulabagal et al. , 2011).

The expression of COX-2 enzymes is the rate limiting step in the production of the prostaglandins that act as pro-inflammatory mediators. COX-2 has been found to be overexpressed in prostate cancer cells and individuals who took non-steroidal anti-inflammatory drugs were found to have a lower incidence of the disease (Lin et al ., 2011). Studies in animal models have shown that pharmacological inhibition or genetic deletions of COX-2 have led to the suppression of tumour growth (Yan et al ., 2006). Cyclooxygenase 2 (COX-2) is frequently evaluated as an anti-cancer and anti-inflammatory target (Rayburn et al. , 2009). The inhibition 6 of COX-2 could affect the production of downstream inflammatory mediators including prostaglandins (Turini and DuBois, 2002).

1.2.1 Prostaglandins

Prostaglandins are bioactive derivatives of unsaturated fatty acids that are involved in the mediation of clotting, anti-clotting, pain, fever, inflammation and anti-inflammation (Payne,

2000). Prostaglandins are produced by the isomerization of prostaglandin H 2 by respective prostaglandin synthases (Figure 1.2) and modulate cellular and tissue responses involved in inflammation (Simmons et al ., 2004). The process begins when phospholipase A 2 (PA 2) is activated to break down membrane phospholipids to arachidonic acid. Arachidonic acid is the first precursor molecule in the production of the prostaglandins. The COX-1 and -2 act on arachidonic acid in two steps to produce prostaglandin H 2. The first step involves the cyclo- oxygenation of arachidonic acid which produces an arachidonic acid radical which is then acted on the by the peroxidase segment of the enzyme to produce prostaglandin H 2 (PGH 2) (Simmons et al ., 2004). PGH 2 then diffuses to the different prostaglandin H 2 isomerases in the cell to produce the functional terminal prostaglandins. The terminal prostaglandins produced have various functions. However, there are three prostaglandins that are known to be pro- inflammatory, prostaglandin D 2 (PGD 2), prostaglandin E 2 (PGE 2) and prostaglandin F2α (PGF 2α)

(Ricciotti and Fitzgerald, 2011). Prostaglandin D2 and prostaglandin F are responsible for vasodilation and permeability of vessels (Arima and Fukuda, 2011). However, prostaglandin E 2

(PGE 2) is particularly important due to the roles it plays in inflammation. Prostaglandin E 2 is a

7 product of microsomal prostaglandin E synthase-2, an enzyme that is mostly expressed during an inflammatory event.

Figure 1.2 Prostaglandin synthetic pathway. The damage to a cell or cells as a result of an injury leads to the activation of phospholipase A 2 (PA 2) which converts membrane phospholipids to arachidonic acid. Arachidonic acid is converted to prostaglandin H 2 by the action of the cyclooxygenases (COX1/COX2). The terminal prostaglandins are produced by specific synthases microsomal or cytosolic prostaglandin E synthase (mPGEs-1/2, cPGEs) (Adapted from Sampey et al. , 2005).

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PGE 2 has pleotropic roles in inflammation that are known to be vasodilation, stimulation of the central nervous system for the conduction of pain and its pyrogenetic effect on the hypothalamus. PGE 2 is involved in the activation of T cells that recruit more immune cells to the site of inflammation during an infection or autoimmune response (Takanishi and Rosenberg,

2013). The production of the PGE 2 has another interesting role in the activation process, it is responsible for the induction of COX-2 expression leading to a positive feedback effect that allows more PGE 2 to be produced as shown in Figure 1.3. The COX-2 induction of expression by PGE 2 leads to a positive feedback mechanism that allows more PGE 2 to be produced by the macrophages and the effector cells (Hernandez et al ., 2011). If the induction and production cycle is not properly regulated and or terminated the large quantities of PGE 2 produced can exacerbate the inflammatory response.

Figure 1.3: The relationship between cyclooxygenase and prostaglandin E 2. The toll-like receptor 4 pathway activates the expression of cyclooxygenase-2 (COX-2) in the macrophage (M Φ) and inflammatory effector cell (IEC). COX-2 expression leads to prostaglandin E 2 production that has multiple effects on the inflammatory system including induction of COX-2 expression. (Adapted from Hernandez et al. , 2011). 9

PGE 2 and its metabolite PGF 2α have another physiological role in the gonads as well that plays a part in disease, particularly in females. The prostaglandins were first isolated from human seminal vesicles, scientists believed the lipid derived molecules were localized in the prostate, hence the name prostaglandin (Zhuang et al ., 2003). The prostaglandins were later discovered in other locations and organs such as the uterus. The prostaglandins, PGE 2 and PGF α, were found to play an important role in the uterus. PGE 2 and PGF α are highly expressed during pregnancy, labour and the menstrual cycle (Sales and Jabbour, 2003). PGE 2 is responsible for muscle contraction and relaxation to allow menstrual flow. The over-expression of the bioactive fatty acids can lead to a condition known as dymensnorrhea. Dymensnorrhea is a pain disorder of the uterus that can result from the overproduction of PGE 2 and PGF α within the uterine muscles (Arif Zaidi et al ., 2012). As mentioned previously PGE 2 mediates pain by activating the neurons that transmit pain signals and overproduction of the prostaglandins leads to uterine muscle cramping due to oxygen deprivation.

The role of PGE 2 in diseases such as cancer and dysmenorrhea, therefore, should not be underestimated. PGE 2 has been shown to modulate the production of other inflammatory mediators. Milano et al. , (1995) speculated that PGE 2 production influences the production of the free radical, nitric oxide (NO) in a murine macrophage cell line.

1.2.2 Inducible nitric oxide synthase

Nitric oxide (NO) is a free radical that mediates neurotransmission, vascular homeostasis and host defence in the body. The reactive nitrogen species (RNS) is produced from the breakdown

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L-arginine by nitric oxide synthases to citrulline as illustrated in Figure 1.4 (Chae et al., 2012).

There are three isoforms of the nitric oxide synthase that are able to catalyse the reaction with L- arginine, namely endothelial nitric oxide synthase, neuronal nitric oxide synthase and inducible nitric oxide synthase. Endothelial NOS (eNOS) and neuronal NOS (nNOS) are calcium- dependent, constitutively expressed and responsible for low levels of NO production. On the other hand, inducible NOS (iNOS), is calcium-independent, not expressed under normal physiological conditions, but can be induced by an immune reaction to injury (Liu et al ., 2003).

Nitric oxide activates protein kinase pathways such as the mitogen activating protein kinase (MAPK) involved in inflammation making it a key enzyme in the inflammatory response

(Lee et al ., 2011). MAPK are involved in the cascade responsible for induction of expression of inflammatory cytokines. NO from eNOS and nNOS is released from the cell that produced it and diffuses into its target cells through the lipid membranes where the radical binds to guanylate cyclase. The guanylate cyclase produces large quantities of the messenger cyclic guanidine monophosphate (cGMP) that effects muscle contraction and inhibits platelet aggregation (Jerca et al ., 2002). NO produced by iNOS is in large quantities that can be cytotoxic to any invading pathogens while acting on effector cells to inhibit platelet aggregation and effect muscle contraction. Unregulated production of NO by iNOS during or after an inflammatory event can lead to an excess of the signalling radical circulating the site of injury (Cheenpracha et al ., 2010).

Excessive production of the radical NO has been associated with diseases such as rheumatoid arthritis, diabetes and hypertension. The enzyme iNOS, produces up to 1000 times more NO than its constitutive isoforms, eNOS and nNOS, for a relatively extended period of time (Park et al. , 2013). The increased NO production by iNOS could relate to other mediators such as prostaglandin whose overproduction are associated with development of disease. A link 11 between the production of NO and the prostaglandins has been established as well that shows that NO is involved in increasing COX-2 activity. NO has been shown to nitrosylate the cysteine residues of COX-2 thereby enhancing its catalytic activity (Kim et al ., 2005). The enhanced

COX-2 activity could increase the amount of PGH 2 available for the production of PGE 2.

Therefore, the improper regulation of the expression of iNOS and NO production can create the chronic microenvironments that are ideal for disease pathogenesis and progression of inflammatory conditions.

Aside from being an effector molecule in inflammation, NO is a highly reactive free radical. The radical state means the molecule has a lone electron that allows it to react with other molecules in order to reach the octet state to be stable. NO radical is usually short-lived as it is converted to the more stable form of nitrites by nitric oxide reductase in the cell. However, if the turnover of the enzyme is lower than the generation of the radical it can lead to protein, lipid and deoxyribose nucleic acid (DNA) damage (Selvakummar et al. , 2007). The damage to the biomolecules is as a result of the abstraction of an electron by the radical. Due to this activity,

NO is classified as part of a group of non-biological molecules that are generated by the body during cellular respiration and bioprocesses called reactive oxygen species (Halliwell, 2001).

1.3 Reactive oxygen species and oxidative stress

Reactive oxygen species (ROS) are atoms or group of atoms that has one or more unpaired electrons or molecular species that are capable of generating free radicals (Kunwar and

Priyadarsini, 2011). Inflammation generates large quantities of reactive oxidative species (ROS) that when overproduced and not eliminated can lead to damage at the site of inflammation which

12 can lead to recruitment of more immune cells (Miguel, 2010). ROS come from both normal metabolism and external sources (Lai et al. , 2011). The oxidants are produced by the body for physiological function, such as NO which is required for neurotransmission or as by-products of cellular processes such as superoxide produced by nicotinamide adenosine diphosphate (NADP) oxidase during the electron transport chain (Hamilton et al ., 2004). Free radicals can be generated during an inflammatory response called the respiratory burst. The respiratory burst is when immune cells such as phagocytes and macrophages release noxious molecules such NO, hypochlorous and hydroxyl radicals in order to kill harmful bacteria and viruses (Biller-

Takahashi et al ., 2013). The free radical species can come from the external environment as well in form of smoke, ultraviolet radiation, pesticides and air-pollution (Masoko and Eloff, 2007).

The different sources of free radicals in the cells are shown in Figure 1.4.

Exogenous sources Endogenous sources

O2 Cigarette smoke

Oxidants .- O2 UV radiation

H2O2 Free radicals in the cell

HOCl OH.

L-arginine NO. NOO.-

Figure 1.4 Endogenous and exogenous sources of free radicals in the body . UV radiation – -. ultra violet radiation O 2 – molecular oxygen, O 2 - superoxide ion, H2O2 - hydrogen peroxide, HOCl – hypochlorous acid, OH . – hydroxyl radical, NO . – nitric oxide, NOO -. – peroxynitrite

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ROS produced are usually quenched by the enzymatic and non-enzymatic reactions in the body. For instance, the superoxide and hydrogen peroxide radicals are broken down to water and oxygen by the enzymatic actions of superoxide dismutase and catalase respectively as illustrated in Figure 1.4. Glutathione (GSH) and selenium are some of the non-enzymatic mechanisms that conjugate to the radicals or quench ROS by donating an electron (Starlin and

Gopalakrishnan, 2013). However, these mechanisms can be overwhelmed or ineffective, particularly in chronic inflammatory conditions where there the free radicals can be perpetually generated (Patel et al. , 2010). High levels of oxidation may result in oxidative stress which is an imbalance between production of ROS and the capacity of the normal detoxification systems to remove the radicals (Bulbul et al ., 2011). Oxidative stress is usually harmful to the cells due to the high reactivity of the radical species that cause it.

ROS and reactive nitrogen species are free radicals, which means the molecules have a lone electron in the outer shell. The lone electron makes radicals highly reactive as they seek stability by abstracting an electron from nearby molecules (Das Sarma et al ., 2010). The reactivity and instability of ROS give them ability to react with each other as they seek stability but can instead produce even more potent free radical species. An example of the result of two radicals reacting is peroxynitrite which is produced by the combination of superoxide and nitric oxide as shown in Figure 1.5. Peroxynitrite is highly reactive with lipid and protein molecules which can lead to the formation of lipid peroxides that can chain react to produce more ROS

(Sakat et al ., 2010). Other ROS and reactive nitrogen species that are inclusive of the hydroxyl, hypochlorous, superoxide or nitric oxide radicals are able to abstract electrons from DNA, lipids and proteins. The removal of the electrons from functional biomolecules can result in loss of structure which can disrupt the proper function of the cells. ROS can facilitate the formation of 14

DNA adducts and protein cross linkages especially in transmembrane proteins as the biomolecules seek stability themselves. In conditions of oxidative stress, the probability of adducts, cross linkages and lipid peroxides forming is increased exponentially and could be the cause of disease (Khansari et al ., 2009).

Figure 1.5 Reactive oxygen species and their role in the induction of disease . Superoxide is produced during the respiratory chain from oxygen and can be broken down by superoxide dismutase (SOD) in the presence of metal ions to hydrogen peroxide (H 2O2). H 2O2 is either broken down by catalase to water or converted to the hydroxyl radical by the action of - glutathione peroxidase. NO radical can react with O 2 to peroxynitrite. Under normal physiological conditions the reactive nitrogen species (RNS) and reactive oxygen species (ROS) are required for signalling but overproduction can lead to interaction with proteins, DNA and lipids. (Adapted from Bellance et al ., 2009).

Oxidative stress is considered one of the major factors in the induction of many chronic and degenerative diseases such as cancer, Alzheimer’s and cardiovascular diseases. The excess 15

ROS produced that cannot be neutralised create a microenvironment that facilitates normal cells to transform and transformed cells to metastasise to other parts of the body (Pala and Gurkan,

2008). Free radicals have been implicated in the lesions which could be responsible for the memory loss associated with Alzheimer’s disease (Das Sarma et al ., 2010). The role ROS play in atherosclerosis is that the interaction of the radicals with the proteins and lipids that make the endothelia of the vessels allows plaques to form. The plaques attract immune cells and platelets that attempt to repair the lesion which could result in obstruction of the blood vessels (Lobo et al ., 2010).

1.4 Inhibition of inflammatory mediators

Inflammation, acute or chronic, could be resolved by inhibiting inflammatory mediators, quenching of radicals and prevention of oxidation stress. Inhibition can be reversible or irreversible depending on the pathway, molecule or process being targeted. Inhibition can target expression or production of the mediator or its activity directly (Szliszka et al ., 2011).

In order to inhibit inflammation at the level of expression of the mediators, NF Κβ would be an attractive target since it is responsible for the up-regulation of the expression of the inducible enzyme systems. Preventing or down-regulating NF Κβ translocation to the nucleus would result in less COX-2 and iNOS expression which could reduce or eliminate the inflammatory response (Lee et al ., 2011). Modulating expression of the inducible enzyme systems by inhibiting NF Κβ translocation would probably down-regulate inflammation in chronic conditions such as rheumatoid arthritis and atherosclerosis. The potential of inhibiting the translocation of NF Κβ for anti-inflammatory effect would probably prevent the adverse

16 effects associated with inhibiting constitutive forms of the enzymes. Wu et al. , (2013) showed that the Gynura bicolor extract inhibited the dissociation of NF Κβ from its inhibitor I Κβ . The inhibition of translocation of NF Κβ led to a lower expression of iNOS and COX-2. However, despite the potential of targeting translocation of NF Κβ as an anti-inflammatory mechanism, inhibiting the process might have far-reaching consequences due to the ubiquitous nature of the nuclear factor.

Although, targeting inflammatory mediators has been a means of therapy for inflammatory conditions, inhibiting the cyclooxygenases is the current medical intervention in inflammatory conditions. Most inflammatory drugs inhibit both isoforms despite COX-2 being the enzyme that is directly involved in inflammatory events (Simmons et al ., 2004). COX-1 being the constitutive form is essential for normal physiological function as it is responsible for the production of prostaglandins, thromboxanes and leukotrienes that are not pro-inflammatory

(Simmons et al ., 2004). Thromboxanes and prostacyclins are important to physiology even when there is no inflammatory response. For example, thromboxanes are involved in the clotting of blood and prostacyclins are anti-inflammatory, therefore, inhibition of COX-1 might actually retard resolution of injury (Rao, 2010). On the other hand, COX-2 inhibition would be desirable in the therapy of inflammation and to prevent the aetiology of disease due to chronic inflammation. COX- 2 has been found in large concentrations at tumour sites in gastric, pancreatic and oesophageal cancers (Rajeswari and Kriushnapriya, 2011). The selective inhibition of COX-2 activity would be ideal as it is induced when the inflammatory response is activated. As the conversion of arachidonic acid by COX-2 is considered the rate limiting step in the production of prostaglandins, inhibition of the enzyme would have an impact on the overall

17 inflammatory response. Inhibiting the enzyme has an influence on the production of prostaglandin E 2, which is heavily implicated in development of cancer (Marnett, 2009).

