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 i 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. i 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. ii 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. iii 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 iv 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 v 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……………………………………………………………….......