BIOACTIVE POLYSACCHARIDES AND SMALL MOLECULES FROM THE NATIVE NORTH AMERICAN FUNGUS ECHINODONTIUM TINCTORIUM by Mehreen Zeb (MPhil Biochemistry/Molecular Biology), Quaid-e-Azam University, Islamabad, Pakistan, 2013 (Doctor of Pharmacy, PharmD), Riphah International University, Islamabad, Pakistan, 2009 DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HEALTH SCIENCE UNIVERSITY OF NORTHERN BRITISH COLUMBIA December, 2021 © Mehreen Zeb, 2021 ABSTRACT Mushrooms, the fruiting bodies of fungi, are known to be powerful sources of nutraceuticals and pharmaceuticals but there are limited studies focusing on exploring the medicinal value of mushrooms native to North America. Here, I describe the isolation of two novel bioactive polysaccharides from the aqueous extracts of the fungus Echinodontium tinctorium: an immunostimulatory complex polysaccharide (EtISPFa) of 1354 kDa, and a growth-inhibitory β-glucan of 275 kDa. In addition, six small molecules including a phenol derivative, a new diphenylmethane derivative and three lanostane-type triterpenes were isolated from the organic extracts of E. tinctorium. The molar mass of these isolated small molecules (labelled 1-6) was determined to be 124, 260, 506, 498, 496, and 440 g/mol respectively. Phase separation, Sephadex LH-20 size exclusion, Sephadex DEAE ion exchange chromatography, Sephacryl S-500 HR size exclusion, silica column chromatography, and HPLC were used for bioactivity-guided purification. Chemical structures and linkages of EtISPFa and EtGIPL1a polysaccharides were determined by gas chromatography mass spectrometry (GCMS) and nuclear magnetic resonance (NMR). Final structures of small molecules were determined by Fourier transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS), NMR, and X-ray crystallography. Immuno-stimulatory activity of EtISPFa was assessed by immunoassay in Raw 264.7 murine macrophage cells and growth-inhibitory activity of EtGIPL1a and small molecules were assessed by MTT growth-inhibitory assay in cancer cell lines. The mechanism of growth inhibition was assessed via apoptosis and cell cycle assays. EtISPFa stimulated the immune response by inducing TNF-α and other inflammatory cytokines in murine macrophage cells. In contrast, EtGIPL1a showed promising anti-proliferative activity against U251 glioblastoma cells and on ii ten other cancer cell lines. EtGIPL1a induced apoptosis in U251 cells with an increased cleaved caspase-3 apoptotic marker and significant DNA fragmentation in cell cycle analysis. Amongst the small molecules, compounds (2), (4) and (5) caused growth-inhibition in U251 cells; compound (4) also showed promising effects on multiple other cancer cell lines; all its bioactivities are reported here for the first time. The crystal structures of compounds (2), (4) and (5) have also been reported for the first time. Molecular targets of (1), (2), (4) and (5) by MolTarPred were predicted and warrants further experimental investigation. iii PREFACE This current PhD dissertation has yielded two manuscripts where Chapter 1 and Chapter 2 have been published as: Paper 1/ Chapter 1: Zeb, M., & Lee, C. H. (2021). Medicinal properties and bioactive compounds from wild mushrooms native to North America. Molecules, 26(2), 1-24. Paper 2/ Chapter 2: Zeb, M., Tackaberry, L. E., Massicotte, H. B., Egger, K. N., Reimer, K., Lu, G., Heiss, C., Azadi, P., & Lee, C. H. (2021). Structural elucidation and immuno-stimulatory activity of a novel polysaccharide containing glucuronic acid from the fungus Echinodontium tinctorium. Carbohydrate Polymers, 258, 1-9. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................. ii PREFACE .....................................................................................................................................iv LIST OF TABLES ......................................................................................................................... x LIST OF FIGURES .................................................................................................................... xii LIST OF ABBREVIATIONS .................................................................................................. xvii ACKNOWLEDGEMENT ...................................................................................................... xviii Chapter 1: Introduction and Literature Review ........................................................................ 1 1.1 Medicinal mushrooms ..................................................................................................... 1 1.2 Mushrooms native to North America .............................................................................. 2 1.3 Medicinal properties of mushrooms native to North America ........................................ 3 1.3.1 Anti-bacterial and anti-viral activities ......................................................................... 4 1.3.2 Anti-proliferative activity ............................................................................................ 9 1.3.3 Anti-inflammatory activity ........................................................................................ 10 1.3.4 Immuno-stimulatory activity ..................................................................................... 11 1.3.5 Anti-oxidant activity .................................................................................................. 15 1.3.6 Anti-fungal activity ................................................................................................... 15 1.3.7 Other bioactivities ..................................................................................................... 15 1.4 Bioactive compounds from mushrooms native to North America ................................ 16 1.4.1 Large molecular weight compounds ......................................................................... 17 1.4.2 Small molecules......................................................................................................... 21 1.5 Overview of strategies used for natural products discovery.......................................... 22 1.6 Mushrooms from North America as a source for drug discovery ................................. 26 1.7 Conclusions ................................................................................................................... 30 1.8 Echinodontium tinctorium ............................................................................................. 31 1.9 Research Hypothesis ..................................................................................................... 32 1.10 Research Objectives ...................................................................................................... 33 1.11 References ..................................................................................................................... 33 Chapter 2: Structural elucidation and immuno-stimulatory activity of a novel polysaccharide containing glucuronic acid from the fungus Echinodontium tinctorium ...... 45 ABSTRACT .................................................................................................................................. 45 2.1. Introduction ................................................................................................................... 45 2.2. Materials and methods ................................................................................................... 46 2.2.1. Materials and reagents ............................................................................................... 46 2.2.2. Collection and extraction of the mushroom .............................................................. 47 2.2.3. Immuno-stimulatory assay ........................................................................................ 47 v 2.2.4. Purification of immuno-stimulatory compound(s) from E. tinctorium ..................... 48 2.2.5. Molecular size distribution and ultrapurification using HPLC ................................. 49 2.2.6. Enzyme digestion and heat denaturation of polysaccharides from E. tinctorium ..... 49 2.2.7. Monosaccharide composition analysis ...................................................................... 50 2.2.8. Methylation and linkage analysis .............................................................................. 50 2.2.9. Structural elucidation by spectral analysis ................................................................ 51 2.2.10. Estimation of glucan content in EtISPFa................................................................... 51 2.3. Results and discussion ................................................................................................... 52 2.3.1. Extraction and assessment of E3 water extract for immuno-stimulatory activity ..... 52 2.3.2. Purification of immuno-stimulatory polysaccharide from E. tinctorium .................. 53 2.3.3. Cytokine and chemokine secretion in Raw264.7 cells induced by 4A ..................... 59 2.3.4. Glucan content analysis ............................................................................................. 61 2.3.5. Role of polysaccharide component in contributing to the immuno-stimulatory activity. .................................................................................................................................. 61 2.3.6. Monosaccharide composition analysis ...................................................................... 63 2.3.7. Glycosyl linkage analysis by GC–MS ....................................................................... 64 2.3.8. Structural analysis by FTIR ....................................................................................... 66 2.3.9. NMR analyses of EtISPFa ......................................................................................... 67 2.4. Conclusion ..................................................................................................................... 76 2.5. References ..................................................................................................................... 76 Appendix A. Supplementary data.................................................................................................. 79 Chapter 3: Structural elucidation of an anti-proliferative polysaccharide from the fungus Echinodontium tinctorium ........................................................................................................... 86 ABSTRACT .................................................................................................................................. 86 3.1. Introduction ................................................................................................................... 86 3.2. Materials and methods ................................................................................................... 88 3.2.1. Materials, reagents and cell lines............................................................................... 88 3.2.2. Collection and extraction of the mushroom .............................................................. 89 3.2.3. Anti-proliferative assay ............................................................................................. 89 3.2.4. Purification of anti-proliferative polysaccharide from E. tinctorium ........................ 90 3.2.5. Molecular size distribution and ultrapurification using HPLC ................................. 90 3.2.6. Monosaccharide composition analysis ...................................................................... 91 3.2.7. Methylation and linkage analysis .............................................................................. 91 3.2.8. Structural elucidation by spectral analysis ................................................................ 91 3.2.9. Growth-inhibition in U251 glioblastoma cells .......................................................... 92 3.2.10. Flow cytometry analysis ........................................................................................ 