Regulation of prostaglandin production in the tissues is important for both maintenance of function and prevention of disease. PGE 2 is implicated in the aetiology of cancer by creating a suitable microenvironment for the development of disease. The prostaglandin has been found to be overexpressed at tumour sites (Lawlor et al ., 2010). PGE 2 promotes the inhibition of apoptosis. Metastasis of transformed cells and cell proliferation are influenced by the prostaglandin as well. PGE 2 induces angiogenesis, the development of new blood vessels that carry nutrients and oxygen to the neoplasm. All these factors are hallmarks of carcinogenesis and account for why PGE 2 is implicated in the aetiology of diseases such as cancer (Ghosh et al .,

2007, Lam et al ., 2004). Therefore, inhibition of prostaglandin E2 production would be most beneficial for the prevention of cancer. Microsomal prostaglandin E 2 synthase is a possible target for the resolution of inflammation. Inhibition of PGE 2 could influence the regulation of other mediators in the response. PGE 2 has been shown to influence the induction of COX-2 and iNOS expression in immune cells during an inflammatory event (Milano et al ., 1995).

The inhibition of nitric oxide production might another possible target. iNOS produces larger proportions of NO in a shorter period of time compared to the constitutive forms of the enzyme. NO radical has a dual role in the immune response, first as an inflammatory effector molecule that activates other pathways and then as a free radical species that can be toxic to pathogens. Both roles allow NO to be important in terms of chronic inflammation. A novel anti- inflammatory that would target iNOS specifically would probably significantly reduce occurrence of chronic inflammation and oxidative stress. The ideal target for iNOS inhibition

18 would probably be its expression after activation of the immune system to discontinue the downstream effects of the NO radical.

Prevention of oxidative stress might have an anti-inflammatory effect or prevent the commencement of chronic inflammation which may lead to disease. Antioxidants could an effective way to eliminate the free radicals that cause oxidative stress (Kumar et al ., 2010).

Antioxidants are molecules that are required in small quantities to prevent or delay the oxidation of an oxidisable substance (Charles, 2013). Oxidative stress usually comes about when the endogenous antioxidant systems are overwhelmed by reactive oxygen species and free radical generation within the cell. Therefore, antioxidants from external sources are required to restore the oxidative balance. Exogenous antioxidant compounds are beneficial in biological systems which have insufficient amounts of endogenous antioxidants. The removal of inflammatory signalling molecules such as NO might result in the down-regulation of the inflammatory response. Studies have found that there are interactions between antioxidant agents and inflammatory mediators involved in inflammatory processes (Pegg, 2009). An example of how all the inflammatory mediators and their effects play a role in the aetiology of disease is the development and progression of rheumatoid arthritis. The prevention of oxidative stress or increasing the antioxidant capacity within the cells could result in the alleviation of inflammation, if not its prevention.

1.5 Rheumatoid arthritis

Rheumatoid arthritis is a chronic systemic inflammatory disease that is typically characterised by the inflammation synovial membranes of bones (Naredo et al ., 2005, Li et al ., 2004). Symptoms

19 of the disease include pain, aching, stiffness, and swelling in or around the joints due to the inflamed synovial membranes. Rheumatoid arthritis usually has an external immune trigger that results in the release of a primary auto-antigen usually through precipitation of protein. The auto-antigen elicits an autoimmune reaction that usually involves an inflammatory response

(Scher and Abrahamson, 2013). An auto-antigen is a normal protein constituent that is able to elicit an immune response from the host as a result of decreased tolerance or recognition of self

(Larman et al ., 2010). Inflammation of synovial membranes (synovitis) leads to the accumulation of migrated immune cells acting against the auto-antigen. The immune response leads to the production of more auto-antigens as more protein molecules precipitate out of solution. The auto-antigens can lead to a persistent state of inflammation at the joints sites. The formation of the auto-antigens is the basis for the in vitro albumin denaturation assay, one of the techniques that was employed in this study. The albumin represents the protein that is denatured during inflammation to form the auto-antigens that activate the immune system against the synovial membranes (Sakat et al ., 2010). The recruited immune cells produce large quantities of the inflammatory mediators and ROS that have been previously described. Pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1β as well as PGE 2,

COX-2, and inducible nitric oxide synthase (iNOS) all are overexpressed at the site of inflammation in arthritic patients (Akaogi et al ., 2008). The over-expression of inflammatory mediators may be responsible for the perpetual state of inflammation that results in swollen joints.

PGE 2 is implicated in the activation of the Th1 pathway which has been implicated in the occurrence of rheumatoid arthritis (Yao et al. , 2009). The ROS produced during the inflammatory event are thought to be responsible for the activation of the matrix metallo- 20 proteinases such as collagenase (Sakat et al ., 2010). Collagenase breaks down the collagen at the joints leading to the tissue damage or bone deformity commonly seen in arthritic patients. If the condition is not treated or managed by medication, it can lead to deformation of the affected limbs as the synovial membranes are slowly eroded (Yanni et al ., 1994). Rheumatoid arthritis can be managed using disease modifying anti-rheumatic drugs (DMARD) and non-steroidal anti- inflammatories (NSAIDs) (Lubrano and Scarpa, 2012). Since the arthritic condition is chronic, medication is required regularly and in relatively high doses. Unfortunately, long term use of

DMARD and the anti-inflammatories has been found to have adverse effects on the body

(Aletaha et al ., 2003).

1.6 Current treatment for inflammation and oxidative stress

Analgesics relieve pain as a symptom, without affecting its cause. Current treatment of inflammatory diseases involves mainly interrupting the synthesis or action of critical mediators that drive the host’s response to injury (Tung et al. , 2008). Narcotic, steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) are currently are the current treatment options against inflammation. However, the available drugs have reduced efficacy against inflammatory conditions due to adverse effects and relatively high potency. For example, steroidal drugs are in use as anti-inflammatories due to their specific mechanisms of action that are considered to be responsible for their adverse effects as well. Steroidal anti-inflammatories inhibit basal physiological function such as leukotriene inhibition. The side effects include hypertension due to analogy of the steroidal drugs to the steroid hormones. The non-steroidal drugs have relatively fewer and less adverse effects than the steroidal that include gastrointestinal bleeding

21 and improper clotting of blood (Burk et al. , 2010). Therefore, there is a need for treatment options that have both of these characteristics. The selective inhibition of COX-2 can offer a possible solution to the anti-inflammatory agent that has these characteristics.

Selective inhibition of the expression and activity of COX-2 could result in the reduction of the inflammatory response without affecting the constitutive COX-1 (Miguel, 2010). The currently available COX-2 selective inhibitors are celecoxib and roxecib (Kaur et al ., 2010).

These clinical drugs have been found to be effective selective COX-2 inhibitors. However, they have also been considered cardio-, nephro- and hepatoxic effects due to the inhibition of the production of the baseline concentration of COX-2 that is required for normal physiological function (Kaur et al ., 2010). Two synthetic COX-2 inhibitors, rofecoxib and valdecoxib, have been withdrawn from the market by the Food and Drug Administration (FDA) due to cardiovascular complications and skin reactions (McDaid et al. , 2010). Therefore, selective inhibition of COX-2 should be low enough not to interrupt the baseline physiological function and high enough to relieve inflammation and possibly prevent disease. The selective inhibitors developed could play another role of quenching free radicals that attenuate chronic inflammation. In fact, aspirin has been shown to possess antioxidant activity and this has been speculated to another mechanism of action besides the inhibition of COX enzymes (Shi et al .,

1999). The antioxidant activity of an anti-inflammatory drug could imply that the reverse could be true and an antioxidant supplement could serve a dual role as an anti-inflammatory.

Although presently, there are no antioxidants in use clinically for the treatment of inflammation the strong link between oxidative stress and inflammation might imply that antioxidant supplements might play a role in alleviating or preventing inflammatory conditions.

Studies have been shown that vitamin E, a known antioxidant, was able to inhibit COX-2 (Akkol 22 et al ., 2012). Examples of currently available antioxidants are the synthetic compounds, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). The antioxidants are used as food additives and supplements to prevent and or remove oxidative species. However, despite being effective as antioxidants, the two compounds have been found have adverse effects that include carcinogenesis, liver and kidney toxicity (Seo et al ., 2012).

Despite the adverse effects of NSAIDs, studies conducted have postulated that regular long-term use of low dose NSAIDs has been associated with a significant reduction in the incidence of numerous cancers by approximately 40% (Lam et al ., 2004). Therefore, the ultimate goal would be to obtain anti-inflammatory agents that are effective with minimal to no adverse effects when administered over a long-term period. The agents would have the dual role of treating inflammation and preventing development of chronic and degenerative diseases.

Plants have been considered to be sources of the new anti-inflammatory agents due to their extensive use in folk medicine globally to treat various ailments (Boukhatem et al ., 2013)

1.7 Ethnopharmacology

A large proportion of the developing world’s population still relies on traditional medicine for their health care due to their accessibility, the relatively higher cost of conventional and synthetic drugs (Saleem et al ., 2011). Ethnobotanical and ethnopharmacological information on the uses of the plants appears to be passed down from one generation to the other through anthropology.

The accumulation of information on their uses can aid in the investigation on them as sources of lead compounds, complimentary or alternative medicines (Hendra et al. , 2011). Plant remedies are perceived to have advantages over synthetic drugs such as their non-narcotic nature, being

23 easily biodegradable, have minimal environmental hazards and adverse effects and are relatively affordable (Umapathy et al. , 2010).

Plants have become the focus of study in the search of new therapeutic agents as to date up to 60 % of the current clinical drugs are natural products (Gribling et al. , 2009). The search has been necessitated by the use of complementary and alternative medicines (CAM) by cancer patients to alleviate the adverse effects of chemotherapy (Ye et al ., 2009). Natural products have proved to be the most reliable source of new therapeutic agents particularly as anti-proliferative agents. Medicinal plants have become important in the health of individuals and their value lies in their ability to produce compounds that could have a therapeutic effect in the body (Chandhur et al ., 2011). The compounds include alkaloids, tannins, flavonoids and phenolic compounds that are produced by the plants as secondary metabolites. Secondary metabolites are complex substances that are synthesized from relatively simple molecules such as glucose and water. The plants produce the secondary metabolites in order to counteract or adapt to their environment.

Therefore, the type and structure of the metabolites are influenced by various factors that include the climate, soil quality, temperature, the stage of development and pathogens of the plant

(Ramakrishna and Ravishankar, 2011). Phytochemicals could serve as prototypes to develop more effective and less toxic medicines. The low toxicity of the plant derived anti- inflammatories has been attributed to the compounds being biological in nature and, therefore, metabolism by the body would be easier (Kaur et al ., 2010). Several phytoconstituents from

Aloe vera , Torreya nucifera and Thymus vulgaris have been found to have COX-2 inhibitory activity (Miguel, 2010).

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1.8 Treatment of pain using herbal remedies

Management of inflammation related diseases has become important in rural communities that rely mostly on alternative treatments from medicinal plants. Medicinal plants are used extensively in traditional medicine in the treatment of inflammatory disorders (Matthew et al .,

2013). The consumption of fruits and vegetables has been found to lower the incidence of diabetes, atherosclerosis, arthritis and acquired immune deficiency syndrome (AIDS), the diseases that are associated with chronic inflammation as well (Iwalewa et al. , 2007). Hence, the role of plants in the treatment and management of inflammation, as well as the possible prevention of diseases such as cancer, should not be underestimated.

Plant phenols, vitamins, carotenoids, phytoestrogens and terpenoids are some of the phytoconstituents that have been found to possess anti-inflammatory activity which could justify their use in folk medicines. Figure 1.6 gives an indication of the proposed mechanisms of action for the various phytochemicals thought to be responsible for the anti-inflammatory activity of plants. All the possible mechanisms of action of plant compounds illustrated in Figure 1.6 show the potential of phytochemicals in the prevention of diseases such as cancer. The carcinogen could be a potentiator such a free radical or inflammatory mediator.

Terpenoids have been shown to inhibit COX activity and scavenge free radicals, while phenols interfere with NF Κβ gene promotion and prevent lipid peroxidation (Salminen et al .,

2008). Phytoconstituents such as phenols and terpenoids are ubiquitous in plants although the actual structures might be specific to a particular species. Novel anti-inflammatory agents from plants have the potential to have multiple targets, as a phytoconstituent or a mixture, for the dispersion of inflammation. As mixtures, several phytochemicals might be present and act on

25 more than one target at a time which could result in faster and more efficient anti-inflammatory activity. However, some terpenoids and phenolic compounds have been shown to increase the activity of inflammatory mediators such as the NO radical instead of inhibiting it (Gouveia et al .,

2011).

Due to the probability of finding novel anti-inflammatory agents from medicinal plants that are already in use by herbalists, this study sought to evaluate the anti-inflammatory activity of selected Zimbabwean plant species that are used as pain remedies.

Figure 1.6: Proposed mechanisms of action of phytochemicals against inflammation . Phytochemicals interacting with various inflammatory processes. Scavengers of reactive oxygen species (ROS) or nitric oxide (NO), inhibitors of nuclear factor kappa beta (NF Κβ ) translocation, phospholipase A2, cyclooxygenase (COX) enzyme expression and activity, lipooxygenase (LOX) expression and activity and tumour necrosis factor (TNF) and interleukin 1 β expression. 26

The reliance of the rural communities on herbal remedies has not only shown a need for cheaper drugs but has raised the question of the efficacy of the decoctions as well. The herbal remedies are used based on the anthropological knowledge of a community that is passed from one generation to the other. These communication methods can be prone to manipulation and misinformation (Li et al. , 2004). Hence, there is a need to validate some of the claims of the herbalists and custodians of ethnomedicinal uses of plants and to document the use the medicinal plants with inflammatory activity. The study was conducted to ascertain the anti-inflammatory activity of selected plants in Zimbabwe used to manage pain, fever and inflammatory related ailments.

Medicinal plants can prove a source of biologically and pharmacologically active drugs

(Iwueke et al ., 2006). Some of the plants used in the treatment of inflammation in Zimbabwe are

Cassia abbreviata , Combretum zeyheri , Diospyros mespiliformis , Lippia javanica , Parinari curatefolia and Amaranthus spinosus (Chinemana et al ., 1985, Gelfand et al ., 1985). The plants are from across the plant kingdom. However, some families and genera of plant species are more prominent in their use in the treatment of inflammation. The Combreteceae family,

Parinari and Brachystegia genera are used extensively across Africa as herbal remedies to treat pain and inflammatory related ailments (Maroyi, 2013).

1.8.1 Combreteceae species

The Combreteceae family is a large family that is comprised of about 600 species that are spread over 20 genera with the Terminalia and Combretum genera being the largest with over 500 27 species put together (Lima et al. , 2012). Terminalia paniculata and Terminalia chebula are used in Asian folk medicine to treat bronchitis and wounds and as antioxidants (Kaur et al. , 2012).

Combretum species have been extensively studied due to their global use in folk medicine (Lima et al. , 2012). The Combretum species are used in the treatment of venereal disease, pneumonia, diarrhoea and cancer. The plant species in genus Combretum have a broad spectrum of uses that include the treatment of pain as indicated by the information in Table 1.2.

The interest in the Combretum genus might be due to the presence of bioactive constituents such as the combrestatins. Phytochemical studies have shown that species from the genus have had several unusual phytoconstituents that have been isolated from them. The phytoconstituents are considered to be unique to the Combretum species such as hydrophenanthrenes and a substituted bibenzyl that was isolated from C. molle (Lima et al. , 2012).

Table 1.2 Medicinal uses of Combretum species.

