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Cytotoxic Activity of Indian, Indonesian and Uk Plant

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Cytotoxic Activity of Indian, Indonesian and Uk Plant CYTOTOXIC ACTIVITY OF INDIAN, INDONESIAN AND UK PLANT EXTRACTS ON BREAST CANCER CELLS A thesis submitted for the degree of Doctor of Philosophy by Susan Jane Wagland Department of Life Sciences, College of Health and Life Sciences, Brunel University London October 2015 Abstract Many plants in biodiversity hotspots have not yet been screened for biological activity in humans. Plants synthesise secondary metabolites that may be unique to each species or induced in response to specific conditions. Assam in North East India forms part of the Indo-Burma biodiversity hotspot for conservation and is a rich source of medicinal plants for traditional Indian medicine. This study concerns the potential cytotoxic effects of three Assamese medicinal plant extracts, from Clitoria ternatea root, Mucuna pruriens seed and Cheilocostus speciosus rhizome. The aim of the study was to characterise the cytotoxic activity, to elucidate the biological mechanism and to isolate the bioactive agents for further investigation. Clitoria was prioritised for study, as its crude extract was found to have a dose related, cytotoxic effect that was 2-3 fold more effective on breast cancer cells than non-cancer cells. Two bioactive fractions of Clitoria were isolated in bioassay-led fractionation with high performance liquid chromatography. The fractions demonstrated a selective dose related cytotoxic effect over a range of 100 to 700 ng/µL. In live cell observation, crude extract and a bioactive fraction were cytostatic at non-lethal treatment doses, and crude extract reduced cells’ motility by 50%. Both crude extract and fractions caused structural changes in the actin cytoskeleton which could be contributing to the cytotoxic and cytostatic effects and reduced motility. Liquid Chromatography-Mass Spectrometry analysis indicates that the two isolated bioactive agents are novel homoisoflavones, polyphenolic secondary metabolites, which were also detected in extracts made from Indonesian sourced Clitoria root and are likely to be in extract made from UK sourced Clitoria root. Results suggest that bioactive components of Clitoria extract are worthy of further study and may provide lead compounds for new cytotoxic or antiproliferative anti-cancer therapies. i Declaration I, Susan Jane Wagland, declare that this thesis is my own work. Signed ___________________________________ Date___________________ ii Table of Contents Table of Contents .........................................................................................................iii List of Figures ............................................................................................................... x List of Tables ........................................................................................................... xvii Glossary.........................................................................................................................xx Acknowledgments ..................................................................................................... xxiii Chapter 1 Introduction .............................................................................................. 1 1.1 Breast cancer ................................................................................................. 1 1.2 Breast cancer therapies .................................................................................. 3 1.2.1 Introduction .............................................................................................. 3 1.2.2 Surgery .................................................................................................... 4 1.2.3 Radiotherapy ........................................................................................... 4 1.2.1 Chemotherapy ......................................................................................... 5 1.2.2 Targeted treatments ................................................................................ 8 1.2.2.1 Hormone therapy ......................................................................................... 11 1.2.2.2 Biological therapy ........................................................................................ 14 1.2.2.3 New developments in targeted treatments .................................................. 16 1.3 Cell death and chemotherapy ....................................................................... 18 1.4 The cytoskeleton in cancer ........................................................................... 22 1.4.1 Introduction ............................................................................................ 22 1.4.2 Microtubules in cell division ................................................................... 23 1.4.3 Drugs that target microtubules ............................................................... 24 iii 1.4.4 Actin filaments in cell motility ................................................................. 28 1.4.5 Actin filaments in cell division................................................................. 33 1.4.6 Drugs that target the actin cytoskeleton ................................................. 34 1.4.7 The need for new drugs ......................................................................... 36 1.5 Sources of new drugs ................................................................................... 37 1.5.1 Introduction ............................................................................................ 37 1.5.2 Target-based and phenotypic drug discovery ........................................ 38 1.5.3 Natural product drug discovery .............................................................. 40 1.6 Traditional medicine systems ........................................................................ 45 1.6.1 Traditional Chinese Medicine ................................................................. 45 1.6.2 Indian traditional medicine ..................................................................... 47 1.7 Aims and objectives of this study .................................................................. 48 1.7.1 Clitoria ternatea ..................................................................................... 48 1.7.2 Mucuna pruriens .................................................................................... 50 1.7.3 Cheilocostus speciosus ......................................................................... 51 1.7.4 Specific aims ......................................................................................... 52 1.7.5 Objectives .............................................................................................. 52 1.7.6 How this thesis is organised .................................................................. 53 Chapter 2 Materials and methods .......................................................................... 55 2.1 Preparation of plant extracts ......................................................................... 55 2.1.1 Sources of plant material ....................................................................... 55 2.1.2 Preparation of extracts ........................................................................... 56 iv 2.2 Tissue culture ............................................................................................... 57 2.2.1 Cell lines ................................................................................................ 57 2.2.2 Cell maintenance ................................................................................... 58 2.2.3 Passaging .............................................................................................. 59 2.2.4 Cell storage, freezing and recovery ....................................................... 60 2.2.5 Cell counting .......................................................................................... 60 2.3 Cell doubling times ....................................................................................... 61 2.4 Cell viability assays ....................................................................................... 61 2.4.1 MTT protocol ......................................................................................... 62 2.4.2 PrestoBlue protocol ............................................................................... 63 2.4.3 Methylene blue assay protocol............................................................... 63 2.4.4 Dilution of biochanin A for cell viability assays ....................................... 64 2.4.5 Calculation of estimated half maximal inhibitory concentration from cell viability assays ..................................................................................................... 64 2.4.6 Method development for cell viability assays ......................................... 65 2.4.6.1 Establishing optimum seeding density for MTT and PrestoBlue assays ......... 65 2.4.6.2 Establishing vehicle concentration ............................................................... 67 2.4.6.3 Establishing optimum incubation time for PrestoBlue assay ......................... 68 2.5 Cell counting assay ....................................................................................... 69 2.6 Metaphase spread preparation and counting ................................................ 70 2.7 Fluorescence microscopy ............................................................................. 71 2.7.1 Protocol ................................................................................................. 71 v
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