93 3.3. Results and Discussion .................................................................................................. 93 3.3.1. Extraction and assessment of methanolic extract from E. tinctorium for antiproliferative activity .............................................................................................................. 93 3.3.2. Purification of anti-proliferative polysaccharide (EtGIPL1a) from E. tinctorium .... 94 3.3.3. Estimation of molecular size and purification by HPLC........................................... 98 3.3.4. Assessing EtGIPL1a against a panel of human cancer cell lines ............................ 100 3.3.5. Growth inhibition in U251 glioblastoma cells ........................................................ 101 vi 3.3.5.1. Morphological changes in glioblastoma cell lines (U251) .............................. 101 3.3.5.2. Flow cytometry analysis of EtGIPL1a ............................................................ 102 3.3.5.3. Molecular markers involved in differentiation and apoptosis ......................... 106 3.3.6. Monosaccharide composition analysis .................................................................... 106 3.3.7. Glycosyl linkage analysis by GC-MS ..................................................................... 107 3.3.8. Structural analysis by FTIR ..................................................................................... 110 3.3.9. NMR analyses of EtGIPL1a .................................................................................... 111 3.4. Conclusion ................................................................................................................... 115 3.5. References ................................................................................................................... 116 Chapter 4: Lanostane triterpenoids, phenol and diphenylmethane derivatives from the fungus Echinodontium tinctorium ............................................................................................ 126 ABSTRACT ................................................................................................................................ 126 4.1. Introduction ................................................................................................................. 126 4.2. Materials and methods ................................................................................................. 128 4.2.1. Materials and reagents ............................................................................................. 128 4.2.2. Collection and extraction of the mushroom ............................................................ 128 4.2.3. Approach 1: Two solvent phase separation ............................................................. 129 4.2.3.1. Extraction and phase separation ...................................................................... 129 4.2.3.2. Assessment of growth-inhibitory activity ....................................................... 129 4.2.3.3. Purification by Sephadex LH-20 SEC ............................................................. 129 4.2.3.4. HPLC analysis and purification....................................................................... 129 4.2.3.5. ESI-MS analysis .............................................................................................. 130 4.2.4. Approach 2: Sequential phase extraction ................................................................ 131 4.2.4.1. Sequential phase extraction and assessment for growth-inhibitory activity.... 131 4.2.4.2. HPLC analysis of phase separated layers ........................................................ 131 4.2.4.3. Approach 2a: Purification of EA layer by Sephadex LH-20 SEC .................. 131 4.2.4.4. HPLC analysis of Sephadex LH-20 purified compounds ............................... 131 4.2.4.5. Approach 2b: Purification of EA layer by silica flash column chromatography 132 4.2.4.6. TLC method development ............................................................................... 132 4.2.4.7. Purification by Silica Flash Column (SFC) ..................................................... 132 4.2.4.8. Additional purification of Post SFC fractions ................................................. 133 4.2.4.9. HPLC analysis and purification of Post SFC fractions ................................... 133 4.2.4.10. Structural elucidation of compounds from EA layer ....................................... 134 4.2.4.11. Crystallization of compound (2) ...................................................................... 134 4.2.4.12. Crystallization of compound (3) ...................................................................... 135 4.2.4.13. Purification and HPLC analysis of phase separated hexane layer................... 135 4.2.4.14. Purification and HPLC analysis of phase-separated chloroform layer............ 136 4.2.5. Approach 3: Direct extraction method .................................................................... 137 4.2.5.1. Solid-liquid extraction and assessment for growth-inhibitory activity ........... 137 4.2.5.2. HPLC analysis and purification of hexane extract .......................................... 137 4.2.5.3. Structural elucidation by spectroscopy ............................................................ 137 4.2.5.4. Crystallization of compound (4) ...................................................................... 138 4.2.5.5. Crystallization of compound (5) ...................................................................... 138 vii 4.2.5.6. Melting point determination ............................................................................ 139 4.2.5.7. HPLC-MS analysis of Diethyl ether extract (DEE) ........................................ 139 4.2.5.8. Growth-inhibitory activity and flow cytometry analysis of purified compounds… ................................................................................................................... 139 4.2.5.9. Molecular target prediction of compounds (1-5)............................................. 140 4.3. Results and Discussion ................................................................................................ 140 4.3.1. Approach 1-Two Solvent Phase Separation ............................................................ 140 4.3.1.1. Extraction and assessment of growth-inhibitory activity ................................ 140 4.3.1.2. Purification by Sephadex LH-20 and HPLC ................................................... 141 4.3.1.3. Mass spectrometric analysis of HPLC-purified compound............................. 143 4.3.2. Approach 2: Sequential phase extraction ................................................................ 144 4.3.2.1. Sequential phase separation and assessment for growth-inhibitory activity ... 144 4.3.2.2. Purification of EA layer by Sephadex LH-20 SEC ......................................... 145 4.3.2.3. HPLC and ESI-MS analysis ............................................................................ 146 4.3.2.4. Purification of EA layer by SFC and HPLC ................................................... 147 4.3.2.5. Chemical characterization of compound (1) ................................................... 150 4.3.2.6. HPLC and ESI-MS analysis of (1) .................................................................. 150 4.3.2.7. Structural elucidation of (1) by FTIR and NMR ............................................. 151 4.3.2.8. Chemical characterization of compound (2) ................................................... 156 4.3.2.9. HPLC and ESI-MS analysis of (2) .................................................................. 156 4.3.2.10. Structural elucidation by FTIR and NMR spectroscopy ................................. 158 4.3.2.11. Crystallization and X-ray crystallography of (2)............................................. 163 4.3.2.12. Assessment of growth-inhibitory activity of (2) ............................................. 164 4.3.2.13. Flow cytometry analysis of compound (2) ...................................................... 165 4.3.2.14. HPLC-MS analysis of additional fractions from SFC ..................................... 168 4.3.2.15. Chemical characterization of compound (3) ................................................... 169 4.3.2.16. HPLC, ESI-MS and FTIR analysis of (3) ....................................................... 169 4.3.2.17. Crystallization of compound (3) ...................................................................... 172 4.3.2.18. HPLC analysis of hexane layer ....................................................................... 172 4.3.2.19. Purification and HPLC analysis of compounds from chloroform layer .......... 173 4.3.2.20. Purification by Sephadex LH-20 SEC ............................................................. 174 4.3.3. Approach 3: Direct extraction method .................................................................... 179 4.3.3.1. Extraction and assessment of growth-inhibitory activity of extracts .............. 179 4.3.3.2. HPLC analysis and purification....................................................................... 180 4.3.3.3. Characterization of compound (4) ................................................................... 186 4.3.3.4. HPLC and ESI-MS analysis of compound (4) ................................................ 186 4.3.3.5. Structural elucidation by FTIR and NMR spectroscopy ................................. 187 4.3.3.6. Crystallization and crystal data of compound (4)............................................ 193 4.3.3.7. Growth-inhibitory effects of (4) on cancer cells ............................................. 194 4.3.3.8. Flow cytometry analysis of compound (4) ...................................................... 195 4.3.3.9. HPLC and ESI-MS analysis of PK5 ................................................................ 197 4.3.3.10. Structural elucidation of (5) by NMR spectroscopy........................................ 199 4.3.3.11. Crystallization and crystal structure of compound (5) .................................... 204 4.3.3.12. Growth inhibitory activity of (5) ..................................................................... 205 4.3.3.13. HPLC analysis of PK5a ................................................................................... 206 4.3.3.14. Melting point determination ............................................................................ 208 viii 4.3.3.15. HPLC and ESI-MS analysis of DEE layer ...................................................... 208 4.3.3.16. Target prediction of compounds 1-5 ............................................................... 209 4.4. Conclusion ................................................................................................................... 211 4.5. References ................................................................................................................... 211 Chapter 5: Discussion................................................................................................................ 242 5.1. Overview of Echinodontium tinctorium ...................................................................... 242 5.2. Overview of project ..................................................................................................... 242 5.3. Key findings of the biomolecules isolated from E. tinctorium ................................... 243 5.4. Biosynthesis of molecules using Biosynthetic Gene Cluster ...................................... 245 5.4.1. Biosynthesis of isolated polysaccharides ................................................................ 247 5.4.2. Biosynthesis of small molecules isolated from E. tinctorium ................................. 249 5.4.2.1. Biosynthesis of Orcinol (1) ............................................................................. 249 5.4.2.2. Biosynthesis of diphenylmethane derivative (2) ............................................. 251 5.4.2.3. Biosynthesis of lanostane-type triterpenes ...................................................... 