Plant species Condition treated Combretum hereroense Headache, infertility in women Combretum molle Stop bleeding after childbirth, headache leprosy, convulsions Combretum imberbe Stomach-ache Combretum platypetalum Infertility in women Combretum zeyheri Embrocation Combretum glutinosum Hypertension (Adapted from Lima et al ., 2012)

Combretum platypetalum is a flowering shrub that grows up to 30 cm from a woody root stock that is native to Sub-Saharan Africa. The plant has red flowers that bloom between August and

October with pink to bright red four-winged fruit as in Figure 1.7. C. platypetalum plants are

28 found at burnt roadsides and in the transitional habitats between grassland and woodland. The plant species was chosen for the study because of its use as a remedy for various pain related ailments such dysmenorrhea (Martini, 2001). Although other Combretum genus have been studied extensively for their inflammatory and antioxidant activities (Lima et al ., 2012,

Bhatnagar et al ., 2010, Gouveia et al ., 2011). Combretum platypetalum that was used in this study, to the best of our knowledge, have not yet been scientifically evaluated for its efficacy as anti-inflammatory agents in traditional medicine and the possible mechanisms of action for the herbal remedies. The anti-inflammatory activities and antioxidant activities of C. platypetalum were, therefore, investigated in this study.

Figure 1.7 Image of Combretum platypetalum growing in a burnt up area (Burt Wursten,

2004) 29

1.8.2 Parinari curatellifolia

Parinari curatellifolia is prominent plant in folk medicine in Sub-Saharan Africa. The plant decoctions are used to treat various ailments (Moshi et al ., 2010). P. curatellifolia is a mushroom shaped evergreen tree with white to pink flowers as the picture of the plant is shown in Figure 1.8. The fruit is edible with a brown to golden colour. The bark and fruit are used as an enema to relieve constipation in Burundi, the roots are used to treat venereal diseases in South

Africa and the leaves are used to treat coughs in Mozambique (Bruschi et al ., 2011). Its use in the treatment of inflammatory ailments and inflammation related condition is common throughout the Sub-Saharan region. Parinari curatellifolia mixed with Maytenus senegalensis is used as a pain remedy in Tanzania (Moshi et al. , 2010). Its documented use in Zimbabwe is that the roots are used in the treatment of diarrhoea. Its prominent therapeutic use in inflammatory ailments in neighbouring countries prompted its inclusion in the study.

Ogunbolude et al. , (2009) investigated the antioxidant activity of P. curatellifolia seeds to determine if that could the mechanism for its anti-diabetic activity. The antioxidant activity of the plant could contribute to its anti-inflammatory activity. However, the anti-inflammatory and antioxidant activity of Parinari curatellifolia leaf extracts was yet to be investigated.

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Figure 1.8 Image of Parinari curatellifolia leaves. (Rob Burret, 2011).

1.8.3 Other plant species used to treat inflammation

The Gymnosporia genus (which can also classified as the Maytenus genus) species are used in

African countries to treat pain, fevers and gastrointestinal diseases in Sub-Saharan Africa particularly in Kenya, Senegal and Zimbabwe (da Silva et al ., 2011). Maytenus heterophylla is used to relieve dysmenorrhea in Zimbabwe and M. senegalensis (G. senegalensis ) is a toothache remedy in India (da Silva et al ., 2011). Phytochemical studies on the genus have shown the presence of tannins, saponins and triterpene compounds that are known to be biologically active.

Isolated compounds were found to be anti-bacterial, anti-leukemic and active against viral

31 proteases. Few studies have been conducted on the possible mechanisms of action of the plant species as anti-inflammatory agents. Gymnosporia senegalensis was investigated for its anti- inflammatory activity in this study. The plant species is ubiquitously distributed throughout the savannah regions if tropical Africa (da Silva et al. , 2011).

Brachystegia species are common to the miombo woodland that are used primarily used as an energy source for cooking and heating in rural homes (Maquia et al ., 2013). Brachystegia boehmii is used as dymensnorrhea remedy in Mali (Sanogo, 2011). A decoction of the B. boehmii bark is used as a remedy for venereal disease (Maroyi, 2013). Despite knowledge of the medicinal use of B. boehmii , few studies have been conducted on the anti-inflammatory and antioxidant activities of the plant species.

1.9 Bioassay model systems

The study of the activity of medicinal plant extracts employs the use of model systems when conducting laboratory based evaluations. The model systems represent the possible scenarios that could occur in a biological system. The study conducted used several model systems to evaluate the antioxidant activity through free radical scavenging, nitric oxide production in a murine macrophage cell line and the activation of nitric oxide production in the cell line by redox cycling compounds.

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1.9.1 Antioxidant assays

There are various methods that are used to evaluate the antioxidant capacity of a compound and formulations or mixtures of compounds in body fluids or in food. The methods include electron resonance, chemiluminescence and analytical methods (Prakash et al .). The analytical methods are usually preferred due to their accessibility, sensitivity and affordability. Analytic methods employ measuring the antioxidant activity of a sample by its ability to quench (that is to donate an electron) a known radical. However, the same sample can give different results when tested against different radical species (Prakash et al.).

The molecule, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) is a stable radical that is used to measure antioxidant activity. The lone electron on the radical molecule allows it to absorb maximally at 517 nm which shifts to 540 nm when the radical is reduced by accepting an electron or hydrogen as shown in Figure 1.9 (Bahar et al. , 2013).

Figure 1.9 DPPH radical quenching. The DPPH radical is purple and absorbs maximally at 517 nm, when it accepts a hydrogen from a donor the absorption maxima of the now yellow to colourless solution shifts to 540 nm. (Adapted from Bahar et al. , 2013).

33

The tetramethoxy azobismethylene quinone (TMAMQ) radical shown in Figure 1.10 was generated from syringaldazine by laccase. The radical generated was shown to be stable enough to use to test antioxidant capacity of substances. The principle of the assay is based on the colour change from purple to colourless (Prasetyo et al ., 2010).

Figure 1.10 Structure of Tetramethoxy azobismethylene quinone (TMAMQ) molecule.

The antioxidant activity of a substance is considered as the rate constant of the substance and a given radical, while the antioxidant capacity of the substance is the number of moles of the radical that are scavenged. There are several mechanisms that antioxidants employ for scavenging free radicals that include the hydrogen atom transfer (HAT) and the single electron transfer (SET) (Charles, 2013). The stable radicals described above that were used in the study fall under the SET mechanism of action. Free radical scavenging activity against the radical would suggest a possible antioxidant mechanism.

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1.9.2 RAW 264.7 cell line

The RAW 264.7 cell line was used in the study as a model system for human macrophages.

RAW 264.7 cell line is a line that was developed by transforming murine (mouse) macrophages derived from a male mouse, with the Abelson murine leukemic virus (attc.org). The RAW cell line gets its name from the last names of the scientists who developed the cells, Ralph, Raschke and Watson (RAW) and is shown in Figure 1.11. The macrophage cell line is used as a model system to screen for anti-inflammatory agents. The cells can be externally activated by antigens such as the Escherichia coli endotoxin, lipopolysaccharide (LPS) to produce inflammatory mediators and cytokines. The enzymes, effector free radicals and signalling pathways can be investigated using various detection techniques.

Figure 1.11 RAW 264.7 cells seen under a light microscope. The cells shown were passaged overnight and are typical of what can be seen under a microscope during a cell count. (Adapted from phe-culturecollections.org).

35

The enzymes that are frequently measured in activated RAW cells are COX-1 and 2 and the iNOS due to their importance in the search for new anti-inflammatory agents. The expression and activity of iNOS can be determined through reverse transcriptase polymerase chain reaction

(RT-PCR) and the Griess reagent assay respectively. The transcription factor NF Κβ can be assayed for as well for its role in the expression of COX and NOS enzymes. NF Κβ nuclear activity has been found to be influenced by the oxidative state of the cell. Compounds such as menadione are able induce oxidative stress in cells by producing free radicals (Criddle et al .,

2006).

1.9.3 Menadione

Menadione is a naphthoquinone compound that has the ability to induce oxidative stress through redox cycling (Malorni et al. , 1993). Menadione undergoes redox cycling through the action of

NADPH-P450 reductase to produce a semi-quinone radical. The semi-quinone radical cycles back to menadione by reacting with molecular oxygen to yield superoxide radicals as shown in

Figure 1.12 (Criddle et al. , 2006). The superoxide radicals are able to induce oxidative stress on the cell. The oxidative stress can act as a means of liberating NF κβ which is responsible for activating the expression of iNOS (Chen and Cederbaum, 1997). The activities iNOS and cyclooxygenase-2 (COX-2) are regulated by NF Κβ (Khan et al. , 2011).

36

Figure 1.12 Menadione redox cycle. Menadione is reduced by quinone reductase to produce superoxide radicals through redox cycling to a semi-quinone then to a hydroquinone. (Adapted from Criddle et al ., 2003)

1.10 Rationale of study

Inflammation plays a role in the pathogenesis of several chronic disorders such as rheumatoid arthritis, cancer, neurodegenerative disease and premature aging. . The current treatment options to reduce the amount of prostaglandin E produced by the cells are non-steroidal anti- inflammatory drugs (NSAIDs) (Li et al. , 2005). NSAIDs inhibit the production of all the prostaglandins which include the thromboxanes required for blood clotting. Chronic use of

NSAIDs has been found to have adverse effects such as gastrointestinal irritation and

37 nephrotoxicity (Payne, 2000). Plants have been used globally in the relief of pain and inflammation. One of the NSAIDs currently in use, aspirin, was synthesised from a plant derived compound that was extracted from the Salix species (Roberson, 2008). Natural COX-2 selective inhibitors have been proposed to have lesser side effects as they have been found to be less selective than the current synthetic inhibitors (Kaur et al. , 2010). Literature indicates that the anti-inflammatory activities of the plant species selected for this study were yet to be evaluated scientifically. Inflammation is a complex process that can be resolved by targeting different reactions. The study conducted sought to determine some of the mechanisms of action of selected plant extracts that are used to relieve pain and inflammation.

1.11 Hypothesis

Zimbabwean medicinal plants used in the treatment of pain and inflammation could possess compounds that can inhibit enzymes and processes that are involved in causing pain and inflammation.

1.12 Objectives

1.12.1 Main objective

The main objective of the study was to seek to confirm the inhibitory effect of Zimbabwean medicinal plants on enzymes and processes involved in pain and inflammation.

38

1.12.2 Specific objectives

The specific objectives of the study were to:

1) Identify, document and collect plant samples used traditionally to reduce pain related ailments and to prepare extracts of the selected medicinal plants.

3) Determine anti-inflammatory activity of the prepared plant extracts by evaluating their effect of on the enzymatic activity of cyclooxygenase-1 and -2.

4) Isolate phytoconstituents from Combretum platypetalum and evaluate their effect on COX-2 activity.

5) Determine the antioxidant activity of the five most potent plant extracts by measuring their free radical scavenging activity.

6) Evaluate the effect of five selected plant extracts on albumin denaturation and membrane stability.

7) Determine the effect of the potent plant extracts from Combretum platypetalum and Parinari curatellifolia on nitric oxide production in lipopolysaccharide (LPS) activated RAW 264.7 cell line.

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1.13 Overview of study

Figure 1.13 shows an overview of the study and how it was conducted. The study started with screening for anti-inflammatory activity. The plants that were found to have anti-inflammatory activity against the cyclooxygenase enzymes were further investigated.

40

Screening of 8 Zimbabwean medicinal plant extracts anti- inflammatory activity using cyclooxygenase enzymes

Fractionation of Combretum platypetalum to isolate Determination of antioxidant activity of 6 phytoconstituents plant extracts using DPPH and TMAMQ radicals Determination of the anti- inflammatory activity of isolated phytoconstituents from Combretum platypetalum

Determination of membrane Determination of protein stabilization activity of 5 denaturation inhibition activity plant extracts using the of 5 plant extracts using the sheep erythrocyte albumin denaturation

stabilization assay inhibition assay

Nitric oxide (NO) production assays in RAW 264.7 cells

Activation of NO production in RAW cells using ROS generating

compounds

Determination of NO production in the presence of redox compounds and 4 plant extracts

Determination of NO Determination of NO production in LPS activated production in LPS activated RAW cells in the presence RAW cells in the presence of Combretum platypetalum of Parinari curatellifolia

41

Figure 1.13 Flowchart of the study conducted. The flowchart shows the steps taken and relationships between the different assays conducted.

CHAPTER TWO

Materials and Methods

2.1 Chemicals

The chemicals used were obtained from Sigma-Aldrich (Darmstadt, Germany) and local reputable sources. The list of reagents includes Dulbecco’s modified eagle medium (DMEM) supplemented with foetal bovine serum, antibiotics, indomethacin, iodine, potassium iodide, menadione, carbon tetrachloride, hydrogen peroxide, diphenyl picrylhydrazine (DPPH), bovine serum albumin (BSA), dextrose, sodium citrate, citric acid and sodium chloride. The assay kit used in the COX Enzyme Inhibitor Screening Assay Kit® was obtained from Cayman Chemical

Co. (Tallinn, Estonia). Tetramethoxyazobismethylene quinone (TMAMQ) was a kind gift from

Prof. G. Nyanhongo of the Institute of Environmental Biotechnology (Petersgasse, Austria).

2.2 Cell lines

The RAW 264.7 cell lines that were used in this study were obtained from Sigma-Aldrich

(Darmstadt, Germany).

2.3.1 Plant selection and collection

The plant samples, Parinari curatellifolia , Combretum molle , Combretum zeyheri , Combretum platypetalum , Gymnosporia senegalensis Brachystegia boehmii , Amaranthus spinosus and 42

Cassia abbreviata , used in this study were selected based on literature by Gelfand et al. , (1985) and Chinemana et al. , (1985) that documented the uses of plants as herbal pain remedies by traditional medical practitioners in Zimbabwe. The plant samples were collected from Norton

(17.88° S, 30.70° E, and 1360 m above sea level) in Mashonaland West and Centenary (16.80°

S, 31.11° E, 1156 m above sea level) in Mashonaland Central provinces in Zimbabwe. The plant samples collected were authenticated by Mr Christopher Chapano, a taxonomist at the National

Herbarium located at the Harare Botanical Gardens, Harare, Zimbabwe. Voucher specimens were made and stored in the Biomolecular Interactions Analyses Laboratory at the Department of Biochemistry, University of Zimbabwe, Harare, Zimbabwe.

2.3.2 Plant extraction

The plant leaf samples as shown in Table 2.1 below were dried in an oven (Jeio Tech, Seoul,

Korea) at 50 oC. The dried leaves were ground using a two speed blender (Cole-Palmer, Vernon

Hills, USA). The plant powder was weighed to determine the amount of solvent required for extraction of plant compounds. The ethanol extract was prepared by adding ethanol at a ratio of

4 ml to 1 g of plant leaf material. The mixture was shaken for 5 mins and allowed to sit for 1 hour at room temperature. The water extract was prepared by adding water at a ratio of 10 ml of water to 1 g of plant material. The mixture was stirred overnight using a magnetic stirrer

(Minor-2 Voss Instruments LTD Malden, Essex). The mixtures were then filtered through a muslin cloth and filtered twice using Whatman no.1® (Sigma-Aldrich, Darmstadt) paper into a clean glass vial. The extract was dried to a constant weight under an airstream. The residue was

43 then reconstituted in absolute ethanol or dimethyl sulphoxide (DMSO) and stored at 4 °C and at room temperature respectively.

44

Table 2.1: Zimbabwean plant used for pain and inflammation that were collected and extracted

Family Botanical Name Local (Shona) Parts used Major name Ethnomedical uses Amaranthaceae Amaranthus spinosus Mhowa Leaves Detoxicant and L. fevers Adjanohoun et al. , (1991) Leguminosae Cassia abbreviatta Muremberembe Leaves Abdominal pain Oliv. Chinemana et al. , (1985) Chrysobalanaceae Parinari Muhacha Leaves Coughs Peni et curatellifolia Planch al ., (2010) ex Benth Leguminosae Brachystegia Mupfute Leaves Back pain boehmii Taub Gelfand et al ., (1985) Combretaceae Gymnosporia Chizhuzhu Leaves Chest pain and senegalensis Loes. pneumonia Arnold and Gulumian, (1984) Combretaceae Combretum zeyheri Mubhondo Leaves Coughs, Sond diarrhoea and candidiasis Raffo, (1999) Combretaceae Combretum molle Muruka Leaves Malaria and pain Engl. & Diels. Gronhaug et al. , (2008) Combretaceae Combretum Bepu Leaves Swelling, platypetalum Welw. dysmenorrhoea ex M.A. Lawson and pneumonia Martini, (2001)

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46

2.4.1 Qualitative analysis phytoconstituents from Combretum platypetalum

The phytoconstituent analyses of the plant sample, Combretum platypetalum , was conducted due to the COX-2 selective activity of the crude extract. Alkaloids, flavonoids, saponins, tannins and cardiac glycosides were determined according to the method by Kaur and Arora (2009).

