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In Search of Novel Immuno-Modulatory Compounds from British Columbia Wild Mushrooms and Their Effectiveness in Inflammatory Micro-Circulation of Mice

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In Search of Novel Immuno-Modulatory Compounds from British Columbia Wild Mushrooms and Their Effectiveness in Inflammatory Micro-Circulation of Mice IN SEARCH OF NOVEL IMMUNO-MODULATORY COMPOUNDS FROM BRITISH COLUMBIA WILD MUSHROOMS AND THEIR EFFECTIVENESS IN INFLAMMATORY MICRO-CIRCULATION OF MICE by Sumreen Javed Doctor of Pharmacy (Pharm-D), Riphah International University, Pakistan, 2013 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOCHEMISTRY UNIVERSITY OF NORTHERN BRITISH COLUMBIA July 2017 © Sumreen Javed, 2017 Abstract Natural products have been an integral component of people’s health and health outcomes for thousands of years. In particular, several mushroom species have demonstrated beneficial therapeutic potential. The goals of this research are to explore the immuno-stimulatory and anti- inflammatory potential of wild mushrooms native to the North Central region of British Columbia. Out of 42 mushroom extracts examined, four exhibited strong immuno-stimulatory activity as assessed by induction of tumor-necrosis factor alpha (TNF-α) production in macrophage cells. Out of thirty-three extracts tested, nineteen demonstrated potent anti- inflammatory activity as determined by inhibition of lipopolysaccharide-induced TNF-α production in macrophage cells. Sodium hydroxide extract of Echinodontium tinctorium exhibited potent anti-inflammatory activity and was selected for further study. A small molecular weight (~5-25 kDa) carbohydrate was successfully purified using sequential size-exclusion and ion-exchange chromatography. GC-MS analysis showed that the polysaccharide has glucose (89.7%) as the major back-bone monosaccharide, and also the presence of other monosaccharides such as mannose (3.1%), galactose (2.8%), fucose (2.4%), and xylose (2.0%). The study also revealed the presence of 1,3-linked glucose, 1,6-linked glucose, 1,3-linked galactose and 1,3,6-linked glucose linkages. Both the semi-purified anti-inflammatory compound(s) from E. tinctorium and the methanol extract of Inonotus obliquus can ameliorate histamine-induced vasodilation in the 2A arterioles (gluteus maximus muscle) in mice. This is the first study to demonstrate the anti-inflammatory activity of purified compounds and extracts from mushroom in an animal microcirculation model using intravital microscopy. i Table of Contents Abstract .................................................................................................................................. i Table of Contents .................................................................................................................. ii List of Figures ..................................................................................................................... vii List of Tables ...................................................................................................................... viii Acknowledgement ................................................................................................................ x Chapter 1: Background and Objectives ................................................................ 1 1.1 The Havoc of Cancer ................................................................................................ 1 1.2 Natural Products to Treat Cancer .............................................................................. 3 1.3 Mushrooms use-Historical Perspective..................................................................... 4 1.4 Mushrooms & Modern Medicines ............................................................................ 6 1.5 British Columbia Fungi............................................................................................. 7 1.6 Immunotherapy & Cancer ......................................................................................... 7 1.7 Mushrooms with potential immuno-modulatory activity ....................................... 10 1.7.1 Lentinula edodes .....................................................................................................11 1.7.2 Inonotus obliquus ....................................................................................................11 1.7.3 Ganoderma lucidum................................................................................................11 1.7.4 Schizophyllum commune. ....................................................................................... 12 1.7.5 Trametes versicolor ................................................................................................ 12 1.7.6 Miscellaneous Mushrooms .................................................................................... 13 1.8 Mushrooms, Inflammation and Micro-circulation .................................................. 14 1.9 Research Objectives ................................................................................................ 16 1.9.1 Specific Aims ................................................................................................... 16 1.10 Thesis Structure ...................................................................................................... 17 ii Chapter 2: Immuno-modulatory Studies........................................................18 Immuno-stimulatory Potential ........................................................................................ 19 Introduction to Immuno-stimulation concepts .................................................................... 19 2.1 An overview of the immune system.............................................................................. 19 2.11 Innate immune response .............................................................................................. 19 2.1.2 Adaptive Immune Response .................................................................................. 20 2.2 Immunotherapy-Role of Macrophages ......................................................................... 22 2.3 Immunotherapy-Role of TNF-α .................................................................................... 24 2.4 Immuno-modulatory compounds from wild mushrooms ............................................. 26 2.4.1 Immuno-modulatory polysaccharides: ................................................................... 27 2.4.2 Fungal immuno-modulatory proteins (FIPs) ......................................................... 27 2.4.3 Immuno-modulatory lectins ................................................................................... 28 2.4.4 Immuno-modulatory terpenes and terpenoids ....................................................... 28 2.5 Materials & Methods .............................................................................................. 29 2.5.1 Mushroom Collection & Identification ........................................................... 29 2.5.2 Chemical extraction ......................................................................................... 31 2.5.3 Assessing chemical fractions for immuno-stimulatory activity ...................... 34 2.5.4 Enzyme-Linked Immunosorbent Assay (ELISA) ............................................ 34 2.5.5 Data Analysis ................................................................................................... 35 2.6 Results ........................................................................................................................... 36 2.6.1 Mushroom Collection and Identification: .............................................................. 36 2.6.2 Mushroom Extraction ............................................................................................ 38 2.6.3 Immuno-stimulatory Assay .................................................................................... 41 iii 2.7 Discussion ..................................................................................................................... 45 Anti-inflammatory Studies ............................................................................................... 50 2.8 Introduction ............................................................................................................. 50 2.8.1 Inflammation .......................................................................................................... 50 2.8.2 Anti-Inflammatory Activity/Compounds from Mushrooms .................................. 51 2.9 Material & Methods ................................................................................................ 55 2.9.1 Tumor Necrosis Factor Alpha Inhibition after LPS treatment ......................... 55 2.9.2 Tumor Necrosis Factor Alpha Inhibition after Histamine treatment ............... 56 2.9.3 Chemical Extracts-Treatment .......................................................................... 56 2.9.4 Enzyme-Linked Immunosorbent Assay (ELISA) ............................................ 57 2.10 Results ..................................................................................................................... 57 2.10.1 Tumor Necrosis Factor Alpha Inhibition after LPS treatment ......................... 57 2.10.2 Tumor Necrosis Factor Alpha Inhibition after Histamine treatment ............... 57 2.11 Discussion ............................................................................................................... 64 2.12 Conclusion .............................................................................................................. 68 Chapter 3: Purification and Characterization of Anti-Inflammatory Compound(s) from Echinodontium tinctorium ....................................................72 3.1 Introduction ............................................................................................................
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