251 5.5. Chemical synthesis of isolated molecules ................................................................... 252 5.5.1. Chemical synthesis of isolated small molecules ..................................................... 252 5.5.2. Chemical synthesis of polysaccharides ................................................................... 253 5.6. Compounds produced by phylogenetically related versus unrelated fungi ................. 254 5.6.1. Phylogenetically unrelated fungi ............................................................................. 254 5.6.2. Phylogenetically related fungi ................................................................................. 256 5.7. Future studies on isolated compounds ......................................................................... 257 5.8. Glioblastoma and molecular mechanisms of apoptosis............................................... 260 5.9. Molecular target prediction and their importance ....................................................... 261 5.10. Experimental approaches to identify molecular targets of isolated compounds ......... 263 5.11. References ................................................................................................................... 264 ix LIST OF TABLES Chapter 1 Table 1.1. Mushrooms native to North America studied for bioactivities ...................................... 5 Table 1. 2. Immuno-stimulatory compounds from mushrooms .................................................... 13 Table 1. 3. Bioactive molecules from mushrooms native to North America ................................ 19 Table 1. 4. Methodologies for purification and characterization of large molecules .................... 24 Table 1. 5. Methodologies for purification and characterization of small molecules ................... 25 Table 1. 6. Medicinal properties of North American mushrooms ................................................. 27 Chapter 2 Table 2. 1. Cytokines induction by EtincFa in Raw 264.7 murine macrophage cells................... 60 Table 2. 2. Monosaccharide composition of EtISPFa ................................................................... 64 Table 2. 3. Glycosyl linkage analysis of EtISPFa by partially methylated alditol acetates .......... 65 Table 2. 4. NMR chemical shift assignments for the residues found in EtISPFa ......................... 70 Chapter 3 Table 3. 1. IC50 of EtGIPL1a against human cancer cell lines .................................................... 101 Table 3. 2. Monosaccharide composition of EtGIPL1a .............................................................. 107 Table 3. 3. Glycosyl linkage analysis of EtGIPL1a by partially methylated alditol acetates ..... 109 Table 3. 4. NMR chemical shift assignments for the residues found in EtGIPL1a that were not found in EtISPFa ......................................................................................................................... 113 Chapter 4 Table 4.1. Small molecules isolated from E. tinctorium and related species .............................. 127 Table 4. 2. Parameters for mass spectrometry analysis ............................................................... 130 Table 4. 3. Quantitative estimation of E2, L1 and L2 layers after phase separation ................... 140 Table 4. 4. Quantitative estimation of phase separated layers..................................................... 145 Table 4. 5. 13C and 1H Chemical shifts of (1) .............................................................................. 153 Table 4. 6. 13C and 1H NMR chemical shifts of (2) .................................................................... 159 Table 4. 7. 13C-1H multiple bond couplings (HMBC) ................................................................. 163 Table 4. 8. Quantitative estimation of extracts and compounds from direct extraction method . 180 Table 4. 9. 13C Chemical shift data of compound (4) .................................................................. 189 Table 4. 10. 1H Chemical shift data of compound (4) ................................................................. 189 Table 4. 11. IC50 of compound (4) on multiple cell lines ............................................................ 195 Table 4. 12. 1H NMR chemical shift data of compound (5)........................................................ 200 Table 4. 13. Chemical shift data of compound (5) from 13C NMR ............................................. 201 Table 4. 14. Small molecules isolated from E. tinctorium .......................................................... 208 Table 4. 15. Predicted molecular targets of compounds from E. tinctorium using MolTarPred 211 Chapter 5 Table 5. 1. Host-derived compounds from phylogenetically unrelated fungi ............................. 256 x Supplementary Table S 1. Calculating the number (Mn) and weight average molecular weight (Mw) of EtISPFa ....................................................................................................................................................... 84 Table S 2. Quantitative estimation of material recovered from each purification step ................. 85 Table S 3. Calculating the number (Mn) and weight average molecular weight (Mw) of EtGIPL1a ..................................................................................................................................... 125 xi LIST OF FIGURES Chapter 1 Fig. 1. 1. Classification of bioactive compounds isolated from mushrooms. ............................... 17 Fig. 1. 2. Research methodology towards the discovery of bioactive compound(s) from natural products. ........................................................................................................................................ 23 Chapter 2 Fig. 2. 1. Chemical extraction of E. tinctorium and assessment of the water extract E3 for immuno-stimulatory activity on macrophage cells (A) Chemical extraction scheme to obtain water extract E3 from E. tinctorium. (B) Dose-dependent immuno-stimulatory assay shows induction of TNF-α production in Raw 264.7 macrophage cells by E3 extract from 0.1-1 mg/mL. ....................................................................................................................................................... 53 Fig. 2. 2. Purification of the immuno-stimulatory polysaccharide (EtISPFa) from E. tinctorium. (A) Summary of the purification scheme used. 1C extract from E. tinctorium was purified using Sephadex LH-20 size-exclusion chromatography (column-2). Fractions collected from column-2 were assessed for immuno-stimulatory activity (solid line in B and C), carbohydrate content (dotted line in B) and protein content ............................................................................................ 54 Fig. 2. 3. Purification of the immuno-stimulatory polysaccharide from E. tinctorium (EtISPFa) 55 Fig. 2. 4. HPLC BioSEC-5 full elution profile of (A) 4A and (B) 4B. The collected two peaks in (A) and (B) were assessed for immuno-stimulatory activity shown in (C). .................................. 57 Fig. 2. 5. (A) HPLC BioSEC-5 profile of purified Peak 1 EtISPFa. (B) HPLC BioSEC-5 purified Peak 1 EtISPFb. ............................................................................................................................. 58 Fig. 2. 6. Effect of cellulase on immuno-stimulatory activity of EtISPFa (A) and EtISPFb (B) from E. tinctorium. ........................................................................................................................ 62 Fig. 2. 7. Effect of fucosidase (A) and galactosidase (B) on immuno-stimulatory activity of EtISPFa from E. tinctorium.. ......................................................................................................... 62 Fig. 2. 8. Effect of heat denaturation on immuno-stimulatory activity of EtISPFa from E. tinctorium. ..................................................................................................................................... 63 Fig. 2. 9. FTIR spectrum of EtISPFa. ............................................................................................ 67 Fig. 2. 10. 1H-NMR spectrum of EtISPFa. .................................................................................... 68 Fig. 2. 11. Partial multiplicity-edited 1H-13C-HSQC NMR spectrum of EtISPFa. ....................... 71 Fig. 2. 12. Partial 1H-1H COSY spectrum of EtISPFa................................................................... 72 Fig. 2. 13. Partial 1H-13C HMBC spectrum of EtISPFa ................................................................ 73 Fig. 2. 14. Proposed representative structure of the immuno-stimulatory polysaccharide EtISPFa ....................................................................................................................................................... 74 Chapter 3 Fig. 3. 1. Chemical extraction of E. tinctorium and assessment of the ethanol extract 1B for antiproliferative activity against HeLa cells. (A) Chemical extraction scheme to obtain ethanol extract from E. tinctorium. (B) Dose-dependent anti-proliferative MTT assay ............................ 94 Fig. 3. 2. Purification of the anti-proliferative polysaccharide (EtGIPL1a) from E. tinctorium. (A) Summary of the purification scheme used. (B) 1B extract from E. tinctorium was purified using Sephadex LH-20 size-exclusion chromatography (column-1). Active fractions (F1-7) were pooled, lyophilized and ran through DEAE-Sephadex anion exchange chromatography (column2) as shown in (C).......................................................................................................................... 96 xii Fig. 3. 3. Purification of the anti-proliferative polysaccharide from E. tinctorium (EtGIPL1a) using Sephacryl S-500 (column-3). Collected fractions were assessed for cell viability (A), carbohydrate (B) and protein content (C).. .................................................................................... 97 Fig. 3. 4. HPLC BioSEC-3 full elution profile of (A) L1a (B) Peak 1 (EtGIPL1a), and (C) Peak 2. The collected two peaks in (B) and (C) were assessed for anti-proliferative activity as shown in (D). ............................................................................................................................................ 99 Fig. 3. 5. Morphological changes induced by EtGIPL1a after treatment at 13.7-220 nM for 48 h in U251 cells. ............................................................................................................................... 102 Fig. 3. 6. Flow cytometry analysis for apoptosis induced by EtGIPL1a at 1.76 µM for 48 h in SW480 cells (A) and at 27 nM for 24 h in U251 cells (B).. ........................................................ 103 Fig. 3. 7. Apoptosis induced by treatment with 27 nM EtGIPL1a compared to 40 µM resveratrol for 48 h in U251 cells .................................................................................................................. 104 Fig. 3. 8. Cell cycle analysis of EtGIPL1a at 27 nM for 48 h on U251 cells (A & B) and % cell population in G1, S, G2/M and subG0 phases of cell cycle (C).. ............................................... 105 Fig. 3. 9. Western blot analysis of EtGIPL1a on expression of cleaved caspase 3 in U251 cells. The three bold bands represent three batches of EtGIL1a at 12.5 µg/mL treatment for 48 h. .... 106 Fig. 3. 10. FTIR spectrum of EtGIPL1a. ..................................................................................... 111 Fig. 3. 11. 1H-NMR spectrum of EtGIPL1a. ............................................................................... 111 Fig. 3. 12. Partial multiplicity-edited 1H-13C-HSQC NMR spectrum of EtGIPL1a ................. 112 Fig. 3. 13. Proposed representative structure of the anti-proliferative polysaccharide EtGIPL1a ..................................................................................................................................................... 