Alkaloids were determined by extracting 1 g of powdered sample with 5 ml methanol and 5 ml of 2 M HCl and then treating the filtrate with Wagner’s reagent (2 % iodine, 6 % potassium iodide). The samples were scored positive based on turbidity or precipitation.

Flavonoids were confirmed by heating 1 g sample with 10 ml ethyl acetate over steam bath (40 oC) for 5 min. The filtrate was treated with 1 ml dilute ammonia. A yellow coloration indicated the presence of flavonoids.

The presence of tannins were confirmed by boiling 0.5 g powdered sample in 20 ml distilled water and then adding 3 drops of 5 % FeCl3 to the filtrate. The development of a brownish-green or blue black coloration indicated the presence of tannins.

The presence of saponins were confirmed by boiling 1 g of powdered sample in 10 ml distilled water for 15 min and after cooling the extract was shaken vigorously. The formation of froth confirmed the presence of saponins.

The presence of cardiac glycosides were confirmed by extracting 2 g sample in 10 ml methanol. The methanol extract was treated with 2 ml glacial acetic acid containing 1 drop 5 %

FeCl 3 solution. The solution was carefully transferred to the surface of 1 ml concentrated

H2SO 4. The formation of a reddish brown ring at the junction of two liquids was indicative of cardenolides/cardiac glycosides.

The terpenoids were identified according to the method by Edeoga et al. , (2005). The presence of terpenoids was confirmed by extracting 2 g sample in 8 ml ethanol and filtering and 47

5 ml of the extract was mixed in 2 ml of chloroform, and 3 ml of concentrated H 2SO 4 were carefully added to form a layer. A reddish brown colouration of the inter face was formed to show positive results for the presence of terpenoids.

2.4.2 Isolation of phytoconstituents from Combretum platypetalum

The alkaloids, flavonoids, saponins and tannins were isolated from C. platypetalum leaves according the method by Harborne, (2005). The alkaloids were isolated by mixing 1 g of powdered leaf sample with 1 ml of 10 % (v/v) ammonia solution and extracting with 5 ml ethanol for 10 minutes in a water bath at 40 °C. The extract was then filtered through Whatman

No. 1 ® (Sigma-Aldrich, Darmstadt) and the filtrate was concentrated by air drying under a fan and stored at 4 °C after reconstitution in methanol. .

The flavonoids were isolated by heating 1 g of dried and ground leaves with 5 ml of methanol in a water bath at 40 °C for 10 minutes. The extract was filtered and concentrated to a quarter of its original volume and stored at 4 °C.

The tannins were isolated from dried ground leaves of C. platypetalum by treating 1 g sample with 10 ml 2 M hydrochloric acid and hydrolysing in a boiling water bath for 30 minutes.

The solution was filtered and mixed thoroughly with 1 ml ethyl acetate. The ethyl acetate layer was then discarded. Five drops of amyl alcohol were added and shaken thoroughly. The alcoholic layer was retained and stored at 4 °C.

Saponins were isolated according to the method by Obadani and Ochuko, (2001), by extracting 1 g of sample in 5 ml methanol and heating in a water bath at 40 °C for 10 minutes.

The extract was filtered and evaporated to 1 ml, mixed with 0.5 ml water and then extracted thrice with 3 ml of n-butanol. The n-butanol phase was evaporated and concentrated to 1 ml.

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The isolated phytoconstituents were used in the COX activity assays in section 2.5.3 and in the nitric oxide production assay in section 2.9.

2.5 Cyclooxygenase Activity Assays

The cyclooxygenase enzymes are responsible for the conversion of arachidonic acid to prostaglandin H 2 (PGH 2). PGH 2 was converted to prostaglandin E 2 and prostaglandin F 2α. The amount of the PGE 2 and PGF 2α present in the sample was quantified by enzyme linked immunosorbent assay (ELISA). The principle of the assay is depicted in Figure 2.1.

Figure 2.1 COX catalysed reaction. Arachidonic acid is converted to PGH 2 by the COX isoforms. PGH 2 reacts with stannous chloride to produce PGE 2 and PGF 2 that is measured by an ELISA.

2.5.1 COX Activity Assay

The effect of the plant extracts on the cyclooxygenase enzymes was determined using a COX

Inhibitor Screening ® kit (Cayman Chemical Co., Tallinn, Estonia). The assay was conducted according the manufacturer’s instructions. The reaction buffer was pre-equilibrated in a water bath at 37 oC. Aliquots of 20 µL each of COX-1 and -2 were transferred to 500 µL micro tubes and placed in boiling water for 3 minutes to inactivate the enzymes. The background tubes were setup by transferring 970 µL of reaction buffer, 10 µL of heme and 10 µL of each enzyme that was inactivated by boiling to the appropriate tubes and vortexing. COX-1 and COX-2 100 % initial activity were setup by transferring 950 µL of reaction buffer, 10 µL of heme, 10 µL of 49

COX-1 or COX-2 and 20 µL of the solvent of the extract and vortexed. The COX-1 and COX-2 inhibitor tubes were set up by transferring 950 µL of reaction buffer, 10 µL of heme, 10 µL of

COX-1 or COX-2 and 20 µL of ethanol extract or the positive control, indomethacin at a final concentration of 100 µg/ml. All the test tubes were incubated in a water bath at 37 oC for 10 minutes. The reaction was initiated by addition of 10 µL of arachidonic acid to all the reaction tubes were mixed by vortexing. The tubes were incubated at 37 oC for 2 minutes. The reaction was stopped by adding 50 µL of 1 M HCl to all the reaction tubes and 100 µL of saturated stannous chloride was added to each tube after removal from the water bath and mixed. The tubes were incubated at room temperature for 5 minutes. The prostaglandins produced were measured by the enzyme immunosorbent assay given in section 2.5.2.

2.5.2 Enzyme Immunosorbent Assay

The prostaglandin screening standards were prepared by two fold serial dilution. Eight clean test tubes were appropriately labelled 1 to 8. Into tube 1, a volume of 800 µL of EIA buffer was added, 200 µL of the bulk standard was transferred in the tube and the mixture was mixed thoroughly. An aliquot of 500 µL was taken from tube 1 and added to 500 µL of EIA buffer in tube 2. The mixture was mixed thoroughly. An aliquot of 500 µL was taken from tube 2 and added to 500 µl of EIA buffer in tube 3. The mixture was mixed thoroughly. The dilutions were repeated up to tube 8. The reaction tubes were diluted in the ratios 1: 100, 1: 2000 and 1: 4000.

The 1: 2000 and 1: 4000 dilutions were used in the EIA. The setup of the plate is shown in Table

2.2. The plate was incubated for 18 hours at room temperature on an orbital shaker (Boelker,

Germany). The plate was emptied and rinsed 5 times with wash buffer. Aliquots of 200 µL were added to all the wells and 5 µL of tracer was added to the total activity well. The plate was

50 covered with a plastic film and allowed to develop in the dark for 60 to 90 minutes. The plate was then read at the dual wavelength of 405 and 450 nm using a microplate reader (Biotek,

Winooski, USA).

Table 2.2: Summary of pipetting into EIA plate

Well EIA buffer Standard Tracer (µL) Antiserum (µL) (µL) /sample (µL) Blank - - - - Total activity - - 5 - NSB 100 - 50 - B0 50 - 50 50 Standard/ - 50 50 50 sample

2.5.3 IC 50 determination of Combretum platypetalum and indomethacin

The IC 50 is the concentration of an inhibitor that is able to decrease enzymatic activity by 50 %.

Combretum platypetalum plant extract and the indomethacin were serially diluted two fold in their respective solvents with final concentrations of 0.1 mg/ml to 5 mg/ml and 0.15 mg/ml to

2.5 mg/ml respectively. Each concentration of C. platypetalum extract and indomethacin was incubated with COX-2 as in section 2.5.1. The inhibition activity was determined as in section

2.5.2.

2.5.4 COX activity assay with phytoconstituents isolated from Combretum platypetalum

Phytoconstituents that were isolated from Combretum platypetalum according to the method in section 2.4.2 were evaluated for their effect on COX-2 catalytic activity. The isolated phytoconstituents were each incubated with COX-2 as was done in section 2.5.1. The respective

51 solvents of each of the phytoconstituents were used as the negative controls. The effect of each phytoconstituent on COX-2 was measured using the ELISA method in section 2.5.2.

2.6 Antioxidant assays

Free radical species play an important role in inflammatory events, as mediators, effectors and by-products of reactions that occur during inflammation. Stable radical species are used as models for free radicals that are produced in vivo . The radical species were tested in vitro to investigate the potential of the plant extracts as free radical scavengers.

2.6.1 Diphenyl picryl hydrazine (DPPH) free radical scavenging assay

The antioxidant activity of the plant extracts was determined by the 1, 1-diphenyl-2- picrylhydrazyl (DPPH) assay according to the method by Govindappa et al ., (2011). The DPPH radicals are scavenged by the plant extracts and the antioxidant activity. The plant extracts were serially diluted two fold from 1000 to 15.25 μg/ml. A 500 μL aliquot of the test extracts was mixed with 500 μL of 100 μM methanolic solution of DPPH. The mixture was incubated for 30 min in the dark at ambient temperature. The absorbance of the test solution was measured using a Spectramax Plus® UV-Vis microplate spectrophotometer (Molecular Devices Inc., California,

USA) at 517 nm. Percentage inhibition was calculated using the formula:

Abscontrol - Abssample Percentage scavenging = x 100 % Abs control

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2.6.2 Tetramethoxyazobismethylene quinone (TMAMQ) antioxidant assay

The tetramethoxyazobismethylene quinone (TMAMQ) free radical assay was conducted according to the method developed by Prasetyo et al ., (2010) with modifications. A 0.04 mg/ml stock of TMAMQ was prepared in analytical grade acetonitrile and diluted in the ratio 1:2 in absolute ethanol. A 56 μM stock solution in ethanol was used to prepare dilutions of 48, 40, 20,

10, 8, 4 and 2 μM in absolute ethanol. The absorbance was read at 520 nm using a Spectramax

Plus® UV-Vis microplate spectrophotometer (Molecular Devices Inc., California, USA). The absorbance values were used to construct a calibration curve to determine the linearity and stability of the TMAMQ radical at the different concentrations. The 56 μM stock solution was mixed with an equal volume of each ethanol extract and incubated for 1 hour at 30 °C. The absorbance of the samples was read at 520 nm using a Spectramax Plus® UV-Vis microplate spectrophotometer (Molecular Devices Inc., California, USA). The free radical scavenging activity for the radical was calculated using the formula below:

Abscontrol - Abssample Percentage scavenging = x 100 % Abs control

2.7 Protein assays

Two protein assays, the albumin denaturation inhibition and sheep erythrocyte membrane stabilisation, were employed as in vitro model systems to investigate alternative mechanisms of action for the potential anti-inflammatory activity of five plant extracts. These were that are used across Zimbabwe to treat pain and inflammatory symptoms.

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2.7.1 Albumin denaturation inhibition assay

The albumin denaturation assay seeks to eliminate the use of live specimens in drug discovery process. Bovine serum albumin when heated, denatures and expresses antigens associated with type III hypersensitive reaction and which are related to diseases such as rheumatoid arthritis.

NSAIDs were shown to stabilize bovine serum albumin (BSA) from denaturation. The albumin denaturation inhibition assay was conducted according to the method by Kumar et al ., (2011). A solution of 0.2 % w/v of bovine serum albumin (BSA) was prepared in Tris buffered saline and pH was adjusted to 6.8 using glacial acetic acid. Stock solutions of the extracts were diluted to 5 different concentrations between 1 µg/ml and 500 μg/ml using methanol as a solvent. A 50 μL aliquot of each extract is transferred to tubes using 1ml micro pipette. All the reaction tubes had

5 ml of 0.2 % w/v BSA added to them. The control consisted of 5 ml 0.2 % w/v BSA solution with 50 μL methanol. The standard is 100 μg/ml of indomethacin in methanol with 5ml 0.2 % w/v BSA solution. The test tubes were heated at 72 °C for 5 minutes and then cooled for 10 minutes. The absorbance of these solutions was measured by a Spectramax Plus® UV-Vis microplate spectrophotometer (Molecular Devices Inc., California, USA) at a wavelength of 660 nm. The percentage inhibition of precipitation was determined on a percentage basis relative to the control using the formula:

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2.7.2 Membrane stabilization anti-inflammatory assay

The erythrocyte membrane stabilization assay was done according to the method by Sachar et al ., (2011). The erythrocyte membrane stabilisation assay was conducted using sheep blood.

Blood was collected from a healthy sheep. The collected blood was mixed with an equal volume of sterilized Alseiver solution (2 % dextrose, 0.8 % sodium citrate, 0.05% citric acid and 0.42 % sodium chloride in 100 ml of water). The blood was centrifuged at 3000 rpm and the packed cells were washed with isosaline (0.85%, pH 7.2). A 10 % v/v suspension of the erythrocytes was made with isosaline. The assay mixture contained the extracts at various concentrations, 1 ml phosphate buffer (0.15 M, pH 7.4), 2 ml of hypo saline (0.36%) and 0.5 ml of the 10 % red blood cell suspension. Indomethacin was used as the reference drug. Instead of hypo saline, 2 ml of distilled water were added to the negative control. All the assay mixtures were incubated at

37°C for 30 min and centrifuged. The haemoglobin content in the supernatant solution was estimated using a Spectramax Plus ® UV-Vis microplate spectrophotometer (Molecular Devices

Inc., California, USA) at 560 nm. The percentage haemolysis was calculated by assuming the haemolysis produced in the presence of distilled water is 100 %. The percentage of erythrocyte membrane stabilization or protection was calculated by using the formula:

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2.8 Nitric oxide production Assays

Nitric oxide is produced by immune cells including macrophages when the inflammatory response is activated. Macrophage cell lines such as the RAW 264.7 cell line can be activated by external triggers to produce nitric oxide in vitro .

2.8.1 Cell culture of RAW 264.7 cell line

Macrophages play a central role in the initiation and propagation of the inflammatory process and serve as an interface between innate and adaptive immunity. A 1 ml aliquot of RAW cells was taken from vial and added to 10 ml of DMEM media and incubated at 37 °C and 5 % CO 2.

The media from the original vial was decanted and fresh media was added and the vial was

® incubated at 37 °C and 5 % CO 2 in Shell Lab CO 2 incubator (Sheldon manufacturing Inc.,

Cornelius, USA). The confluent cells underwent trypinisation in order to conduct the cell count with the Trypan blue ® exclusion assay. A 200 µL aliquot of the trypsinised cells was mixed with

100 µL of Trypan blue ®. The number of cells was determined using a haemocytometer. Viable cells would exclude the dye while dead cells would take up the dye appearing blue.

2.8.2 Determination of the effect of compounds on nitric oxide production

The total number of RAW cells counted as in section 2.8.1 and diluted to obtain a final cell concentration of 2 x 10 5 cells/ml in the assay well of a twelve well plate. A volume of 1.9 ml of

DMEM was placed in a well and 1 ml of the cell suspension was added and mixed by gentle flushing. A 100 µL aliquot of the test compound (100 µM) was added to a final concentration of

® a 100 µM. The plate was incubated at 37 °C and 5 % CO 2 in a Shell Lab CO 2 incubator

56

(Sheldon manufacturing Inc., Cornelius, USA) for 24 hrs. The contents of each well was centrifuged at 1500 rpm in a PLC-02 ® benchtop centrifuge (Gemmy Industrial Corp., Taipei,

Taiwan) for 10 mins. The effect of the compounds on nitric oxide production was determined on the cell free supernatants by the nitrite quantification assay described in section 2.8.4.

Menadione and hydrogen peroxide were selected for combination assays with 4 plant extracts.

RAW 264.7 cells were cultured and counted as in section 2.8.1. A 1.8 ml volume of DMEM was added to all the wells of a 6 well plate followed by 1 ml of the diluted cell suspension. A 100 µL volume of compounds was added to the appropriate wells followed by the extract or isolated

® phytoconstituents. The plate was incubated at 37 °C and 5 % CO 2 in a Shell Lab CO 2 incubator

(Sheldon manufacturing Inc., Cornelius, USA) for 24 hrs. The contents of the wells were centrifuged at 1500 rpm for 10 minutes in a PLC-02 ® benchtop centrifuge (Gemmy Industrial

Corp., Taipei, Taiwan) for 10 mins. The cell free supernatants were measured by the nitrite quantification assay described in section 2.8.4.