115 Chapter 4 Fig. 4. 1. HPLC solvent gradient for analysis and purification. B is % of acetonitrile and C is % of 0.2 % formic acid in water. ..................................................................................................... 130 Fig. 4. 2. HPLC solvent gradient for analysis and purification. B, C and D refers to acetonitrile, water and methanol...................................................................................................................... 134 Fig. 4. 3. Dose dependent growth-inhibitory effect (n=3) of L2 at 0.1-1 mg/mL for 48 h on HeLa cells (A), RIE-1 and SW480 cells (B). ........................................................................................ 141 Fig. 4. 4. A) Purification strategy using two solvent phase separation. B) Sephadex LH-20 SEC elution profile for growth-inhibitory small molecule(s) .............................................................. 142 Fig. 4. 5. HPLC spectrum of Post Sephadex LH-20 purified F20 (A) and HPLC purified compound (B) at 238 nm. ............................................................................................................ 143 Fig. 4. 6. ESI-MS FIA of HPLC purified compound at fragmentor voltage 40-400 V in UV (A) and MSD scan signal (B). ............................................................................................................ 143 Fig. 4. 7. ESI-MS spectrum of F20 in scan mode (A) and SIM mode (B). ................................. 144 Fig. 4. 8. A) Purification methodology for sequential phase separation of E. tinctorium. B) Sequential phase separated layers treated at 0.4 mg/mL for 48 h were assessed for growthinhibitory activity in HeLa cells .................................................................................................. 145 Fig. 4. 9. Sephadex LH-20 (70 mL column) elution profile of EA layer. ................................... 146 Fig. 4. 10. A) HPLC spectrum of Post Sephadex LH-20 bioactive fractions, B) ESI-MS of peak retained at 8 min. ......................................................................................................................... 147 Fig. 4. 11. TLC visualization by UV and multiple stains. ........................................................... 147 Fig. 4. 12. A) A TLC quadrant test of EA-and methanol-eluted fractions from silica column chromatography visualized under UV light, B) Full TLC on pooled UV visible fractions 4-9 visualized under UV light, C) Growth-inhibitory fractions of EA elution from silica column chromatography ........................................................................................................................... 148 xiii Fig. 4. 13. HPLC spectrum of SCC purified bioactive fractions 6-9 (A), and HPLC purity check of peak 1 (B), peak 3 (C) and peak 3 (D). ................................................................................... 149 Fig. 4. 14. HPLC spectrum of approach (iv) fractions F14-16 (A), F17-18 (B), and F19-22 (C). ..................................................................................................................................................... 150 Fig. 4. 15. HPLC DAD spectrum of (1) at 220 (A) and 280 nm (B)........................................... 151 Fig. 4. 16. ESI-MS spectrum of (1) in scan mode (A) and SIM (B). .......................................... 151 Fig. 4. 17. FTIR spectrum of (1). ................................................................................................ 152 Fig. 4. 18. 1H NMR spectrum of (1). ........................................................................................... 152 Fig. 4. 19. 13C NMR spectrum of (1). .......................................................................................... 153 Fig. 4. 20. 13C-1H HSQC spectrum of (1).................................................................................... 153 Fig. 4. 21. 13C-1H HMBC spectrum of (1). ................................................................................. 154 Fig. 4. 22. 1H-1H COSY spectrum of (1). .................................................................................... 155 Fig. 4. 23. 1H-1H COSY and 13C-1H HMBC structural correlations. .......................................... 155 Fig. 4. 24. 1H-1H NOESY spectrum of (1). ................................................................................. 156 Fig. 4. 25. HPLC DAD spectrum of (2) at multiple wavelengths. .............................................. 157 Fig. 4. 26. ESI-MS spectrum of (2) in scan mode m/z = 100-1000 (A) and SIM mode (B). ...... 157 Fig. 4. 27. FTIR spectrum of (2). ................................................................................................ 158 Fig. 4. 28. 13C NMR of (2). ......................................................................................................... 159 Fig. 4. 29. 1H NMR of (2). .......................................................................................................... 159 Fig. 4. 30. 13C-1H HSQC spectrum of compound (2). ................................................................. 160 Fig. 4. 31. 13C-1H HMBC spectrum of compound (2)................................................................. 161 Fig. 4. 32. 1H-1H COSY spectrum of compound (2). .................................................................. 162 Fig. 4. 33. 2D NMR structural co-relations of compound (2). .................................................... 162 Fig. 4. 34. 1H-1H NOESY spectrum of compound (2). .............................................................. 163 Fig. 4. 35. A) Crystals of compound (2) under microscope, B) ORTEP style image of compound (2). ............................................................................................................................................... 164 Fig. 4. 36. Growth inhibition caused by (2) in HeLa cells (A) and U251 cells (B) .................... 165 Fig. 4. 37. Apoptosis induced by 50 µM and 100 µM of compound (2) in U251 cells (A), and % apoptotic cells at 24 h (B) and 48 h (C) time intervals. ............................................................... 166 Fig. 4. 38. A) Cell cycle analysis of compound (2) at 100 µM for 48 h, B) % population of cells in G1, S and G2/M phase of cell cycle. ....................................................................................... 167 Fig. 4. 39. HPLC profile of fraction 9 of SFC. ............................................................................ 168 Fig. 4. 40. HPLC DAD spectrum of peaks at 16 min (A) and 18 min (B). ................................. 168 Fig. 4. 41. ESI-MS scan mode (A) and SIM (B) of peak at 16 min. ........................................... 169 Fig. 4. 42. HPLC DAD spectrum of compound (3) at 230, 240 and 260 nm.............................. 170 Fig. 4. 43. High resolution HRESI-MS (A) and low resolution ESI-MS (B) of compound (3) in scan mode, and SIM mode (C). ................................................................................................... 171 Fig. 4. 44. FTIR spectrum of compound (3)................................................................................ 172 Fig. 4. 45. Crystals of compound (3) from vapor diffusion method (A) and slow evaporation (B). ..................................................................................................................................................... 172 Fig. 4. 46. HPLC spectrum of phase separated hexane layer. ..................................................... 173 Fig. 4. 47. TLC quadrant test (A) and post SFC collected fractions (B). .................................... 173 Fig. 4. 48. HPLC profile of chloroform layer.............................................................................. 173 Fig. 4. 49. Sephadex LH-20 SEC profile of chloroform layer .................................................... 174 Fig. 4. 50. HPLC DAD spectrum of pooled fractions F14-16 and F4a-c.................................... 175 Fig. 4. 51. UV DAD spectrum of peak at 11.5 min. .................................................................... 175 xiv Fig. 4. 52. ESI-MS spectrum of peak at 11.3 min in scan mode (A) and SIM mode (B). .......... 175 Fig. 4. 53. UV DAD spectrum of peak at 14.4 min. .................................................................... 176 Fig. 4. 54. ESI-MS ion chromatogram of peak at 14.4 min. ....................................................... 176 Fig. 4. 55. HPLC spectrum of bioactive fractions 17-20 of Sephadex LH-20 column. .............. 177 Fig. 4. 56. HPLC DAD spectrum of peak at 4.9 min at 230 and 210 nm. .................................. 177 Fig. 4. 57. ESI-MS spectrum of peak at 4.9 min in scan mode (A) and SIM mode (B). ............ 178 Fig. 4. 58. HPLC DAD spectrum of peak at 6.4 min. ................................................................. 178 Fig. 4. 59. ESI-MS spectrum of peak at 6.4 min in scan mode (A) and SIM mode (B). ............ 179 Fig. 4. 60. A) Methodology for direct extraction of small molecules from E. tinctorium, B) Hexane and DEE extracts at 0.6 mg/mL for 48 h were assessed for growth-inhibitory activity on HeLa cells.. .................................................................................................................................. 179 Fig. 4. 61. HPLC DAD profile of hexane layer from direct extraction method at 210 and 230 nm. ..................................................................................................................................................... 180 Fig. 4. 62. Semi preparative HPLC DAD profile of hexane layer. ............................................. 181 Fig. 4. 63. HPLC DAD profile of PK1. ....................................................................................... 181 Fig. 4. 64. MSD spectrum of PK1 in scan (100-1000 m/z) and SIM mode. ............................... 182 Fig. 4. 65. HPLC DAD spectrum of PK2. ................................................................................... 182 Fig. 4. 66. HPLC DAD spectrum of PK2 on Phenomenex Luna C18(2) column....................... 183 Fig. 4. 67. HPLC DAD spectrum of PK2 repurification on Agilent Poroshell C18 column. ..... 183 Fig. 4. 68. HPLC profile of freshly collected PK2b for purity check at MSD1 scan mode, MSD2 SIM mode and UV at 210 nm...................................................................................................... 184 Fig. 4. 69. MSD spectrum of PK2 in scan mode and SIM mode. ............................................... 184 Fig. 4. 70. HPLC DAD spectrum of PK3. ................................................................................... 185 Fig. 4. 71. ESI-MS spectrum of PK3 in scan mode (A) and SIM mode (B). .............................. 185 Fig. 4. 72. UV DAD profile of compound (4) at 210 and 230 nm. ............................................. 186 Fig. 4. 73. MS spectrum of compound (4) in scan mode 100-1000 m/z (A) and SIM mode (B). ..................................................................................................................................................... 187 Fig. 4. 74. FTIR spectrum of compound (4)................................................................................ 187 Fig. 4. 75. 1H NMR of compound (4). ........................................................................................ 188 Fig. 4. 76. 13C NMR of compound (4). ...................................................................................... 189 Fig. 4. 77. 13C-1H HSQC spectrum of compound (4). ................................................................. 190 Fig. 4. 78. 13C-1H HMBC spectrum of compound (4)................................................................. 191 Fig. 4. 79. 2D COSY and HMBC structure correlations of compound (4). ................................ 191 Fig. 4. 80. 1H-1H COSY spectrum of compound (4). .................................................................. 192 Fig. 4. 81. 1H-1H NOESY spectrum of compound (4). ............................................................... 