2.8.3 LPS activated Nitric oxide production inhibition assay

Cells such as macrophages can express iNOS, the enzyme which is responsible for the production of large amounts of NO when stimulated by antigens such as the Escherichia coli endotoxin, lipopolysaccharide (LPS) (Makchuchit et al. , 2010). The total number of RAW cells counted were diluted to a final cell concentration of 2 x 10 5 cells/ml in the assay well of a twelve well plate. A 1.87 ml volume of DMEM was placed in a well and a 1 ml aliquot of the cell suspension was added and mixed. LPS was added to a final concentration of 100 ng/ml in the appropriate wells. The plant extracts were added to the appropriate wells in combination with

57

LPS and alone to a final concentrations of 250 to 15.6 µg/ml. The control wells contained media

® and cells only. The reaction plate was incubated at 37 °C and 5 % CO 2 in a Shell Lab CO 2 incubator (Sheldon manufacturing Inc., Cornelius, USA) for 24 hrs or 48 hrs. The contents of the wells were centrifuged at 1500 rpm in a PLC-02 ® benchtop centrifuge (Gemmy Industrial

Corp., Taipei, Taiwan) for 10 mins. The cell free supernatants were measured for the amount of

NO produced by each experiment using the nitrite quantification assay described in section 2.8.4.

2.8.4 Nitrite quantification assay

The effect of the plant extracts on the production of nitric oxide was determined by quantifying the amount of nitrites in the samples. NO is converted to nitrite ions in the presence of oxygen.

The principle of the assay is that the nitrite ions react with sulphanilamide in acidic conditions to a diazonium salt that in turn reacts with naphylethyldiamine (Giustarini et al ., 2008). The reaction produces a pink azo product that absorbs maximally at 540 nm as illustrated in Figure

2.2. The standard curve was prepared by two fold serially diluting 100 µM solution of sodium nitrite in a microplate to a final concentration range of 20 to 1.25 µM. The contents of each sample well were centrifuged at 1500 rpm for 10 minutes. The cell free supernatants of the test samples were added to wells of the microplate. An equal volume of Griess reagent (1 % sulphanilamide and 0.1 % N, (1-N-naphyl) ethyldiamine in 2.5 % phosphoric acid) was added to the standard wells and test solutions and the plate was incubated for 10 minutes in the dark at room temperature. The reaction and standard curve samples were read in a Spectramax Plus ®

UV-Vis microplate spectrophotometer (Molecular Devices Inc., California, USA) at 540 nm.

58

Figure 2.2 Schematic diagram of the determinaion and quantification of nitrite ions by Griess assay . Nitric oxide is converted to nitrites in the presence of oxygen. Sulphanilamide reacts with the nitrite ions to form a diazonium salt that reacts with N-(1- Naphthyl)ethylenediamine to form a pink azo dye that absorbs at 540 nm. (Adapted from Tarpey et al ., 2003)

2.9 Statistical analysis

Data analyses were performed using GraphPad version 5 and 6 Instat software ® (GraphPad

Prism Inc. San Diego, CA, USA). Levels of significance were determined using ANOVA using 59 the Dunnet post-test where all columns of treatments were compared to the control. All data were expressed as mean ± standard deviation. P ≤ 0.05 values or less were considered to indicate statistical significance.

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CHAPTER THREE

Results

3.1 The effect of plant extracts on COX inhibitory activity

The anti-inflammatory activity of the plants selected shown in Table 2.1 was initially screened using the COX enzyme activity inhibitory assay as indicated above in section 2.6. Table 3.1 below shows the results that were obtained.

Eight plant extracts were tested for their effect against COX-1 and COX-2 enzymes using the

COX activity assay. Indomethacin, an NSAID, was used as the positive control. The results, shown in Table 3.1, show the effect of the plant extracts tested on the cyclooxygenase enzymes.

The five plants that were most effective at inhibiting the activity of the COX enzymes were

Combretum platypetalum , Combretum molle , Combretum zeyheri , Parinari curatellifolia and

Gymnosporia senegalensis in that order. The extracts inhibited COX-1 with percentage inhibitions of 42 %, 93 %, 97 %, 74 % and 77 % and for COX-2 at 85 %, 42 %, 68 %, 110 % and 121 %. However, C. platypetalum was the only extract that was able to selectively inhibit

COX-2. The plant extract was more selective of COX-2 than COX-1 by 0.5. The IC 50 s of

Combretum platypetalum and indomethacin were determined and are shown in Figure 3.1. The

IC 50 s for C. platypetalum and indomethacin were also determined using the COX activity assay.

The graphs shown in Figure 3.1 indicate the IC 50 values for indomethacin and the C. platypetalum extract respectively.

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Table 3.1: Inhibitory activity of plant extracts on cyclooxygenase enzymes

Enzyme isoform Extract [Prostaglandin] % Inhibition Selectivity* (mg/ml)

COX-1 Indomethacin 0.4 70 0.99 Combretum platypetalum 6.1 42 0.5 Combretum molle 0.7 93 1.4 Combretum zeyheri 0.3 97 2.3 Parinari curatellifolia 0.6 77 7.7 Gymnosporia senegalensis 0.6 74 Brachystegia boehmii 1.1 48 Amaranthus spinosus 1.8 Nr ‡ Cassia abbreviate 1.3 Nr

COX-2 Indomethacin 0.3 71 Combretum platypetalum 0.7 85 Combretum molle 3.6 68 Combretum zeyheri 2.8 42 Parinari curatellifolia 1.8 -10 § Gymnosporia senegalensis 2.0 -21 Brachystegia boehmii 3.2 -98 Amaranthus spinosus 3.7 Nr Cassia abbreviate 4.0 Nr

*Selectivity was determined by (% inhibition of COX-1 ÷ % inhibition of COX-2) ‡ Nr means the results were not in range of the standard curve. §– signifies the activation of the COX enzymes.

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A 100 IC 50 = 414 µg/ml r2= 0.999 75

50 % Activity % 25

0 0 1 2 3 4 Log [indomethacin] µg/ml

200 B

IC 5 0 = 571 µg/ml 150 r2= 0.999

100 % Activity % 50

0 2.0 2.5 3.0 3.5 4.0 Log [ C. platypetalum ] µg/ml

Figure 3.1 Percentage activity of COX-2 against various inhibitor concentrations of indomethacin and Combretum platypetalum . The IC 50 values for indomethacin (A) and Combretum platypetalum ethanol extract (B) were determined from the graphs by graphical software (GraphPad Prism, San Franscisco, USA).

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The graphs in Figure 3.1 were used to calculate the IC 50 values for indomethacin and Combretum platypetalum using graphical software. The IC 50 value for the C. platypetalum ethanol extract at

517 µg/ml was greater than that of the positive control, indomethacin which had an IC 50 value of

414 µg/ml. The value indicates that indomethacin is able to inhibit 50 % of COX-2 catalytic activity at lower concentration than the extract.

3.2 Fractionation of Combretum platypetalum and the effect of isolated phytoconstituents on

COX-2

The Combretum platypetalum ethanol extract was found to be a potent COX-2 selective inhibitor. Phytochemical analysis was conducted on the plant sample to determine the phytoconstituents present. The phytoconstituents that were found to be present were evaluated for their effect on COX-2 to determine which group was responsible for the inhibitory activity of

C. platypetalum ethanol extract.

The presence of the five major phytoconstituent groups were then tested for in the leaves of C. platypetalum . The plant sample was evaluated for the presence of tannins, flavonoids, saponins, alkaloids, terpenoids and cardiac glycosides. The phytoconstituents were then isolated from the leaves of C. platypetalum .

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Table 3.2: Phytoconstituent analysis of Combretum platypetalum leaf extract

Phytoconstituent Test result Inference

Tannins Blue black coloration Present Saponins Froth formation Present Flavonoids Yellow coloration Present Alkaloids Red precipitate Present

Terpenoids Red ring at interface Present Cardiac glycosides Brown ring formed Present

Flavonoids, saponins, tannins, alkaloids, terpenoids and cardiac glycosides were all found to be present in the C. platypetalum leaves. Four of the six phytoconstituents tested were isolated.

The phytoconstituents that were isolated were evaluated for their effect on COX-2.

Table 3.3: Anti-COX-2 activity of Combretum platypetalum phytoconstituents

Enzyme Phytochemical [Prostaglandin](mg/ml) % Inhibition

COX-2 Flavonoids 0.6 72

Saponins 0.2 73

Alkaloids 0.3 59

Tannins 0.2 29

The inhibitory activity of the phytoconstituents isolated from C. platypetalum was determined against COX-2. The values show mean ± SD n=4.

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The isolated C. platypetalum phytoconstituents all showed inhibitory activity against COX-2.

The saponins and flavonoids inhibited COX-2 to the greatest extent with percentage inhibitions of 73 % and 72 % respectively. The alkaloids showed weaker inhibitory activity against COX-2 the saponins and flavonoids with a percentage inhibition of 59 %. The tannins were the least inhibitory of the isolated phytoconstituents with a percentage inhibition of 29%.

3.3 Free radical scavenging activity of plant extracts

Five plants of the plants selected to be evaluated for anti-inflammatory activity were tested for free radical scavenging activity. Tetramethoxyazobismethylene quinone and diphenyl picryl hydrazyl radicals were used as model systems for oxidative states that occur in the presence of free radicals.

3.3.1 Tetramethoxyazobismethylene quinone free radical scavenging activity

The free radical scavenging activity of the plant extracts was evaluated using the tetramethoxyazobismethylene quinone radical. Figure 3.2 represents the free radical scavenging activity of four extracts, Combretum zeyheri , Brachystegia boehmii , Combretum molle and

Parinari curatellifolia ethanol extracts.

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100 C. zeyheri B. boehmii 80 C. molle 60 P. curatellifolia

40

20

0 % TMAMQ radical scavenging radical TMAMQ % 0 50 100 150 [Plant extract]/ µg/ml

Figure 3.2 TMAMQ free radical scavenging activity of Combretum zeyheri , Brachystegia boehmii , Combretum molle and Parinari curatellifolia plant extracts.

The graph was used to determine the lowest concentration that gave the maximal free radical scavenging activity of the plant extracts the TMAMQ radical. The values for the concentrations are shown in Table 3.4.

3.3.2 Diphenyl picryl hydrazyl (DPPH) free radical scavenging activity of plant extracts.

Figure 3.3 illustrates the maximal DPPH free radical scavenging activity of four plant extracts C. molle , P. curatellifolia , C. platypetalum , C. zeyheri and gallic acid, the positive control. The

DPPH radical was incubated with different concentrations of the extracts.

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Figure 3.3 DPPH free radical scavenging activity of Combretum molle , Parinari curatellifolia , Combretum platypetalum and Combretum zeyheri extracts.

The graphs in Figure 3.3 show that there is a point where an increase in the concentration did not result in an increase in free radical scavenging activity reaching a plateau which was the maximal free radical scavenging capacity of that plant extract. The concentration that the extracts reached their individual maximal scavenging activity was interpolated from the graph in

Figure 3.3. P. curatellifolia ethanol extract reached its maximal radical scavenging at the lowest concentration of 16 µg/ml followed by C. zeyheri extract at 25 µg/ml, C. molle at 88 µg/ml and

C. platypetalum at 108 µg/ml.

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Table 3.4: Antioxidant activity of selected plant extracts using TMAMQ and DPPH free radical scavenging assays

Plant extract Concentration of maximal Concentration of maximal

TMAMQ free radical scavenging DPPH free radical scavenging

(µg/ml) (µg/ml)

C. zeyheri 21 25

C. molle 62 88

P. curatellifolia 18 16

B. boehmii 46 54

C. platypetalum 101 108

G. senegalensis 24 32

Ascorbic acid 54 -

Gallic acid - 31

Results shown represent n=2. The value represents the concentration that was able to quench 100 % of the free radicals.

The free radical scavenging activity of six plant extracts was evaluated using two stable radicals, the TMAMQ radical and the DPPH radical. P. curatellifolia extract had the most potent free radical scavenging activity with a value of 16 µg/ml for TMAMQ radical and 18 µg/ml for the

DPPH radical which was more effective than the positive control, ascorbic acid. C. zeyheri and

G. senegalensis extracts were more potent than the positive control as well for the TMAMQ radical in particular. B. boehmii and C. molle had similar free radical scavenging activity as the positive control. However, the C. platypetalum extract was the least potent at quenching both

69 free radicals with a value of 101 µg/ml and 108 µg/ml compared with 54 µg/ml and 31 µg/ml for

TMAMQ radical and DPPH radical respectively.

3.4 The effect of plant extracts on inflammatory processes in vitro

Five plant extracts that were found to be potent inhibitors of the cyclooxygenases were evaluated for their effect on in vitro inflammatory processes such protein denaturation and membrane stabilisation. The two assays, the albumin denaturation inhibition assay and the sheep erythrocyte membrane stabilisation assay are used to measure the potential anti-inflammatory activity of plant extracts.

3.4.1 The effect of plant extracts on protein denaturation.

Five plant extracts namely, C. platypetalum , C. molle , P. curatellifolia , G. senegalensis and B. boehmii were tested for their effect on the denaturation of protein using bovine serum albumin as a model for human albumin. The results of the experiment are shown in Table 3.5.

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Table 3.5: Percentage inhibition of bovine serum albumin denaturation by plant extracts

Compound/plant extract Concentration ( μg/ml) % Inhibition Indomethacin 200 16 100 8 50 9 10 2

C. platypetalum 200 19 100 23 50 9 10 -10

C. molle 200 1 100 -5 50 -16 10 -19

P. curatellifolia 200 23 100 -10 50 -19 10 -84

G. senegalensis 200 -32 100 -11 50 -42 10 -44

B. boehmii 200 -16 100 4 50 7 The results show mean n=2. The minus sign depicts increase in protein denaturation

The albumin denaturation inhibition assay was carried out for the concentrations 10 to 200 µg/ml for the plant extracts and positive control, indomethacin. The denaturation inhibition was evaluated against the control without treatment. The negative control was taken to have 100 % protein denaturation. C. platypetalum ethanol extract was able to inhibit protein denaturation for three of the four concentrations tested with percentage inhibitions ranging from 23 to -10 % which were slightly more potent than indomethacin. The percentage inhibitions for the positive

71 control indomethacin ranged from 16 % to 2 % from the lowest to the highest concentration tested. P. curatellifolia ethanol extract inhibited the protein denaturation at its highest concentration of 200 µg/ml at 23 % although the rest of the increased denaturation. B. boehmii extract was able to inhibit protein denaturation at the lower concentrations but the 200 µg/ml concentration enhanced protein denaturation by 16 %. Plant ethanol extracts of Parinari curatellifolia , Gymnosporia senegalensis and Brachystegia boehmii tested for their effect on protein denaturation had no inhibitory activity and instead enhanced the extent of the protein denaturation.

3.4.2 The effect of plant extracts on sheep erythrocyte membrane stability

The erythrocyte membrane was used as a model for the lysosomal membrane that is involved in inflammation. Stabilisation of the membrane would imply anti-inflammatory activity by inhibiting the release of proteolytic enzymes. The results are represented in Table 3.6.

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Table 3.6: Percentage stabilisation of erythrocyte membrane by plant extracts

Plant extract Concentration ( μg/ml) % Stabilisation C. platypetalum 500 114 ± 7 250 63 ± 7 125 50 ± 9 62.5 12 ± 8

P. curatellifolia 500 40 ± 4 250 43 ± 5 125 42 ± 4 62.5 4 ± 1

B. boehmii 500 82 ± 2 250 77 ± 1 125 65 ± 1 62.5 57 ± 2

C. molle 500 112 ± 1 250 105 ± 0 125 49 ± 1 62.5 26 ± 2

G. senegalensis 500 64 ± 2 250 73 ± 0 125 83 ± 1 62.5 85 ± 2

Indomethacin 350 40 ± 2 250 30 ± 3 125 21 ± 2 62.5 15 ± 0 The results shown represent mean ± standard deviation. n=4.

The erythrocyte membrane stabilisation assay was conducted with concentrations 500 to 62.5

µg/ml for the extracts and 350 to 62.5 µg/ml for the positive control, indomethacin. The percentages were all calculated relative to the negative control which was considered to 0 % stable. The order of potency for the stabilization of the erythrocyte membranes by the five extracts at the greatest concentration of 500 µg/ml was C. platypetalum > C. molle > G.

73 senegalensis > B. boehmii > P. curatellifolia at 114 %, 112 %, 82 %, 64 % and 40 % respectively. Four out the five ethanol extracts C. platypetalum , C. molle , G. senegalensis and B. boehmii were able to stabilise the sheep erythrocyte membrane to a greater extent than the control, indomethacin, at the same concentration of 250 µg/ml. P. curatellifolia extract showed similar stabilization activity with the control. However, the G. senegalensis extract, the stability of the erythrocyte membrane increased with decrease in concentration of the extract with the lowest concentration 62.5 µg/ml increasing the stability by 85 %. G. senegalensis extract was considered to the extract that had the best stabilizing effect on the membrane despite C. platypetalum extract being the most potent.

3.5 The effect of plant extracts on nitric oxide production induced by selected compounds in RAW 264.7 murine macrophage cells

There are compounds that have the ability to produce reactive oxygen species when metabolised in the cell. Four compounds, menadione, β-naphthoflavone, hydrogen peroxide and carbon tetrachloride, were selected to induce oxidative stress in RAW 264.7 murine macrophage cells based on their ability to produce ROS in cells. The oxidative stress was required to induce the production of nitric oxide in the murine macrophages.

The nitrite concentration is measured when evaluating NO production by cells. The nitrite ions are measured as they give an indication of NO produced, this due to the fact that NO radical has a short half-life and it is metabolized to the nitrite ion by the reductase. The Griess assay is then used to measure the amount of nitrites present from a sodium nitrite standard curve.

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3.5.1 Activation of nitric oxide production

The results shown in Figure 3.4 show the compounds that were tested for their effect on the production of NO in RAW 264.7 cell lines. The compounds were selected based on their known effects to produce reactive oxygen species. The compounds were menadione, hydrogen peroxide, β naphthoflavone and carbon tetrachloride.

8

6 ] mM 2 4

[NaNO 2

0 n th 2 l4 2O tim Me aph H CC n β Uns

Figure 3.4: Effect of selected compounds on nitrite concentration in RAW 264.7 cells. The compounds were tested for their modulation of NO production in the cell line. Results are mean ± standard deviation n= 6. Men – menadione, β naphth – beta naphthoflavone, H 2O2 – hydrogen peroxide, CCl 4 – carbon tetrachloride, Unstim – unstimulated cells.

Four compounds were tested for their effect on NO production in RAW cells. Menadione was able to increase the nitrite concentration in the cells when compared with the baseline concentration of the cells alone although the increase was not statistically significant. However,

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β naphthoflavone decreased the baseline concentration while hydrogen peroxide and carbon tetrachloride had no effect on the nitrite concentration.

3.5.2 The effect of combining menadione and plant extracts on nitric oxide production in

RAW 264.7 cells

Menadione increased nitrite concentration in RAW 264.7. The compound was combined with four ethanol plant extracts from C. platypetalum , C. zeyheri , C. molle and P. curatellifolia . The results of the combination are illustrated in Figure 3.6.

Figure 3.5: Nitrite concentration in the presence of menadione and extracts of C. platypetalum , C. zeyheri , C. molle and P. curatellifolia . * denotes level of significance compared to the control of cells alone (p ≤ 0.05). Unstim – unstimulated cells, C. p - Combretum platypetalum , C. z - Combretum zeyheri , C. m – Combretum molle , P. c - Parinari curatellifolia

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The results illustrated in Figure 3.5 show that all the plant extracts tested increased the nitrite concentration when combined with menadione when compared with the negative control of cells alone. Three extracts, in order of potency, C. platypetalum , C. zeyheri and C. molle ethanol extracts showed significant differences to the control. The ethanol extract of C. platypetalum showed the highest increase with a 5 fold increase from the baseline concentration of cells alone.

C. molle and C. zeyheri ethanol extracts caused 3 fold and 4 fold increase in the nitrite concentration respectively when compared to the cells alone. The P. curatellifolia ethanol extract showed no significant increase of nitrite concentration in the presence of menadione.

3.5.3 Effect of combining H 2O2 and plant extracts on nitric oxide production in RAW 264.7 cells

Hydrogen peroxide can be metabolized in cells to produce free radicals which in turn can induce oxidative stress within the cells. The effect of combining H 2O2 and plant extracts on production of nitric oxide in RAW cells was evaluated. The results are illustrated in Figure 3.6.

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Figure 3. 6 Nitrite concentration in RAW cells the presence of hydrogen peroxide and plant extracts. The results shown are mean ± standard deviation. n=6. * (p ≤ 0.05) versus control. Unstim – unstimulated cells, C. p - Combretum platypetalum , C. z - Combretum zeyheri , C. m – Combretum molle , P. c - Parinari curatellifolia

The incubation of hydrogen peroxide with the cells showed no difference in the nitrite concentrations. C. platypetalum and C. zeyheri ethanol extracts in combination with hydrogen peroxide increased the nitrite concentration almost 3 fold from the baseline concentration of cells alone in the presence of hydrogen peroxide. C. molle and P. curatelllifolia ethanol extracts had no significant difference in the nitrite concentration with the control.

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3.5.4 Effect of menadione and H 2O2 combination with C. platypetalum flavonoids

Flavonoids isolated from C. platypetalum leaves were combined with Menadione and H 2O2 respectively. The effect of the combinations on nitric oxide production is illustrated in Figure

3.7.

400

] ** - 2 300

200 *

100 % increase % [NO

0 n v 2 im e la lav O lav f f 2 f M p H nst . . p U C. p C C + 2 O Men+ 2 H

Figure 3.7 Nitrite concentration in the presence of menadione and H 2O2 in combination with Combretum platypetalum saponins. The results shown represent mean ± standard deviation. n= 8. * (p ≤ 0.05) versus control. Unstim – unstimulated cells, Men – menadione, C. p flav - Combretum platypetalum flavonoids, H 2O2 – hydrogen peroxide

The flavonoids isolated from C. platypetalum leaves were able to increase the nitrite concentration in the presence and absence of menadione and hydrogen peroxide. The

79 combination of C. platypetalum flavonoids and menadione resulted in a significant 1.5 fold increase in NO production, while the flavonoids alone showed a 0.5 fold increase compared the nitrite concentration of menadione and H 2O2 alone respectively.

3.5.5 The effect of menadione and hydrogen peroxide in combination with C. platypetalum saponins on nitric oxide production

Saponins were isolated from C. platypetalum leaves and were combined with menadione and hydrogen peroxide in RAW cells. The results are depicted in Figure 3.8.

Figure 3.8 Nitrite concentration in RAW cells in the presence of menadione and hydrogen peroxide in combination with C. platypetalum saponins. The results shown represent mean ± standard deviation. N= 8. * (p ≤ 0.05) versus control. Unstim – unstimulated cells, Men – menadione, C. p sap- Combretum platypetalum saponins

80

The results shown in Figure 3.9 indicate that C. platypetalum saponins when combined with menadione showed a significant increase in nitrite concentration when compared with the negative control. Hydrogen peroxide had no effect on nitric oxide production in the RAW cells.

A combination of the C. platypetalum saponins and hydrogen peroxide did not significantly increase the nitrite concentration in the RAW cells when combined with hydrogen peroxide.

3.6 The effect of Combretum platypetalum and Parinari curatellifolia extracts on LPS activated RAW 264.7 murine macrophage cells

RAW 264.7 macrophage murine cells were activated for inflammation with the E. coli endotoxin, lipopolysaccharide. LPS is recognised as an antigen and leads to the induction of expression of pro-inflammatory enzymes such inducible nitric oxide synthase. The effects of P. curatellifolia water extract and C. platypetalum ethanol extract on nitric oxide production were evaluated in this study.

3.6.1 The effect of Parinari curatellifolia water extract on LPS activated cells

The graphs shown in Figure 3.10 represent the results obtained when LPS activated RAW 264.7 cells were incubated with P. curatellifolia water extract. The LPS and the extract were combined in the cells simultaneously. The results show the nitric oxide production in RAW cells in the presence of P. curatellifolia water extract.

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Figure 3.9 Nitrite concentration of RAW cells for the simultaneous combination of LPS with Parinari curatellifolia extract after 24 hr incubation. * (p ≤ 0.05) versus control. Unstim – unstimulated cells, LPS – lipopolysaccharide, P. c - Parinari curatellifolia

The results in Figure 3.9 illustrate that LPS was able to significantly increase the nitrite concentration from the baseline concentration of the unstimulated RAW cells. The combination of LPS and P. curatellifolia water extract resulted in a significant decrease in the nitrite concentration when compared to both the unstimulated cells and the LPS activated cells. P. curatellifolia water extract in unstimulated cells significantly decreased the baseline nitrite concentration that was obtained for the unstimulated RAW cells.

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3.6.2 The effect of Combretum platypetalum ethanol extract on LPS activated cells

The results shown in Figure 3.10 represent the cells that were incubated for 24 hours with simultaneous LPS and the plant extract being added simultaneously to the wells. The model mimics the effect of administering the extract before or as the inflammatory event occurs.

M ]/ 2 [NaNO

p p tim LPS C. C. ns U S + P L

Figure 3.10 Nitrite concentration of RAW cells for the simultaneous combination of LPS with Combretum platypetalum extract after 24 hr incubation . * (p ≤ 0.05) versus control. Unstim – unstimulated cells, LPS – lipopolysaccharide, C. p - Combretum platypetalum .

The results in Figure 6.11 indicate that a very low concentration of nitrites was present in the unstimulated cells. The sample with the LPS and the C. platypetalum ethanol extract showed an increase in the amount of nitrites compared to the cells not activated. The increase indicated that the LPS was able to induce NO production in the RAW cells. The combination of LPS and the plant extract led to an increase in the nitrite concentration compared to the LPS and C. 83 platypetalum ethanol extract. The increase in the amount of nitrites could mean that there was an increase nitric oxide production when the endotoxin and ethanol extract were combined.

3.6.3 The effect of Combretum platypetalum ethanol extract on LPS activated RAW 264.7 cells.

The results shown in Figure 3.11 were for cells that were incubated for 24 hours with LPS and the plant extract was subsequently added and the plate was incubated for a further 24 hours. The model mimics the effect of administering the extract after the inflammatory process is initiated by LPS. M ]/ 2 [NaNO

p p m . . sti LPS C C Un PS+ L

Figure 3.11 10 Nitrite concentration of RAW cells for the concurrent combination of LPS with C. platypetalum extract after 48 hr incubation. * (p ≤ 0.05) versus control. Unstim – unstimulated cells, LPS – lipopolysaccharide, C. p - Combretum platypetalum .

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The unstimulated cells in Figure 6.11 had no detectable nitrites formed during incubation as indicated by the negative concentration. The LPS activated cells and the unstimulated cells with

C. platypetalum ethanol extract had similar nitrite concentrations which were higher than the concentration for the negative control of the unstimulated cells. However, the combination of

LPS and the C. platypetalum ethanol extract gave a significantly lower nitrite concentration (p <

0.05) than for the LPS stimulated and unstimulated cells with the ethanol extract.

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CHAPTER FOUR

Discussion

Drug discovery research is relying on lead compounds from natural sources due to the high cost of developing new synthetic drugs (Hostettmann and Terreaux 2000). The potential for medicinal plants to provide novel anti-inflammatory agents is immense as they produce a diverse amount of novel compounds that are biologically active. Plants have served as templates for anti-inflammatories since the advent of the first synthetic anti-inflammatory drug, aspirin.

Aspirin was developed from salicylic acid, a metabolite of salicin which was isolated from the

Salix species. The Salix species were used by Native Americans as an herbal remedy to treat pain and fever (Arihan and Guvenc, 2011). Plant compounds and their metabolites from various other species have been shown to possess anti-inflammatory activity. The study was conducted to determine the anti-inflammatory activity of eight Zimbabwean medicinal plants that are used to treat pain and inflammatory conditions by evaluating the effect of the plant extracts on enzymes and processes involved in inflammation.

4.1 The effect of plant extracts on COX activity

Inhibition of cyclooxygenase enzymes has been the mechanism of action of most of the current anti-inflammatory drugs. However, some of the drugs inhibit both the constitutive (COX-1) and inducible (COX-2) isoforms of the enzymes leading to side effects such as gastrointestinal bleeding (Lee et al. , 2011). Selective inhibition of COX-2 could alleviate the side effects associated with inhibiting the COX-1 isoform while reducing inflammation and that was set as

86 the initial goal for this study. Natural selective COX-2 inhibitors could play role where long term inflammatory treatment is required. COX-2 inhibitors can be cancer chemopreventive as well as prostaglandin E 2 production is down-regulated.

The inhibition of the COX enzymes is an important anti-inflammatory mechanism and the study screened eight plant samples that are used by herbalists to treat pain and inflammation for anti-inflammatory activity using COX enzymes. The ideal anti-inflammatory extract would inhibit COX-2 with minimal or no inhibitory effect on COX-1. Eight ethanol extracts of

Combretum platypetalum , Combretum molle , Combretum zeyheri , Parinari curatellifolia ,

Gymnosporia senegalensis , Cassia abbreviatta , Brachystegia boehmii and Amaranthus spinosus tested for their effect on the two COX isoenzymes. The screening for anti-inflammatory activity from the selected medicinal plant samples using the COX activity assay showed that the three

Combretum species were the most potent inhibitors of COX catalytic activity. Combretum platypetalum , in particular, had potent anti-COX-2 activity compared to its effect on COX-1.

The ethanol plant extract of C. platypetalum was found to inhibit the activity of both

COX-1 and -2 at a percentage of 42 % and 85 % respectively. The extract inhibited COX-2 to a greater extent than COX-1 indicating that the extract is COX-2 selective. A decoction of C. platypetalum is used traditionally to treat dysmenorrhea, which a condition that results in painful menstrual cramps and headaches (Martini, 2001). Prostaglandin E 2 and F 2α have been found to be mediators of the condition with production of the biomolecules being proportional to the symptoms observed in dysmenorrhea (Rao et al. , 2011). As the cyclooxygenases are responsible for producing the precursor molecule of the prostaglandins, inhibition of the enzymes could result in the down-regulation of PGE 2 and PGF 2α. The ability of the extract to selectively inhibit

87

COX-2 as shown in Table 3.1 could be responsible for its use in folk medicine. In fact, other

Combretum species have been shown to have the ability to relax uterine muscles in mice which implicates the down-regulation of PGE 2 and PGF α production (Lima et al. , 2012). COX-2 inhibition must be high enough to inhibit the induced expressed but low enough to not remove the basal concentration of the enzyme required for physiological function. IC 50 value of C. platypetalum ethanol extract showed that although the extract was selective, indomethacin, a non-selective inhibitor had a lower IC 50 value. However, C. platypetalum ethanol extract would still be a better anti-inflammatory agent as it preferentially inhibits COX-2 over COX-1 whereas indomethacin inhibits both the isoforms to a similar extent (Simmons et al ., 2004). Combretum platypetalum ethanol extract could be a source of novel and natural COX-2 selective anti- inflammatory agents.

Phytochemical analysis of C. platypetalum leaves during this study showed the presence of terpenoids, flavonoids and saponins. The phytoconstituents are known to be inhibitors of

COX-2 (Divakarn et al. , 2010). The presence of the phytochemicals in C. platypetalum led to the decision to isolate and test them against COX-2. Flavonoids, saponins, alkaloids and tannins were isolated from C. platypetalum leaves. Flavonoids and saponins inhibited COX-2 to a similar extent with percentage inhibitions of 72 % and 73 % respectively. The similarity in the percentage inhibitions implies that the inhibition might due to a structure or moiety that is common to both molecules. The moiety is most likely to be responsible for the COX-2 selective activity of the C. platypetalum ethanol extract as illustrated in Figure 5.1. Flavonoids are a ubiquitous class of polyphenols that are produced by plants (Garcia-Lafuente et al ., 2009).

Flavonoids are characterized by at least two aromatics rings that have one hydroxyl group each and a heterocyclic pyran. Flavonoids usually have a glycoside chain attached to the core 88 structure. Flavonoids have been shown to inhibit COX-2. Saponins are considered to be plant compounds that have sugar moieties attached to them thought there are groups that are aglycones

(Wina et al. , 2005). Examples of saponins include glycosylated steroids, triterpenoids, and steroid alkaloids (Wina et al ., 2005). Saponins isolated from Wattakaka volubilis inhibited PGE 2 production in RAW 264.7 cells which was attributed to the effect of the phytochemical on COX-

2 (Jadhav et al. , 2013).