192 Fig. 4. 82. Crystallization of compound (4) using method 1 (A), method 2 (B), and method 3 (C). ..................................................................................................................................................... 193 Fig. 4. 83. ORTEP style image of compound (4). ....................................................................... 194 Fig. 4. 84. Effect of compound (4) at 48 h treatment on growth inhibition of HeLa cervical cancer cells .................................................................................................................................. 195 Fig. 4. 85. Apoptosis induced by compound (4) at 4.6, 10 and 40 µM in U251 cells (A), % apoptotic cells at 24 h (B) and 48 h (C)....................................................................................... 196 Fig. 4. 86. Cell cycle analysis of compound (4) at 40 µM for 24 h (A) and 48 h (B). ................ 197 Fig. 4. 87. Repurification of PK5 on Phenomenex Luna C18(2). ............................................... 197 Fig. 4. 88. Repurification of PK5 on Agilent Zorbax Poroshell C18. ......................................... 198 Fig. 4. 89. HPLC DAD spectrum of compound (5). ................................................................... 198 xv Fig. 4. 90. Positive mode ESI-MS of compound (5) in scan mode 100-1000 Da (A) and SIM mode (B). ..................................................................................................................................... 199 Fig. 4. 91. 1H NMR of compound (5).......................................................................................... 200 Fig. 4. 92. 13C NMR spectrum of compound (5). ........................................................................ 202 Fig. 4. 93. 1H-1H COSY spectrum of compound (5). .................................................................. 202 Fig. 4. 94. 1H-1H NOESY spectrum of compound (5). ............................................................... 203 Fig. 4. 95. 1H-13C HSQC spectrum of compound (5). ................................................................. 203 Fig. 4. 96. 1H-13C HMBC spectrum of compound (5)................................................................. 204 Fig. 4. 97. Crystals of (5) obtained from method 1 (A), method 2 (B) and method 3 (C), and ORTEP style image of (5) from X ray crystallography (D). ....................................................... 205 Fig. 4. 98. Growth inhibition caused by compound (5) after 48 h treatment in U251 cells.. ...... 206 Fig. 4. 99. HPLC DAD spectrum of PK5a. ................................................................................. 206 Fig. 4. 100. HPLC DAD spectrum of compound (6). ................................................................. 207 Fig. 4. 101. ESI-MS spectrum of compound (6) in scan mode (A) and SIM mode (B). ............ 207 Fig. 4. 102. HPLC analysis of DEE............................................................................................. 208 Chapter 5 Fig. 5. 1. Schematic representation of biosynthesis of polysaccharides ..................................... 249 Fig. 5. 2. Biosynthesis of orcinol (1) ........................................................................................... 250 Fig. 5. 3. Biosynthesis of orcinol from decarboxylation of orsellenic acid................................. 250 Fig. 5. 4. Mevalonic acid pathway for biosynthesis of triterpenes .............................................. 252 Fig. 5. 5. Collie’s synthesis scheme for orcinol (1) ..................................................................... 252 Fig. 5. 6. Chemical synthesis of diphenylmethane derivative compound (2) ............................. 253 Fig. 5. 7. Structure comparison of A-007, Tamoxifen and compound (2) .................................. 258 Fig. 5. 8. Structures of anti-histamine drugs with diphenylmethane backbone .......................... 259 Fig. 5. 9. Molecular targets of bioactive molecules for apoptosis in glioblastoma ..................... 261 Supplementary Fig. S 1. Optimization of conditions for anion exchange chromatography using multiple buffers. ....................................................................................................................................................... 80 Fig. S 2. Purification of the immuno-stimulatory polysaccharide from E. tinctorium (EtISPFa) using Sephacryl S-500 size exclusion chromatography ................................................................ 81 Fig. S 3. Estimating the peak maxima molecular weight (Mp) of EtISPFa using HPLC BioSEC5. (A) Overlay spectra of dextran standards (25-2000 kDa) with EtISPFa. (B) Retention times (min) of dextran standards were plotted against their molecular weight (kDa). ........................... 82 Fig. S 4. The GC-MS chromatograms from glycosyl composition analysis using TMS derivatization. ................................................................................................................................ 83 Fig. S 5. GC-MS chromatogram.................................................................................................... 83 Fig. S 6. Growth-inhibitory assay for optimization of Sephadex DEAE with buffers at different pH ................................................................................................................................................ 122 Fig. S 7. Estimating the peak maxima molecular weight (Mp) of EtGIPL1a using HPLC BioSEC-3... .................................................................................................................................. 123 Fig. S 8. The GC-MS chromatograms from glycosyl composition analysis using TMS derivatization. .............................................................................................................................. 124 Fig. S 9. GC-MS chromatogram resulting from glycosyl linkage analysis of neutral and uronic acid residues ................................................................................................................................ 124 xvi LIST OF ABBREVIATIONS COSY Correlation Spectroscopy CD3CN Acetonitrile-d DAD Diode Array Detector DEAE Diethylaminoethyl DEPT Distortionless Enhancement by Polarization Transfer DMEM Dulbecco’s Modified Eagle Medium DPBS Dulbecco’s Phosphate Buffered Saline EA Ethyl acetate ELISA Enzyme-linked Immunosorbent Assay EMEM Eagle Minimum Essential Medium ESI-MS Electro Spray Ionization-Mass Spectrometry FBS Fetal Bovine Serum FIA Flow Injection Analysis FTIR Fourier Transform InfraRed Spectroscopy GB Glioblastoma HPLC High Performance Liquid Chromatography HMBC Heteronuclear Multiple Bond Correlation HRESI-MS High Resolution Electro Spray Ionization-Mass Spectrometry HSQC Heteronuclear Single Quantum Coherence MTT 3-(4,5-Dimethylthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide Mp Peak maxima molecular weight Mn Number average molecular weight Mw Weight average molecular weight NOESY Nuclear Overhauser Effect Spectroscopy TNF- α Tumor Necrosis Factor-alpha SEC Size Exclusion Chromatography VWD Variable Wavelength Detector 1 H NMR Proton Nuclear Magnetic Resonance 13 Carbon Nuclear Magnetic Resonance C NMR xvii ACKNOWLEDGEMENT I would like to express my gratitude to my supervisor Dr. Chow Lee for giving me the opportunity to pursue PhD research. I am extremely thankful for his guidance, mentorship, and continuous support. His thoughtful recommendations and timelines helped me achieve my research goals in a timely fashion. I am also extremely grateful to all my committee members, Dr. Kerry Reimer, Dr. Hugues Massicotte, and Dr. Keith Egger, for providing insightful comments and suggestions that helped me shape my dissertation. I greatly acknowledge the assistance of Dr. Reimer in guiding me on structure elucidation and other chemistry related aspects. I very much appreciate the critical discussions with Dr. Wai Ming Li on experiments that helped me grow and think in multiple dimensions. It was a pleasure working and learning chemistry techniques from Dr. Kaila Fadock. I also want to thank the NALS members (Charles Bradshaw and Dr. Hossein Kazemian) for providing all the critical support that was much needed to perform important analysis for my dissertation. I also had the great pleasure of working with former and current Lee lab members including Sumreen Javed, Almas Yaqoob, Victor Liu, Hooi Xian, Noburu Kato, and others. Much thanks to my best PhD cohort, Daman Kandola, Lisa Kyle, and Steinunn Jonathan who have always provided unparalleled support. The endless moral support from my spouse Muhammad Ali Khan and my children Meerab Ali Khan and FariahAli Khan has always been behind all my accomplishments. A very special thanks to my parents Aurangzeb Khan and Tabassum Fayyaz for always believing in me and motivating me to achieve more. My appreciation also goes to my brother Jahanzeb Khan and sisters Aqsa Khan and Romaisa Khan for their encouragement. xviii Chapter 1: Introduction and Literature Review 1.1 Medicinal mushrooms Natural products have been used as medicines for centuries; however, it is only in the last century that researchers have begun to diligently characterize their biological and chemical properties. Mushrooms are natural reservoirs of potent pharmaceuticals and continue to be an interface for drug discovery. Mushrooms belong mostly to phyla Ascomycota or Basidiomycota, and together they constitute the sub-kingdom Dikarya within the Kingdom Fungi. Mushrooms are defined as “epigeous and hypogeous fruiting bodies of macroscopic fungi” (Chang, 2013). Fungi are defined as “achlorophyllous, heterotrophic (saprophytic, parasitic, or symbiotic), eukaryotic and spore-bearing organisms surrounded by a well-defined cell wall made up of chitin, with or without fungal cellulose along with many other complex organic molecules” (Sharma, 1989). According to recent estimates, fungi constitute 2.2 – 3.8 million species worldwide (Hawksworth & Lucking, 2017). These fungal estimates include all the different types of fungi including mushrooms. Earlier estimates indicated the total number of mushrooms to be 140,000-160,000 species, where only 10% have been explored (Hawksworth, 2001; Wasser, 2014). Mushrooms have a long history of medicinal use in various cultures across the globe. They have earned medicinal status long ago in China and other parts of Asia including Japan and Korea. The scope of medicinal mushrooms has expanded to other countries such as the USA and eastern European countries such as Russia (Chang, 1999; Reshetnikov & Tan, 2001; Van Griensven, 2009; Wasser & Weis, 1999; Wasser, 2010). Initially, mushrooms became popular as a folklore remedy. For example, in the sixteenth century in Russia and Europe, Inonotus obliquus (chaga) became famous as a folklore medicine for cancer treatment (Zheng et al., 2010). Later 1 on, scientists became interested in finding the evidence behind the diverse bioactive potential that is contained in mushrooms. This has led to the exploration of untapped mushroom resources for their medicinal benefit and the bioactive compounds that impart these properties. Unlike in Asia and parts of Eastern Europe, there are relatively few studies on the exploration of mushrooms native to North America for medicinal properties. The purpose of this review is to provide up-to-date information on the bioprospecting efforts on mushrooms native to North America. With literature searches and the information gained, the aim is to answer the following questions: (i) Do similar species found in North America compared to the ones found in Asia or Europe exhibit similar bioactivities and bioactive compounds? (ii) Do similar species found in North America and Asia or Europe exhibit distinct bioactivity and produce distinct compounds? (iii) Do new species found in North America produce new compound(s)? and (iv) Based on the answers to (i) to (iii) above, the efforts to explore mushrooms native to North America for medicinal properties warrants new medicinal compounds? 