C. platypetalum flavonoids or saponins

COX-2 Arachidonic acid PGH2 PGF2

Figure 4.1 Proposed mechanism of action of Combretum platypetalum extract on COX-2 catalytic activity . The conversion of arachidonic acid to PGH 2 is catalysed by COX-2. C. platypetalum flavonoids and/or saponins were able to inhibit COX-2 enzymatic activity. Inhibition of COX-2 would lead to reduced production of PGF 2 used as an indicator of COX-2 activity.

Salicin, the compound from which aspirin was derived, is a glycoside molecule that was shown to be an inhibitor of both COX isoforms. Hence, a glycoside moiety could be responsible for the inhibition activity shown by the phytoconstituents as both groups are known to possess the chemical groups. The anti-inflammatory mechanism of action of salicin has not yet been elucidated despite that of aspirin being known (Simmons et al ., 2004). The mechanism of action of glycosides derivatives, such as saponins and flavonoids, against the cyclooxygenases need

89 have to be elucidated. The elucidation of the mechanism of action of the phytochemicals might enable the use of the herbal remedies without engineering synthetic derivatives.

Combretum zeyheri and Combretum molle ethanol extracts were both able to inhibit both

COX isoforms and neither of one of the extracts was COX-2 selective. The ethanol extract of C. zeyheri was able to inhibit COX-2 to the greatest extent compared to the other extracts that were not selective for COX-2. C. molle ethanol extract was able to inhibit COX-2 as well. The ethanol extracts from the three Combretum species in the study were the extracts that were able to inhibit the COX-2 isoform while the other extracts were either activating its catalytic activity or were inactive at the concentrations tested. Previous studies have shown that Combretum species have stable anti-COX activity although the particular enzyme was not specified (Ahmad et al. , 2007). The COX-2 inhibitory activity of the Combretum species could suggest that further phytochemical analysis of the genus could be required to determine the active constituent or constituents. The Combretum species as a whole or Combretum platypetalum in particular, could be a potential source of novel naturally derived COX-2 inhibitors.

On the other hand, the other three extracts, Parinari curatellifolia , Gymnosporia senegalensis and Brachystegia boehmii inhibited COX-1 and not COX-2. The inhibitory activity of the extracts against the COX-1 catalytic activity could account for their use as pain remedies.

The efficacy of the remedies in the individuals is yet to be evaluated. This study has shown that the ethanol extracts inhibit COX-1 at percentages close to hundred percent. The physiological role of COX-1 would suggest that the extracts, although they have anti-inflammatory effects, could potentially have adverse effects on the body that are similar to those of aspirin due to the inhibition of COX-1 instead of COX-2.

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4.2 The effect of plant extracts on free radical scavenging activity

Five ethanol extracts of Parinari curatellifolia , Combretum zeyheri , Combretum molle ,

Brachystegia boehmii and Combretum platypetalum were evaluated for their free radical scavenging activity. The phytochemicals that were identified in C. platypetalum , C. zeyheri and

P. curatellifolia were tannins, flavonoids, terpenoids, saponins, alkaloids and cardiac glycosides.

Phenols and flavonoids are generic names given to class of compounds with great structural diversities and activities. It is expected that the antioxidant phytoconstituents present in P. curatellifolia may have different anti oxidative mechanisms (Ogunbolude et al ., 2009). P. curatellifolia fruit was found to have potent antioxidant activity by Ndhlala et al. , (2006), showing the plant could be a potential source of antioxidants that can be utilized in the prevention of inflammatory related diseases.

Antioxidants such as Vitamin E have been suggested to be used in the prevention of diseases associated with oxidative stress such cardiovascular disease (Pashkow, 2011). The three

Combretum species that were tested showed antioxidant activity, with C. zeyheri and C. molle ethanol extracts having values that were similar to those of the positive controls gallic acid and ascorbic acid. Previous work by Masoko and Eloff (2007), showed that the two species had moderate and strong antioxidant activity respectively. C. molle ethanol extract, however, was found to be a more potent antioxidant in this work. C. zeyheri ethanol extract had a lower value for quenching of the radicals than the C. molle extract. The differences between this study and the previous work could be due to the different locations and collection times of the samples, as work by Bhatnagar et al ., (2012) showed that the antioxidant activity of C. roxburghii fluctuated

91 according to the time of year the samples were collected. In a study by Gouivenia et al ., (2011),

C. duartenum was shown to have potent hydroxyl radical scavenging activity. The Combretum species show potential to be sources of novel antioxidant agents that are natural sources.

The DPPH free radical assay is a common assay in the estimation of the potential antioxidant capacity from plant material (Thaiponga et al ., 2006). Stable free radicals with an oxygen centre are considered to be more efficient at reacting with phenolics resulting in quenching (Osma et al., 2010). This could mean that the plant extracts were able to quench the

TMAMQ radical to a greater extent than the DPPH radical. However, the results showed that the extent of radical scavenging by the extracts was similar for both stable radicals. The similar results could suggest that the assays could be used interchangeably to evaluate the free radical scavenging capacity of a plant extract.

4.3 The effect of plant extracts on protein denaturation

All the plant extracts tested were able to inhibit the precipitation of protein and to stabilize the erythrocyte membrane. The effect of the plant extracts on protein denaturation was evaluated using bovine serum albumin. When a protein is heated, it undergoes denaturation leading to the exposure of antigens that are similar to those expressed associated with diseases such as rheumatoid arthritis (Duganath et al., 2010). The production of auto antigen in certain arthritic disease may be due to denaturation of protein that is responsible for the autoimmune response that leads to rheumatoid arthritis. The mechanism of denaturation may be alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding due to the inflammatory response.

NSAIDs have been found to inhibit the denaturation of protein besides inhibiting the 92 prostaglandin synthesis as shown by the positive control, indomethacin (Govindappa et al.,

2011).

The phytoconstituents contained in the C. platypetalum ethanol extract were probably able to inhibit the denaturation of the protein by interacting with the amino acids that are exposed on heating. Interaction with amino acid residues such as the lysine and threonine could lead to the prevention of precipitation of protein. This could indicate that the inhibition of protein precipitation may be a mechanism of action for the Combretaceae plant that are used traditionally to treat inflammatory conditions.

4.4 The effect of plant extracts on membrane stabilisation

The principle of the stabilization of human red blood cell membrane is the hypotonicity induced membrane lysis that mimics the lysis of the lysosomal membrane (Lavanya et al., 2012). The lysosomal enzymes such as bactericidal proteases that are released during an inflammatory event have been thought to be involved in some disorders as these enzymes are implicated in acute and chronic inflammation (Rahman et al., 2012). NSAIDs can either work by inhibiting the enzymes or they can stabilize the lysosomal membrane to prevent the release of the lysosomal enzymes into the extracellular matrix (Yoganandam et al., 2010). C. molle ethanol extract was shown to have significant anti-inflammatory activity in vitro at its highest concentration tested. Work done by McGaw et al ., (2001) supports the anti-inflammatory activity of the plant species.

However, studies have shown that high concentrations of C. molle extracts can cause acute toxicity in vivo (Yeo et al., 2012). The therapeutic concentration of the extract might prove to have adverse effects especially over a long period. The use of C. molle extract as a potential anti-arthritic agent might require isolation of the active phytoconstituent or phytoconstituents if

93 they are found not be responsible for the toxicity shown in the study by Yeo et al ., (2012). B. boehmii ethanol extract showed potent anti-inflammatory activity as it showed the ability to stabilize the red blood cell membrane more than the positive control, indomethacin.

Decoctions of B. boehmii root parts are used to treat wounds in Tanzania by local application (Haerdi, 1964). The stabilization of the lysosomal membrane could be the mechanism of action of B. boehmii extract when it is used to reduce inflammation during wound healing as less tissue-damaging enzymes are being released to the inflammatory site. The activity of the three plant extracts could be attributed to the presence of compounds that may be specific to the Combretaceae family that are able to stabilize the red blood cell membrane against haemolysis. However, G. senegalensis was the most effective at stabilizing the erythrocyte membrane at the lowest concentration of 62.5 µg/ml with a percentage stabilization of 85 %. G. senegalensis is extensively used in folklore medicine in Africa to treat inflammatory conditions, in Mali it is used to alleviate dysmenorrhea (Sanogo, 2011). In Zimbabwe the antioxidant and anti-inflammatory activity of Combretum species that have tested have been found to be potent indicating that the plant species could be a potential source of new anti-inflammatory agents.

4.5 The effect of combining oxidative compounds and plant extracts on nitric oxide production in RAW murine macrophage cells

Oxidative stress plays an important role in the development of chronic inflammatory conditions.

Menadione was shown to increase the nitrite concentration when incubated with RAW 264.7 cells. The increase in NO production could have been due to the ability of menadione to induce oxidative stress in cells. Menadione has been shown to be able to induce the generation of free radical species in the Hep G2 cell line and pancreatic acinar cells in vitro (Chen and Cederbaum,

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1997, Criddle et al. , 2006). The presence of ROS induces the liberation of NF Κβ , the transcription factor that is associated with the induction of genes that code for iNOS

(Franceschelli et al ., 2011).

The results shown in Figure 3.4 show that the combination of menadione and the C. platypetalum ethanol extract led to an increase in the nitrite concentration when compared to the baseline and to the concentration of menadione alone. The increase in the concentration could be attributed to some of the phytoconstituents present in C. platypetalum ethanol extract acting as pro-oxidants. Flavonoids isolated from C. platypetalum ethanol extract increased the nitrite concentration by 117 % compared to the cells alone. Phytoconstituents, flavonoids in particular, have been found to act as pro-oxidants when in high concentrations (Yordi et al. , 2011). The structure of the flavonoids also plays a role in their pro-oxidant activity as the presence of multiple hydroxyl groups on the B ring of a flavonoid leads to the production of free radicals in the Fenton reaction which could attenuate the state of oxidative stress (Horakova, 2011). The alkaloids, on the other hand, had no effect on the nitrite concentration even in the presence of menadione even though they have been found to act as pro-oxidants when in high concentrations

(Maiza-Benabdesselam et al ., 2007). Therefore, this could indicate that the structure of flavonoids might be responsible for any pro-oxidant activity since the same concentrations were used for both extracts.

The results in Figure 3.8 show that P. curatellifolia ethanol extract had a relatively effect on the production of NO in the RAW cells. The combination of menadione and P. curatellifolia ethanol extract had no significant effect on the nitrite concentration of menadione. The combination of P. curatellifolia ethanol extract and hydrogen peroxide, on the other hand, resulted in a decrease in the nitrite concentration. This could have been due to the antioxidant

95 activity of the P. curatellifolia ethanol extract which was shown to have potent antioxidant activity through free radical scavenging. Hydrogen peroxide had no effect on the NO production, therefore, the decrease in the nitrite concentration could be due to P. curatellifolia ethanol extract removing NO radicals. A study by Boora et al ., (2014) showed that P. curatellifolia ethanol extract was a potent scavenger of NO radical in vitro .

Figure 3.6 shows the results for combining the C. platypetalum , C. zeyheri , C. molle and

P. curatellifolia ethanol extracts with menadione. The extracts increased nitrite concentration in the presence of menadione in that order. However, the trend of increase was probably dependent on the antioxidant activity of the plant extract as the least antioxidant active C. platypetalum ethanol extract showed the greatest increase of 4 fold compared to the most antioxidant active,

P. curatellifolia , which showed the least increase in nitrite concentration in the presence of menadione. C. zeyheri and C. molle ethanol extracts showed intermediate increase although they had similar antioxidant activities in the previous work, C. zeyheri ethanol extract increased the nitrite concentration about 50 % more than the C. molle ethanol extract.

The results shown in Figure 3.8 indicate that the nitrite concentration increased when C. platypetalum and C. zeyheri ethanol extracts were combined with H 2O2. However, when H 2O2 were combined with P. curatellifolia and C. molle ethanol extracts had similar effects on the nitrite concentration as it decreased in the presence of the extracts. The nitrite concentrations for the two extracts in the presence of H 2O2 were also lower than that of the control of the cells alone. The effect of the extracts could have been due to the quenching of the peroxide molecule before oxidative stress could be induced.

The effect of C. platypetalum ethanol extract on the production of NO in RAW cells shows that the extract might not be effective as a chemopreventive agent for inflammatory

96 conditions. C. platypetalum ethanol extract increases NO production in unstimulated cells which in vivo could exacerbate inflammation. However, the ethanol extract could be looked at as a potential source of anti-cancer drugs as the pro-oxidant activity in the presence of an oxidative compound such as menadione could be useful in inducing processes such as apoptosis in proliferating cells. Mangoyi et al ., (2014) showed that the anti-proliferative activity of C. platypetalum extract was modulated by the antioxidant compound, GSH implying that the mode of action of the extract could have been through generation of oxidative components.

4.6 The effect of C. platypetalum and P. curatellifolia extracts on nitric oxide production on

LPS activated RAW 264.7 murine macrophages

Macrophages play important signalling and therapeutic roles when they produce and release nitric oxide during an inflammatory event. The nitric oxide is eventually converted to the more stable nitrite form. Due to the short half-life of NO, the concentrations of the radical produced are usually measured by quantifying the amount of nitrites present in the samples. NO production in the RAW cells was activated using an E. coli endotoxin, lipopolysaccharide. LPS has been shown to induce the expression of iNOS in macrophage cells in cell culture leading to an increase in the production of NO within the cells. The effects of Parinari curatellifolia water extract and Combretum platypetalum ethanol extract on the NO production was investigated in this study.

P. curatellifolia water extract was incubated with the RAW cells in the presence and absence of the endotoxin LPS. The results for the experiment are shown in Figure 3.9. The effect that P. curatellifolia water extract has on nitric oxide production in RAW cells implies that the extract has inflammatory activity by quenching the NO radicals. The proposed mechanism

97 shown in Figure 4.2 proposes that the NO radicals are removed by the water extract before they are converted to the nitrite form. The proposal for the mechanism was based on two pieces of evidence. The first piece of evidence is that there was a decrease in the nitrite concentration in the un-induced RAW cells in the presence of P. curatellifolia water extract indicating a reduction in the baseline NO concentration. The LPS induced cells showed a significant increase in NO production compared to the un-induced cells. The combination of LPS and P. curatellifolia water extract led to a significant decrease in the nitrite concentration compared to the LPS activated cells. The second piece of evidence is that P. curatellifolia water extract was found to be a potent scavenger of the NO radical in a study by Boora et al ., (2014). P. curatellifolia extracts of seeds and pulp were found to possess antioxidant activity against the DPPH radical

(Ogunbolude et al ., 2009, Ndhlala et al ., 2006). However, the extract caused a decrease in the nitrite concentration which is required for normal physiological roles such as neurotransmission, vascular flow and synaptic plasticity (Karimi et al ., 2012). The inhibition of NO production required for the physiological functions could result in toxicity if administered in the wrong doses. Therefore, the use of the P. curatellifolia extract as an herbal remedy might require proper formulation to ensure that either the therapeutic or chemopreventive concentrations are administered. The extract could be fractionated by bio guided fractionation to determine and isolate the active components if there is no loss in potency.

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Figure 4.2 Proposed mechanism of action of effect of Parinari curatellifolia water extract on nitric oxide production in RAW 264.7 murine macrophage cells . Lipopolysaccharide (LPS) activates a G-protein cascade that leads to the dissociation of NF Κβ from its inhibitor molecule IK β. The dissociation of NF Κβ allows nuclear translocation of the transcription factor to activate the expression of iNOS. iNOS catalyses the breakdown of L-arginine to citrulline with NO as a by-product. P. curatellifolia water extract was able to scavenge NO radicals which could reduce its effect on its target cells.

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The low nitrite concentration in the presence of the water extract indicates that the radicals were scavenged as soon as they were produced as NO have a short half-life. The removal of NO radicals after production by the macrophages would mean that the inflammatory reactions that are mediated by NO are terminated which would result in the relief of symptoms.

Fever or pyresis is mediated by PGE 2 that stimulates the hypothalamus to increase the body temperature (Hattapakki and Hukkeri, 2010). The role NO may play in pyresis is that it may increase the catalytic activity of COX-2 by nitrosylation of a cysteine residue on the enzyme.