1.2 Mushrooms native to North America North America, encompassing the northern subcontinent of the Americas that includes Canada, the United States, Mexico, and Greenland, has one of the world’s largest and most diverse ecological systems. It is also home to diverse mushrooms that are relatively unexplored for their therapeutic benefit. Although new species continue to be discovered in North America, about 22-55% of the mushroom species remain unexplored (Hawksworth, 2001). Like elsewhere, mushrooms were recognized as an important source for medicine by people who lived in North America centuries ago. The indigenous people of North America had used Calvatia mushrooms (more commonly known as puffballs) to heal wounds (Burk, 1983). The therapeutic value of 2 Fomitopsis officinalis was also discovered by First Nations peoples of North America, including those in British Columbia (BC) where F. officinalis sporophores were carved as shaman grave guardians (Blanchette et al., 1992). In recent years, there has been increasing reports on mushrooms native to North America possessing medicinal properties. Whether it be Hericium sp., found growing on hardwood and coniferous trees that contains a number of small molecules with antibacterial properties (Song et al., 2020) or Echinodontium tinctorium, native to British Columbia and commonly found as a woody conk on hemlocks, with immuno-stimulatory and anti-inflammatory compounds in its fruiting bodies (Javed et al., 2019; Zeb et al., 2021). Others include Cortinarius armillatus which grows in moist coniferous forests and contains orellanine, a potential toxin against renal carcinoma (Shao et al., 2016; Buvall et al., 2017). Table 1.1 summarizes all the mushrooms found in North America that have been reported to possess medicinal properties and their origin of collection and/or discovery. 1.3 Medicinal properties of mushrooms native to North America Mushrooms are known to possess medicinal properties that provide benefits against a large number of diseases. Some of the important medicinal benefits reported include antimicrobial, antioxidant, anticancer, immune system enhancer, antiviral, anti-hyperlipidemia, radical scavenger, anti-parasitic and anti-inflammatory activity (Wasser, 2017). Amongst these, the most common medicinal properties reported from mushrooms native to North America are anticancer, immuno-stimulatory, anti-inflammatory, antimicrobial and antioxidant as shown in Table 1.1 (Song et al., 2020; Javed et al., 2019; Zeb et al., 2021; Shao et al., 2016, Buvall et al., 2017; Wasser, 2017; Shideler et al., 2017; Stanikunaite et al., 2009; Yaqoob et al., 2020; Liu et al., 2010; Smith et al., 2017; Barad et al., 2018; Pacheco-Sanchez et al., 2006 & 2007). 3 1.3.1 Anti-bacterial and anti-viral activities Antimicrobial resistance is a major healthcare problem worldwide. A recent landmark report indicated that bacterial infections resistant to treatment are likely to grow from 26% in 2018 to 40% by 2050, and such increases are expected to cost thousands of lives, billions of dollars in hospital expenses and gross domestic product, and have a negative social impact on people worldwide (Council of Canadian Academies, 2019). Therefore, it is recommended that efforts to discover new antimicrobial drugs to combat specific antibiotic-resistant pathogens should be strengthened and should include innovative strategies (Council of Canadian Academies, 2019; Strachan & Davie, 2017; Gould et al., 2019). To this end, researchers have focused on unexplored and unique environments and resources, including mushrooms, as avenues to discover novel antimicrobial metabolites. Grifolin, neogrifolin and confluentin isolated from Albatrellus flettii collected in California were found to have potent activity against gram-positive bacteria Bacillus cereus and Enterococcus faecalis (Liu et al., 2010). Another lanostane-type tripterpene isolated from Jahnoporus hirtus also inhibited the growth of Bacillus cereus and Enterococcus faecalis (Liu et al., 2010). Supernatants from culture of Lenzites betulina and Haploporus odorus (Shideler et al., 2017) as well as extracts from Pleurotus ostreatus and P. levis (Adebayo et al. 2018), also have antimicrobial activity. However, the responsible antimicrobial compounds have not been isolated from these mushrooms. In another study using extracts from 75 mushrooms collected in Oxford, Ohio, USA, it was found that a total of 25 species had antibacterial activity against at least one of the bacterial strains assessed (Hassan et al., 2019). From this study, extracts from Ganoderma lucidum and Laetiporus sulphureus were found to have the strongest antibacterial activity (Hassan et al., 2019). 4 Table 1.1. Mushrooms native to North America studied for bioactivities Mushroom Origin Bioactivity Albatrellus flettii Amanita augusta Amanita muscaria Astraeus pteridis Barssia oregonensis Boletus curtisii Smithers, BC Anti-proliferative (Yaqoob et al., 2020), antimicrobial (Liu et al., 2010) Anti-proliferative, immuno-stimulatory, anti-inflammatory (Deo et al., 2019) Anti-proliferative, immuno-stimulatory (Smith et al., 2017) Antituberculosis (Stanikunaite et al., 2007) Antituberculosis (Stanikunaite et al., 2007) ND Cantharellus cibarius Chroogomphus tomentosus Clavulina cinerea Collybia dryophila Coprinellus sp. Coprinus comatus Cortinarius armillatus Echinodontium tinctorium Elaphomyces granulatus Elaphomyces muricatus Flammulina velutipes Fomes fomentarius Haida Gwaii, BC Prince George, BC Linn County, Oregon Clackamas, Oregon Chapel Hill, NC Haida Gwaii, BC Haida Gwaii, BC Haida Gwaii, BC Quebec Seattle, WA, USA Seattle, WA, USA Bioactive component1 Small molecules Extracts Extracts Extracts Extracts ND Anti-proliferative, immuno-stimulatory, anti-inflammatory (Deo et al., 2019) Anti-proliferative, immuno-stimulatory, anti-inflammatory (Deo et al., 2019) Anti-proliferative (Deo et al., 2019) Extracts Anti-inflammatory (Pacheco-Sanchez et al., 2006 & 2007) Anti-proliferative (Wang et al., 2007) Polysaccharide Anti-proliferative (Wang et al., 2007) Extracts (Gu et al., 2006) Protein (Zhang et al., 2017) Small molecule Extracts Extracts Extracts Massachusetts Anti-proliferative, anticancer (Buvall et al., 2017; Shao et al., 2016) Smithers and Anti-inflammatory Javed et al., 2019), Polysaccharides Terrace, BC immuno-stimulatory (Zeb et al., 2021) Oregon and Anti-inflammatory (Stanikunaite et al., Small Bonner 2009 & 2007), antioxidant molecules and County, Idaho (Stanikunaite et al., 2007) extracts Benton Anti-inflammatory, antioxidant, Extracts County, antituberculosis (Stanikunaite et al., Oregon 2007) Seattle, WA, Anti-proliferative (Gu et al., 2006) Extracts USA Prince Anti-proliferative, immuno-stimulatory, Extracts George, BC anti-inflammatory (Smith et al., 2017) 5 Ganoderma applanatum Ganoderma lucidum Ganoderma tsugae Gautieria monticola Geopora clausa Guepina helvelloides Gyromitra esculenta Haploporus odorus Hericium corralloides Hericium sp. Hydnellum sp. Hydnum repandum Hygrophoropsis aurantiaca Hymenogaster subalpinus Hymenopellis furfuracea Hypholoma fasciculare Inocybe sp. Inonotus obliquus Jahnoporus hirtus Laetiporus conifericola Laetiporus sulphureus Terrace, BC and Oxford, Ohio Oxford, Ohio Anti-proliferative, immuno-stimulatory, anti-inflammatory (Smith et al., 2017), antimicrobial (Hassan et al., 2019) Antimicrobial (Hassan et al., 2019) Extracts Haida Gwaii, Anti-proliferative, immuno-stimulatory, BC anti-inflammatory (Deo et al., 2019) Benton Antioxidant (Stanikunaite et al., 2007) County, Oregon Inyo Country, Antioxidant, anti-proliferative California (Stanikunaite et al., 2007) Haida Gwaii, Anti-proliferative, immuno-stimulatory, BC anti-inflammatory (Deo et al., 2019) Prince Anti-proliferative, immuno-stimulatory George, BC (Smith et al., 2017) Calgary, Antimicrobial (Shideler et al., 2017) Canada Prince Immuno-stimulatory, antiGeorge, BC inflammatory (Smith et al., 2017) Minnesota Antimicrobial (Song et al., 2020) Prince Anti-proliferative, immuno-stimulatory George, BC (Smith et al., 2017) Haida Gwaii, Anti-proliferative, anti-inflammatory BC (Deo et al., 2019) Haida Gwaii, Anti-proliferative, anti-inflammatory BC (Deo et al., 2019) Benton Anti-inflammatory, antituberculosis County, (Stanikunaite et al., 2007) Oregon Oxford, Ohio Antimicrobial (Hassan et al., 2019) Extracts Extracts Extracts Extracts Extracts Extracts Extracts Extracts Small molecule Extracts Extracts Extracts Extracts Extracts Haida Gwaii, BC Haida Gwaii, BC Manitoba & Prince George, BC USA Anti-proliferative, anti-inflammatory (Deo et al., 2019) Anti-proliferative, immuno-stimulatory, anti-inflammatory (Deo et al., 2019) Anti-inflammatory (Javed et al., 2019; Van et al., 2009) Extracts Antimicrobial (Liu et al., 2010) Small molecule Haida Gwaii, BC Oxford, Ohio Anti-proliferative, immuno-stimulatory, anti-inflammatory (Deo et al., 2019) Antimicrobial (Hassan et al., 2019) Extracts 6 Extracts Extracts Extracts Lentinellus subaustralis Lentinus edodes Oxford, Ohio Antimicrobial (Hassan et al., 2019) Extracts Quebec Polysaccharide Leucogaster rubescens Pend Oreille County, Oregon Prince George, BC Quebec Anti-inflammatory (Pacheco-Sanchez et al., 2006) Antioxidant (Stanikunaite et al., 2007) Anti-proliferative, immuno-stimulatory, anti-inflammatory (Smith et al., 2017) Anti-inflammatory (Pacheco-Sanchez et al., 2006) Antituberculosis, anti-inflammatory, antioxidant (Stanikunaite et al., 2007) Anti-proliferative (Barad et al., 2018) Extracts Leucocybe connata Marasmius oreades Melanogaster tuberiformis Paxillus involutus Phellinopsis conchata Phellinus conchatus Lane County, Oregon Prince George, BC Oxford, Ohio Extracts Polysaccharide Extracts Polysaccharide Antimicrobial (Hassan et al., 2019) Extracts Oxford, Ohio Antimicrobial (Hassan et al., 2019) Extracts Phellinus conchatus Terrace, BC Extracts Phellinus nigricans Phellodon atratus Pholiota terrestris Piptoporus betulinus Pleurotus djamor Pleurotus levis Terrace, BC Anti-proliferative, anti-inflammatory (Smith et al., 2017) Anti-proliferative, immuno-stimulatory, anti-inflammatory (Smith et al., 2017) Anti-proliferative, immuno-stimulatory, anti-inflammatory (Deo et al., 2019) Antimicrobial (Hassan et al., 2019) Anti-proliferative, immuno-stimulatory, anti-inflammatory (Smith et al., 2017) Anthelmintic activity (Pineda-Alegria et al., 2017) Mexico Antimicrobial, antioxidant (Adebayo et al., 2018) USA & Haida Anti-proliferative, immuno-stimulatory, Gwaii, BC anti-inflammatory (Deo et al., 2019), antioxidant, antimicrobial (Adebayo et al., 2018) Olympia, WA Anti-proliferative (Zhang et al., 2011), antimicrobial (Adebayo et al., 2018) Oxford, Ohio Antimicrobial (Hassan et al., 2019) Extracts Pleurotus ostreatus Pleurotus tuberregium Polyporus badius Polyporus squamosus Pyrofomes demidoffi Haida Gwaii, BC Oxford, Ohio Prince George, BC Mexico Extracts Extracts Extracts Small molecules Extracts Extracts Polysaccharide and extracts Extracts Oxford, Ohio Antimicrobial (Hassan et al., 2019) Extracts Oxford, Ohio Antimicrobial (Hassan et al., 2019) Extracts 7 Ramaria cystidiophora Haida Gwaii, BC Rhizopogon couchii Lebanon State Forest, New Jersey Lebanon State Forest, New Jersey Pend Oreille County, Oregon Lewis County, Washington Lebanon State Forest, New Jersey Jackson County, Oregon Prince George, BC Haida Gwaii, BC Lebanon State Forest, New Jersey Oxford, Ohio Rhizopogon nigrescens Rhizopogon pedicellus Rhizopogon subareolatus Rhizopogon subaustralis Rhizopogon subgelatinosus Royoporus badius Russula paludosa Scleroderma laeve Stereum hirsutum Trametes versicolor Trichaptum abietinum Tricholomopsis rutilans Tyromyces chioneus Oxford, Ohio Anti-proliferative, anti-inflammatory (Deo et al., 2019), antimicrobial (Centko et al., 2012) Anti-inflammatory, antioxidant, antituberculosis (Stanikunaite et al., 2007) Anti-inflammatory, antioxidant (Stanikunaite et al., 2007) Small molecules and extracts Extracts Antioxidant, antituberculosis (Stanikunaite et al., 2007) Extracts Antimalarial (Stanikunaite et al., 2007) Extracts Anti-inflammatory, antioxidant (Stanikunaite et al., 2007) Extracts Anti-inflammatory, anti-proliferative (Stanikunaite et al., 2007) Extracts Immuno-stimulatory (Lee, 2020) Polysaccharideprotein Extracts Anti-proliferative, anti-inflammatory (Deo et al., 2019) Anti-inflammatory, antioxidant, antituberculosis (Stanikunaite et al., 2007) Antimicrobial (Hassan et al., 2019) Antimicrobial (Hassan et al., 2019) Extracts Extracts Extracts Extracts Prince Anti-proliferative, immuno-stimulatory, Extracts George, BC anti-inflammatory (Smith et al., 2017) Haida Gwaii, Anti-proliferative, anti-inflammatory Extracts BC (Deo et al., 2019) Haida Gwaii, Anti-proliferative, immuno-stimulatory, Extracts BC anti-inflammatory (Deo et al., 2019) 1 Refer to Table 1.2 for more information of bioactive small molecules and polysaccharides. Extracts of polypore mushrooms native to North America have been shown to have antiviral activities against viruses that attack honey bees (Stamets et al., 2018). Mycelium extracts from Fomes fomentarius collected in Ithaca, New York and Ganoderma resinaceum culture from 8 Ontario, Canada were found to reduce the levels of honey bee deformed wing virus and Lake Sinai virus in vivo in both laboratory and field studies (Stamets et al., 2018). 1.3.2 Anti-proliferative activity Mushrooms native to North America have been explored for anti-proliferative activity against cancer cell lines. For instance, out of 29 species of mushrooms examined from north-central British Columbia (BC) (Smith et al., 2017) and Haida Gwaii, BC (Deo et al., 2019), 27 species exhibited anti-proliferative activity; 16 out of the 27 species (59%) had their anti-proliferative activity reported for the first time (Deo et al., 2019; Smith et al., 2017). These species include Amanita augusta, Cantharellus cibarius, Chroogomphus tomentosus, Guepinia helvelloides, Gyromitra esculenta, Hydnellum sp., Inocybe sp., Laetiporus conifericola, Leucocybe connata, Phellodon atratus, Pleurotus ostreatus, Ramaria cystidiophora, Russula paludosa, Trichaptum abietinum, Tricholomopsis rutilans, and Tyromyces chioneus. Inspired by such findings, we have further explored BC wild mushrooms for bioactivities. Out of 49 species collected from northern interior BC, we found that 21 species (43%) exhibited potent anti-proliferative activity that has not been previously reported (Lee, 2020, pers. comm.). Another study was conducted on 38 species of mushrooms collected from greater Seattle area, Washington State, USA, and out of these, aqueous extracts from 3 species were identified as anti-proliferative in human estrogen receptor negative (MDA-MB-231, BT-20) and estrogen receptor positive (MCF-7) breast cancer cells. These included Coprinus comatus, Coprinellus sp., and Flammulina velutipes (Gu & Leonard, 2006). Elsewhere, the ethanol and water extracts of Pleurotus tuber-regium from Washington State displayed anti-proliferative effects in HCT-116 colon and HeLa cervical cancer cell lines (Maness et al., 2011). Although in most cases the identity of anti-proliferative compounds from the mushroom species described above remains unknown, there are detailed 9 structural elucidation and mechanistic studies of both bioactive small molecules and polysaccharides isolated from selected species. For example, a growth-inhibitory polysaccharide GIPinv that caused growth inhibition in several cancer cell lines and induced apoptosis in HeLa cancer cells was isolated from Paxillus involutus (Barad et al., 2018). Small molecules grifolin, neogrifolin and confluentin were found to be the major growth-inhibitory compounds in the ethanol extracts of Albatrellus flettii (Yaqoob et al., 2020). It was also discovered that confluentin can inhibit the RNA-binding function of the oncogenic protein insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) (Yaqoob et al., 2020). Elsewhere, an exopolysaccharide isolated from P. tuber-regium inhibited the growth of chronic myelogenous leukemia K562 cells (Zhang & Cheung, 2011). Orellanine, a small molecule originally isolated from a European mushroom species Cortinarius orellanus, has recently been isolated from a North American mushroom species C. armillatus, with cytotoxic potential against renal carcinoma in a dose-dependent manner (Buvall et al., 2017). 1.3.3 Anti-inflammatory activity Mushrooms native to North America have also been explored for anti-inflammatory activity. Out of 29 species examined from north-central BC (Smith et al., 2017) and Haida Gwaii (Deo et al., 2019), 26 (69%) had their anti-inflammatory activity reported for the first time (Deo et al., 2019; Smith et al., 2017). These species include Amanita augusta, Chroogomphus tomentosus, Clavulina cinerea, Guepinia helvelloides, Gyromitra esculenta, Hydnum repandum, Hygrophoropsis aurantiaca, Hypholoma fasciculare, Inocybe sp., Laetiporus conifericola, Leucocybe connata, Phellinus nigricans, Phellodon atratus, Pleurotus ostreatus, Ramaria cystidiophora, Russula paludosa, Trichaptum abietinum, Tricholomopsis rutilans, and Tyromyces chioneus. We have further explored BC wild mushrooms for anti-inflammatory 10 activity. Out of 49 species collected from northern interior BC, we found 34 species (69%) that exhibited potent anti-inflammatory activity not been previously reported (Lee, 2020, pers. comm.). Extracts from Inonotus obliquus collected in BC, like those found in other parts of the world, showed strong anti-inflammatory activity in vitro (Javed et al., 2019; Smith et al., 2017). In addition, Javed et al. (2019) demonstrated for the first time the ability of methanol extracts of I. obliquus to attenuate histamine-induced inflammation in the arterioles of gluteus muscle of mice. A limited number of anti-inflammatory compounds have been isolated from mushrooms native to North America. A polysaccharide called CDP from Collybia dryophila, and CDP-like polysaccharides from Lentinula edodes and Marasmius oreades, can inhibit nitric oxide production in Raw264.7 macrophage cells (Pacheco-Sanchez et al., 2007). Javed et al. (2019) demonstrated that a 5 kDa polysaccharide (AIPetinc) isolated from the NaOH extract of E. tinctorium showed anti-inflammatory activity in vitro as well as in the histamine-induced inflammatory mouse microcirculation model. Elsewhere, the ethanolic extract as well as two small molecules, syringaldehyde and syringic acid, isolated from Elaphomyces granulatus inhibited COX-2 enzyme in Raw264.7 cells; the extract caused 68% inhibition of COX-2 at 50 µg/mL, whereas syringaldehyde and syringic acid were effective with IC50 of 3.5 µg/mL and 0.4 µg/mL respectively (Shao et al., 2016). 1.3.4 Immuno-stimulatory activity Mushroom species are known to have immunotherapeutic properties and over 270 species had been recognized (Ooi & Liu, 2000). Mushrooms are natural immune-modulators that can enhance the host immune system by activating dendritic cells, T cells, NK cells, macrophages, and cytokines. A desired immune status requires an equilibrium in T helper type 1 (Th1) cellular immune response and T helper type 2 (Th2) humoral immune response. Th1 cells are 11 components of cell-mediated immune responses and they produce cytokines interferon-γ (IFNγ), Tumor necrosis factor (TNF)-α, interleukin-12 (IL-12) and interleukin-2 (IL-2) whereas Th2 cells are involved in humoral immune responses and express IL-5, 6, 9, 10, 13, GM-CSF and macrophage-derived chemokines (Mosmann & Sad, 1996; Romagnani, 2000). Th1 immune response is required for cancer treatment. Th2 immune response is usually not associated with cancer. In the context of such immune response patterns, mushrooms which have the ability to stimulate Th1 responses by increasing IFN-γ and IL-2 production are known to possess immunomodulatory activity. Mushroom native to North America have also been explored for immuno-stimulatory activity. Out of 29 species of mushrooms examined from north-central British Columbia (BC) (Smith et al., 2017) and Haida Gwaii, BC (Deo et al., 2019), 20 exhibited immuno-stimulatory activity. Fifteen out of the 20 species (75%) had their immuno-stimulatory activity reported for the first time (Deo et al., 2019; Smith et al., 2017). These species include Amanita augusta, Chroogomphus tomentosus, Clavulina cinerea, Fomes fomentarius, Guepinia helvelloides, Gyromitra esculenta, Hericium coralloides, Hydnum repandum, Hydnellum sp., Hygrophoropsis aurantica, Hypholoma fasciculare, Inocybe sp., Laetiporus conifericola, Leucocybe connata, Phellinus nigricans, Phellodon atratus, and Piptoporus betulinus. Furthermore, out of 49 additional species collected from northern interior BC, 25 (51%) exhibited potent immunostimulatory activity that has not been previously reported (Lee, 2020, pers. comm.). Table 1.2 lists several bioactive compounds characterized from diverse mushrooms from elsewhere with immuno-stimulatory potential. 12 Table 1. 2. Immuno-stimulatory compounds from mushrooms Mushroom name Common name Bioactive compounds Reference Agaricus blazei, Agaricus subrufescens Almond mushroom Cogumelo do Sol (Brazil), Himematsutake (Japan) White button mushroom Stout camphor fungus Caterpillar fungus Polysaccharide (β-D-glucan) (Guggenheim et al., 2014; Kim et al., 2009; Wilbers et al., 2016) Polysaccharide (Zhang et al., 2014) Protein ACA (Sheu et al., 2009) Nucleotide (Adenosine, Cordycepin) Trametes versicolor (Coriolus versicolor) Turkey tail fungus Ganoderma lucidum Reishi, Lingzhi “King of herbs” "soul/spirit mushroom" Ganoderma microsporum - Polysaccharide-protein complexes (PSPC), Polysaccharide-peptide (PSP), Polysaccharide-K (PSK) Protein (Ganoderic acid, Danoderiol, Danderenic acid, Lucidenic acid). Polysaccharide (GLPS- G. lucidum polysaccharide) Protein (Guggenheim et al., 2014; Hsieh et al., 2013) (Guggenheim et al., 2014; Luo et al., 2014; Tzianabos, 2000) Inonotus obliquus Grifola frondosa Chaga Polysaccharide (Baek et al., 2012) Hen of the woods, Maitake Dictyophora indusiata Sparassis crispa Lentinus edodes Veiled Lady Mushroom - Grifolan Polysaccharide (β-Dglucan, D-fraction, MDfraction), Protein low molecular wt. β-D-glucan (Guggenheim et al., 2014; Kodama et al., 2010; Park et al., 2015) (Fu et al., 2015) β-(1,3)-glucan Shiitake mushroom Lentinan (β-(1,3), β-(1-6) D-glucan) Schizophyllum commune - Schizophyllan (β-(1-3), β-(1-6) D-glucan) (Harada & Ohno, 2008) (Abel et al., 1989; Chihara et al., 1987; Wasser & Weis, 1999) (Miyazaki et al., 1995) Tricholoma mongolicum - TML-1 and TML-2 Agaricus bisporus Antrodia camphorata Cordyceps sinensis 13 (Guggenheim et al., 2014; Lin et al., 2009; Zhou et al., 2015) (Lin et al., 2010) (Wang et al., 1996) Pleurotus eryngii King oyster mushroom Polysaccharide (Liu et al., 2015) To date, most of the immuno-stimulatory compounds from mushrooms are polysaccharides or polysaccharide-protein complexes (Giavasis, 2014; Enshasy & Hatti-Kaul, 2014). Immunopotentiator polysaccharides isolated from mushrooms are chemically categorized as βD-glucans or β-D-glucans conjugated with proteins. Researchers believe that immunostimulation mechanism involves the binding of β-D-glucans to membrane receptors on the surface of cells, as they cannot enter the cells due to their large size. β-D-glucans have affinity to bind with different receptors which include β-D-glucan inhibitable receptor (Czop and Austen, 1985), Dectin-1 receptor (Adachi et al. 2004; Brown et al. 2003), Lactosylceramide (LacCer), Toll-like receptor (TLR) 2, and Complement receptor type 3 (CR3) (Ross et al. 1999; Xia et al. 1999). CR3 is also recognized as Mac-1, CD11b/CD18 or αMβ2-integrin. Based on this evidence, CR3 is the major β-glucan receptor potentiating the immunomodulatory responses of polysaccharides; it acts as an adhesion molecule and a receptor for factor I-cleaved C3b (iC3b) on macrophages. When CR3 binds to iC3b, CD11b I-domain binding site recognizes iC3b whereas the lectin site of CR3 recognizes glucans. This interaction results in phagocytosis and cytotoxic degranulation (Chen & Seviour, 2007; Guggenheim et al., 2014). Due to problems such as: (i) high variability of polysaccharides, that is further exacerbated during extraction and purification, (ii) impurities and contaminants, and (iii) difficulty in defining the structure and chemical fingerprint to understand structure-function relationships (Persin et al., 2011), there has been little interest amongst scientists in North America to study and isolate immuno-stimulatory polysaccharides from mushrooms native to North America. Despite such obstacles, we have isolated two immuno-stimulatory compounds from mushrooms 14 native to northern BC. A complex glucuronic acid-rich polysaccharide called EtISPFa was isolated from Echinodontium tinctorium (Zeb et al., 2021), and a polysaccharide-protein complex rich in galactose and mannose called ISPP-Rb was isolated from Royoporus badius (Lee, 2020, pers. comm.). Both EtISPFa and ISPP-Rb are capable of inducing the production of TNF-α and other pro-inflammatory cytokines and chemokines (Lee, 2020; Zeb et al., 2021). 