COX-2 catalyses the reaction which produces the PGE2 precursor molecule, PGH 2. NO was shown to NO is responsible for vasodilation which allows vascular permeability which results in immune cell migration to the site of inflammation (Tousoulis et al ., 2012). The NO scavenging activity of the P. curatellifolia water extract may be responsible for the anti-inflammatory activity of the decoctions that are administered by herbalists in the treatment of headaches and fever.

The effect of the water extract of P. curatellifolia on nitric oxide production was analysed as the water extract would be similar to the decoction that is administered by herbalists. Most herbal remedies are prepared in water. Therefore, the results supports the use of P. curatellifolia as a pain remedy in folk medicine. The P. curatellifolia water extract shows potential as a source of NO radical scavenging agents that could have anti-inflammatory activity.

The simultaneous combination of LPS and the C. platypetalum ethanol extract represented in Figure 3.10. The combination of LPS and the extract had a higher nitrite concentration than that of the LPS alone. The difference implies that the extract had a pro- oxidant effect in combination with the LPS. The results suggest that extract may be unable to prevent an inflammatory event. Instead, the results suggest that the extract might exacerbate

100 inflammation if it is administered prior or at same time that the inflammatory response is activated. The C. platypetalum ethanol extract had poor free radical scavenging activity as shown in Table 3.4. The poor antioxidant activity of the extract may account for its pro-oxidant activity instead. A previous study by Mangoyi et al ., (2014) showed that GSH was able to antagonise the effect of C. platypetalum extract on proliferation of Jurkat T cells. The antagonistic action of GSH suggests that the C. platypetalum ethanol extract was producing oxidative components that were leading to the death of the lymphocytes in culture. The pro- oxidant activity of the C. platypetalum extract that occurs as the macrophages are stimulated suggests that at the concentration tested the extract would increase the level of inflammation in the cells. Therefore, the C. platypetalum ethanol extract might not be suitable as a chemopreventive agent against inflammation.

The results in Figure 3.11 represents the results obtained from the subsequent addition of the plant extract after the LPS was added. C. platypetalum ethanol extract was able to inhibit

LPS induced nitric oxide production. This implies that the plant extract would be effective as an anti-inflammatory agent after the injury. However, the plant extract on its own increased the amount of nitrites in the cells. C. platypetalum ethanol extract appears to have a pro-oxidant effect on the cells on its own, the effect could mean that the extract would ineffective as a means of preventing inflammation. The extract was found in this study to have flavonoids and terpenoids. The two phytoconstituents are thought to attenuate the release and extracellular action of NO. Therefore, the down-regulation of NO production could be attributed to the effect on COX-2. COX-2 expression and iNOS expression are linked to each other, COX-2 has been found to influence expression of iNOS in murine cells. Inhibition of COX-2 by C. platypetalum ethanol extract could responsible for the down regulation of NO production.

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The other mechanism of action of the C. platypetalum extract that could be proposed is that of NO radical scavenging. Boora et al ., (2014) showed that the C. platypetalum extract was able to remove NO radicals from solution. However, the extract did not decrease nitrite concentrations in the unstimulated macrophage cell, instead the nitrite concentration increased.

A study by Gouveia et al ., (2011) showed that Combretum duartenum was able to scavenge NO radicals at low concentrations but there were increased amounts of NO in the presence of the extract at concentrations above 100 µg/ml. Plant extracts are complex mixtures of various components and compounds. Different shifts from antioxidant to pro-oxidant activity are possible due to the presence of constituents that would have different redox properties at varying concentrations (Gouveia et al ., 2011). This study used a final concentration of 250 µg/ml for C. platypetalum ethanol extract which could results in compounds with oxidative potential being in greater concentrations than those with the antioxidant activity.

However, despite the pro-oxidant activity, the C. platypetalum ethanol extract constituents could be useful as a lead for a therapeutic agent against inflammation that can be used to alleviate pain.

4.7 The potential of Zimbabwean plants as sources of anti-inflammatory agents

The study was conducted to evaluate the anti-inflammatory activity of selected Zimbabwean medicinal plants that are in use as herbal remedies for pain and inflammatory related ailments.

The eight plant samples collected from Norton and Centenary were initially screened for their anti-inflammatory activity using COX enzymes. The screening process of the selected plant extracts was conducted using the COX isoforms due to their being the rate limiting step in the formation of prostaglandins, mediators of inflammation. The screening showed that Cassia

102 abbreviata and Amaranthus spinosus were inactive against COX isoforms. The plant extracts were eliminated from the study but this does not imply that the plant extracts do not contain potential anti-inflammatory agents. The mechanism of action of the extracts could have been beyond the scope of this study.

Gymnosporia senegalensis and Brachystegia boehmii ethanol extracts were found to possess anti-inflammatory activities in different assays. B. boehmii ethanol extract was able to inhibit COX-1 activity, had relatively potent antioxidant activity, was a potent inhibitor of protein denaturation and was able to stabilize the erythrocyte membrane as well. G. senegalensis ethanol extract inhibited COX-1, stabilised the erythrocyte membrane and had potent free radical scavenging activity. The G. senegalensis and B. boehmii extracts may warrant further investigation as a possible source of novel anti-inflammatory agents that inhibit new targets, such as the stabilisation of the lysosomal membrane, within the inflammatory process.

Parinari curatellifolia ethanol and water extracts showed anti-inflammatory activity in this study. The P. curatellifolia ethanol extract was a potent inhibitor of COX-1, the most potent free radical scavenging agent, was able to stabilise the erythrocyte membrane. The P. curatellifolia water extract lower the amount of NO in RAW murine macrophage cells. P. curatellifolia extracts have the potential to produce anti-inflammatory agents that work by removing free radicals from the inflammatory environment. The removal of the free radicals could have therapeutic and /or chemopreventive effects. The therapeutic effects could work by lowering the activity of NO in inflammation. The chemopreventive effects would work by removing free radicals that would otherwise react biomolecules and cause disease.

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4.8.1 Conclusions

The plant species that were selected for the study are all used in the treatment of pain and inflammatory related conditions. Five of the eight plant extracts tested were able to inhibit both

COX activities. Combretum platypetalum ethanol extract was a potent COX-2 selective inhibitor which could account for its use as an herbal remedy for dysmenorrhea. Parinari curatellifolia ethanol extract had potent free radical scavenging activity against DPPH and TMAMQ free radicals. Combretum platypetalum ethanol extract had potent pro-oxidant activity when co- administered with oxidative compounds and lipopolysaccharide making it a poor source for chemopreventive anti-inflammatory agents. Parinari curatellifolia water extract was found to be a potent inhibitor of nitric oxide production in RAW cells which could be its mechanism of action as an anti-inflammatory agent in folk medicine. Therefore, Combretum platypetalum and

Parinari curatellifolia plant species could be potential sources of leads for novel anti- inflammatory agents.

4.8.2 Limitations

The limitations of this project was that part of the work that was proposed was not conducted.

The work with the RAW cell lines was not completed as the reagents related to the work did not arrive on time. The study on the effect of plant extracts on recombinant microsomal prostaglandin E 2 synthase-1 due to unavailability the appropriate competent cells for the transformation process.

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4.8.3 Suggested future work

The proposed future work for the project would to evaluate the effect of Combretum platypetalum on the expression of iNOS and COX-2 in RAW 264.7 cells to determine the possible mechanism of action for the down-regulation of nitric oxide production in RAW 264.7 murine macrophage cells.

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CHAPTER FIVE

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APPENDIX

APPENDIX 1

List of publications from the study

Chirisa E., Mukanganyama S. Evaluation of selected Zimbabwean plant extracts for anti- inflammatory and antioxidant activity in vitro . Submitted to Journal of Herbs, Spices and

Medicinal Plants .

Boora F., Chirisa E. and Mukanganyama S. (2014). Evaluation of nitrite radical scavenging properties of selected Zimbabwean plant extracts and their phytoconstituents.

Journal of Food Processing. 2014: 1-7.

Mangoyi R., Chitemerere T., Chimponda T., Chirisa E., Mukanganyama S. (2014). Multiple

Anti-Infective Properties of Selected Plant Species from Zimbabwe

In: Ameenah Gurib-Fakim. Novel Plant Bioresources: Applications in Food, Medicine and

Cosmetics. John Wiley and Sons Ltd. Page 179-190.

123

APPENDIX 2

COX results

COX-1 and -2 Inhibition assay for Parinari curatellifolia , Gymnosporia senegalensis, Brachystegia boehmii 0.136 0.225 0.237 0.355 0.029 -0.023 0.003 -0.039 0.55 -0.002 0.068 0.139 0.2 0.251 0.359 0.029 -0.026 0.004 -0.042 0.055 -0.021 0.069 0.314 0.234 0.223 0.382 0.026 -0.027 -0.001 -0.042 0.048 -0.021 0.057 0.272 0.139 0.229 0.402 0.022 -0.025 -0.01 0.044 -0.016 0.047 0.22 0.234 0.218 0.343 0.022 -0.023 -0.005 0.033 -0.011 0.035 0.14 0.23 0.162 0.313 0.014 -0.022 -0.012 -0.033 0.024 0.004 0.026 0.267 0.283 0.122 0.324 0.014 -0.021 -0.012 -0.021 0.024 0.014 0.026 0.277 0.253 0.159 0.295 0.014 -0.021 -0.012 -0.02 0.024 0.014 0.025

IC 50 0.146 0.18 0.184 0.255 0.023 -0.019 -0.002 -0.029 0.035 0.009 0.038 0.149 0.165 0.192 0.277 0.025 -0.024 0 -0.037 0.036 0.002 0.041 0.246 0.188 0.181 0.293 0.024 -0.024 0 -0.037 0.036 0.001 0.041 0.225 0.13 0.17 0.3 0.014 -0.024 -0.01 -0.038 0.026 0 0.028 0.18 0.191 0.173 0.279 0.013 -0.023 -0.009 -0.025 0.027 0.011 0.025 0.142 0.189 0.156 0.241 0.015 -0.018 -0.009 -0.019 0.027 0.018 0.026 0.216 0.212 0.127 0.246 0.016 -0.017 -0.009 -0.021 0.027 0.016 0.029 0.23 0.202 0.173 0.246 0.025 -0.022 0.001 -0.027 0.035 0.009 0.042

124

APPENDIX 3

Membrane stabilisation

P. curatellifolia , C. molle and C. platypetalum

1 2 3 4 5 6 7 8 9 10 11 0.0492 0.0479 0.047 0.0459 0.0675 0.0505 0.0364 0.0468 0.0467 0.0484 0.0467 0.0478 0.1382 0.1187 0.0888 0.1237 0.0746 0.1134 0.0485 0.0501 0.0724 0.0493 0.0487 0.0539 0.1265 0.1114 0.0828 0.1236 0.0749 0.1083 0.0459 0.0462 0.072 0.0461 0.0482 0.0472 0.1297 0.1142 0.0842 0.1161 0.0759 0.1095 0.0472 0.0475 0.0725 0.0478 0.0469 0.0443 0.0433 0.044 0.0438 0.0471 0.0439 0.046 0.0444 0.0447 0.0465 0.0461 0.0394 0.0418 0.045 0.0456 0.0455 0.046 0.0471 0.0465 0.0466 0.0468 0.0454 0.0483 0.0489 0.0461 0.0445 0.045 0.046 0.0454 0.0454 0.0453 0.046 0.044 0.045 0.0456 0.0477 0.0468 0.0451 0.0442 0.0458 0.0451 0.045 0.0452 0.045 0.0461 0.0449 0.0453 0.0453 0

125

APPENDIX 4

Albumin denaturation inhibition results

1 2 3 4 5 6 7 8 9 10 11 0.0863 0.0678 0.0488 0.0479 0.0466 0.0489 0.0465 0.0505 0.047 0.0494 0.0484 0.0485 0.0624 0.0487 0.0484 0.0482 0.0475 0.0485 0.1717 0.1809 0.1763 0.1659 0.1689 0.0503 0.0475 0.0477 0.0484 0.0479 0.0478 0.0476 0.1474 0.1905 0.1943 0.1658 0.1739 0.0522 0.0502 0.0505 0.0502 0.0493 0.0505 0.0495 0.1546 0.1493 0.1968 0.1755 0.1762 0.0499 0.0456 0.0453 0.0551 0.0452 0.0453 0.0486 0.0459 0.0466 0.0462 0.0463 0.0463 0.0467 0.0487 0.0484 0.0479 0.049 0.0506 0.0486 0.049 0.0501 0.0504 0.0507 0.0528 0.0497 0.0487 0.0479 0.0498 0.048 0.0474 0.0472 0.0542 0.0481 0.0487 0.0493 0.0484 0.0532 0.0472 0.0483 0.0512 0.0478 0.0493 0.0481 0.0487 0.048 0.046 0.047 0.0467

0.5971 0.1574 0.1279 0.0723 0.1787 0.0471 0.047 0.0437 0.0841 0.0486 0.0458 0.0461 0.135 0.1327 0.0806 0.0742 0.1817 0.0489 0.0484 0.0495 0.05 0.0472 0.0499 0.0494 0.1354 0.1347 0.0791 0.0726 0.1796 0.0471 0.0473 0.0478 0.0479 0.047 0.0471 0.0468 0.1379 0.1295 0.0794 0.0801 0.1866 0.0496 0.0511 0.0506 0.0498 0.0505 0.0496 0.0503 0.0439 0.2015 0.087 0.0986 0.1902 0.0479 0.0448 0.0442 0.0447 0.0445 0.0454 0.0451 0.1899 0.1495 0.1865 0.1949 0.199 0.0482 0.0477 0.0482 0.0476 0.0479 0.0508 0.0469 0.1914 0.1495 0.1881 0.1974 0.1935 0.0479 0.0477 0.0463 0.0468 0.0479 0.0467 0.0473 0.1888 0.1508 0.1867 0.1923 0.1971 0.0478 0.0474 0.0498 0.0478 0.0469 0.0483 0.0472

126

APPENDIX 5

TMAMQ

1 2 3 4 5 6 7 8 9 10 11 0.0671 0.0538 0.047 0.0438 0.043 0.0437 0.0445 0.1113 0.047 0.0463 0.0491 0.1229 0.0543 0.0477 0.0466 0.0477 0.0572 0.0712 0.0824 0.0461 0.0466 0.1082 0.1137 0.0533 0.0488 0.0441 0.0472 0.0571 0.0723 0.0845 0.0466 0.0472 0.0928 0.0918 0.0881 0.0543 0.0481 0.0456 0.0476 0.0589 0.0743 0.0938 0.0486 0.049 0.0721 0.0725 0.0705 0.0508 0.0449 0.0407 0.0402 0.038 0.0381 0.0378 0.1237 0.0472 0.055 0.0493 0.0457 0.0432 0.043 0.0472 0.0576 0.073 0.0462 0.0819 0.0469 0.0439 0.0445 0.0438 0.0466 0.0448 0.0442 0.0479 0.0627 0.0771 0.0477 0.0869 0.0463 0.0483 0.0478 0.0478 0.0442 0.0431 0.0423 0.0499 0.0591 0.0754 0.0466 0.0836 0.0481 0.0525 0.051 0.0494

127

APPENDIX 6

Nitrite quantification assay

Parinari curatellifolia

1 2 3 4 5 6 7 8 9 10 11 2.2715 2.5447 3.2 1.4848 1.3883 1.1074 0.6857 0.6205 0.396 0.2933 0.3234 0.3006 0.292 0.3019 0.191 0.2124 0.2049 0.1539 0.2154 0.2041 0.1224 0.1016 0.1068 0.0788 0.0563 0.058 0.0654 0.0652 0.0635 0.063 0.0752 0.0731 0.0766 0.0728 0.0727 0.0778 0.0576 0.058 0.0615 0.0606 0.0594 0.0602 0.0767 0.079 0.0786 0.083 0.073 0.0736 0.0726 0.074 0.0732 0.0745 0.066 0.0678 0.0687 0.0659 0.0688 0.0665 0.0677 0.0752 0.0771 0.0763 0.0773 0.0681 0.0678 0.0717 0.0702 0.0695 0.0656 0.0713 0.0737 0.0761 0.0751 0.078 0.0781 0.0691 0.0689 0.0744 0.0712 0.0693 0.069 0.0708 0.0723 0.075 0.0752 0.0783 0.0794 0.0701 0.0705 0.0731 0.0712 0.0702 0.0701 0.0737 0.0722

128

Standard curve

129