1.3.5 Anti-oxidant activity There have been relatively less studies on anti-oxidant activity of mushrooms native to North America. One study reported ethanolic extracts from 11 species of mushrooms collected from Idaho, Oregon, and New Jersey, to have moderate to weak anti-oxidant activity (Stanikunaite et al., 2007). The ethanolic extract, as well as syringic acid isolated from the fruiting bodies of Elaphomyces granulatus, showed potent antioxidant effect on myelomonocytic HL-60 cells with an IC50 of 41 µg/mL for the extract and 0.7 µg/mL for syringic acid (Stanikunaite et al., 2009). Two edible mushrooms, Pleurotus ostreatus from USA and P. levis from Mexico, also showed antioxidant activity (Adebayo et al., 2018). 1.3.6 Anti-fungal activity Many mushrooms are known to possess antifungal activity (Alves et al., 2013). However, there has been only one study on anti-fungal activity of mushroom native to North America; it shows that a chlorinated orcinol derivative, 2-chloro-1,3-dimethoxy-5-methyl benzene isolated from Hericium sp. collected in Minnesota, USA, has inhibitory effect on Candida albicans and Candida neoformans, suggesting its role as an antifungal agent (Song et al., 2020). 1.3.7 Other bioactivities In addition to the bioactivities described above, mushrooms from North America have also been investigated for antiparasitic, antimalarial and antituberculosis activities. A study conducted 15 in Mexico showed antiparasitic effects of hydroalcoholic extracts from Pleurotus djamor against Haemonchus contortus eggs (Pineda-Alegria et al., 2017), suggesting its metabolites can act as anthelmintics. In the same study, a few small molecules with antiparasitic activity, pentadecanoic, hexadecanoic, octadecadienoic, octadecanoic acid, β-sitosterol, were also isolated (Pineda-Alegria et al., 2017). Stanikunaite and co-workers assessed 22 species of mushrooms native to North America and found Rhizopogon subareolatus to have antimalarial activity (Stanikunaite et al., 2007). They also found the following species to have antituberculosis activity: Astraeus pteridis, Barssia oregonensis, Elaphomyces granulatus, E. muricatus, Hymenogaster subalpinus, Melanogaster tuberiformis, Rhizopogon couchii, R. pedicellus, R. subareolatus, and Scleroderma laeve (Stanikunaite et al., 2007). 1.4 Bioactive compounds from mushrooms native to North America Since there is a relatively limited number of studies on mushrooms native to North America, it is not surprising to find relatively limited number of bioactive compounds isolated from the mushrooms. Here, we summarize all the bioactive compounds that have been isolated from mushrooms native to North America into two general groups; large molecules and small molecules (Table 1.3) (Fig. 1.1). 16 Fig. 1. 1. Classification of bioactive compounds isolated from mushrooms. 1.4.1 Large molecular weight compounds Large molecular weight compounds isolated from mushrooms are typically homo- and heteroglycans, proteins, polysaccharide-protein complexes and nucleic acids-protein complexes (Fig.1.1) (Ferreira et al., 2010). Table 1.3 summarizes the bioactive large molecules that have been isolated from North American wild mushrooms. A 229 kDa growth-inhibitory heteroglycan GIPinv was isolated from the 5% NaOH extract of Paxillus involutus (Barad et al., 2018). GIPinv is made up predominantly of glucose (65.9%), galactose (20.8%) and mannose (7.8%) with traces of fucose (3.2%) and xylose (2.3%) (Barad et al., 2018). It has mixed linkages in the backbone containing (1→6)-Gal, (1→4)-Glc, (1→6)-Glc, (1→3)-Glc, and (1→2)-Xyl, with branching points at (1→2,6)-Man and (1→3,6)-Man. Another growth-inhibitory polysaccharide, an exopolysaccharide called EPS, was isolated from P. tuber-regium (Maness et al., 2011). EPS is 3,180 kDa and consisted mainly of mannose (57.5%) and glucose (42.5%). Another study showed anti-proliferative effect of a 12 kDa 130-amino acid containing glycan-binding protein 17 (Y3). Y3 is a tertiary protein isolated from Coprinus comatus and exhibited selective antiproliferative effects in human T cell leukemia Jurkat cells (Zhang et al., 2017). A 1,234 kDa anti-inflammatory β-glucan polysaccharide CDP consisting of (1→3) and (1→4) glucosidic linkages was isolated from aqueous extract of the fruiting bodies of Collybia dryophila (Pacheco-Sanchez et al., 2006, 2007). Some other CDP-like polysaccharides with antiinflammatory potential, 610 kDa and 1,316 kDa in size, were isolated from the aqueous extracts of Lentinula edodes and Marasmius oreades respectively (Pacheco-Sanchez et al., 2006). In the same study, water-soluble CDP-like polysaccharides were also isolated from multiple mushrooms obtained from Quebec, Canada. These include Agaricus arvensis, Amanita muscaria, A. rubescens, Coprinus atramentarius, C. comatus, Hydnum imbricatum, Lycoperdon pyriforme, Lactarius deliciosus, Leccinum aurantiacum, L. subglabripes, Lepiota americana, Panellus serotinus, Piptoporus betulinus, Polyporus squamosus, Russula variata, Suillus americanus, Tricholoma flavovirens, T. vaccinum (Pacheco-Sanchez et al., 2006). Another relatively small 5 kDa β-glucan called AlPetinc with anti-inflammatory activity was isolated from the 5% NaOH extract of E. tinctorium (Javed et al., 2019); AlPetinc is a heteroglucan composed mainly of glucose (88.6%) with a small amount of mannose (4.4%), galactose (4.0%), xylose (2.3%), and fucose (0.7%). Two immuno-stimulatory polysaccharides have been isolated from mushrooms native to North America. EtISPFa with an estimated size of 1,294 kDa was isolated from the water extract of E. tinctorium (Zeb et al., 2021). It is composed of glucose (66.2%), glucuronic acid (10.1%), mannose (6.7%), galactose (6.4%), xylose (5.6%), rhamnose (3.1%), fucose (1.8%), and arabinose (0.2%). 2-D NMR analysis showed that EtISPFa has a backbone containing mostly of 3-substituted β-glucopyranose with some 4-substituted glucopyranosyl uronic acid (Zeb et al., 18 2021). ISPP-Rb has an estimated size of 1,053 kDa is a polysaccharide-protein complex isolated from Royoporus badius (Lim, 2018). Its polysaccharide component consisted of glucose (49.2%), galactose (11.3%), mannose (10.8%), rhamnose (9.6%), galacturonic acid (8.2%), xylose (5.2%), fucose (2.8%), N-acetyl glucosamine (1.8%), and arabinose (1.2%). The protein component of ISPP-Rb, which is indispensable for its immuno-stimulatory activity, is currently unknown (Lim, 2018). As mentioned briefly in earlier sections, there has been a lack of interest amongst scientists in the West, especially in North America, in studying bioactive polysaccharides for use as medicinal compounds (Persin et al., 2011). However, with recent advances in identifying biosynthetic gene clusters and transcriptomic studies including those in fungi, it is now possible to produce compounds including large polysaccharides using heterologous expression systems by genetic engineering (Almeida et al., 2019; Skinnider et al., 2015; Skellam, 2019; Zhang et al., 2017). Such efforts are expected to enable large scale production of pure bioactive polysaccharides, thereby overcoming some if not all of the problems previously encountered (Persin et al., 2011). Table 1. 3. Bioactive molecules from mushrooms native to North America Types Bioactive compound Mushrooms References Small molecule Syringaldehyde (1) E. granulatus Syringic acid (2) E. granulatus Grifolin (3) A. flettii Neogrifolin (4) A. flettii Confluentin (5) A. flettii 3,11-Dioxolanosta-8,24(Z)-diene26-oic acid1 (6) Erinacerin V1 (7) J. hirtus (Stanikunaite et al., 2009) (Stanikunaite et al., 2009) (Liu et al., 2010; Yaqoob et al., 2020) (Liu et al., 2010; Yaqoob et al., 2020) (Liu et al., 2010; Yaqoob et al., 2020) (Liu et al., 2010) Hericium sp. (Song et al., 2020) 19 4-Hydroxy-2,2-dimethyl chromane6-carbaldehyde1 (8) 4-Chloro-3,5dimethoxybenzaldehyde (9) 2-Chloro-1,3-dimethoxy-5-methyl benzene (10) 4-Chloro-3,5dimethoxyphenylmethanol (11) 3,6-Bis(hydroxyl methyl)-2-methyl4H-pyran-4-one (12) 4-Chloro-3,5-dimethoxybenzoic acid (13) 5-Hydroxy-6-(1-hydroxyethyl) isobenzofuran-1(3H)-one (14) Erinacine (15) Pentadecanoic acid (16), Hexadecanoic acid (17), Octadecadienoic acid (18), Octadecanoic acid (19), β-sitosterol (20) Orellanine (3,3′,4,4′-tetrahydroxy2,2′-bipyridine-1,1′-dioxide) (21) Ramariolide A1 (22) Hericium sp. (Song et al., 2020) Hericium sp. (Song et al., 2020) Hericium sp. (Song et al., 2020) Hericium sp. (Song et al., 2020) Hericium sp. (Song et al., 2020) Hericium sp. (Song et al., 2020) Hericium sp. (Song et al., 2020) Hericium sp. P. djamor (Song et al., 2020) (Pineda-Alegria et al., 2017) C. armillatus (Shao et al., 2016) (Centko et al., 2012) GIPinv1 R. cystidiophora R. cystidiophora R. cystidiophora R. cystidiophora P. involutus CDP1 C. dryophila AlPetinc1 EtISPFa1 CDP-like polysaccharide1 E. tinctorium E. tinctorium L. edodes CDP-like polysaccharide1 M. oreades (Pacheco-Sanchez et al., 2006 & 2007) (Javed et al., 2019) (Zeb et al., 2021) (Pacheco-Sanchez et al., 2006) (Pacheco-Sanchez et al., 2006) (Maness et al., 2011) Ramariolide B1 (23) Ramariolide C1 (24) Ramariolide D1 (25) Large molecule EPS1 ISPP-Rb1 Y31 P. tuberregium R. badius C. comatus 1 New compounds. 20 (Centko et al., 2012) (Centko et al., 2012) (Centko et al., 2012) (Barad et al., 2018) (Lim, 2018) (Zhang et al., 2017) 1.4.2 Small molecules Small molecules isolated from mushrooms are usually chemically characterized as quinones, isoflavones, cerebrosides, amines, catechols, sesquiterpenes, triacylglycerols, steroids, organic germanium, and selenium (Fig. 1.1) (Ferreira et al., 2010). Amongst the handful of small molecules isolated from North American mushrooms, most are terpene derivatives as shown in Table 1.3. Two small molecules, syringaldehyde and syringic acid, with molar mass of 183 and 199 respectively, were isolated from 95% ethanol extract of the fruiting bodies of E. granulatus (Stanikunaite et al., 2009). Grifolin (m/z = 329), neogrifolin (m/z = 329) and confluentin (m/z = 327), known to exhibit growth-inhibitory and antibacterial activities, were isolated from the ethanol extract of the fruiting bodies of A. flettii (Liu et al., 2010; Yaqoob et al., 2020). A lanostane-type triterpene named 3, 11-dioxdanosta-8,24(Z)-diene-26-oic acid with molar mass of 469 was isolated from J. hirtus; this triterpene effectively inhibited the growth of two grampositive bacteria: Bacillus cereus and Enterococcus faecalis (Liu et al., 2020). The compound canthin-6-one and its thiomethylated derivative 5-methyl-thiocanthin-6-one, were isolated from Boletus curtisii, but to date there has been no activity reported for these small molecules (Pacheco-Sanchez et al., 2007). Many small molecules were isolated from Hericium sp. which included two new compounds, an erinacerin V alkaloid with molar mass of 258 and an aldehyde derivative of 4-hydroxy chroman, 4-chloro-3,5-dimethoxybenzaldehyde with molar mass of 207 (Song et al., 2020). Seven known compounds were also isolated from Hericium sp., including 2-chloro-1,3dimethoxy-5-methyl benzene, (4-chloro-3,5-dimethoxyphenyl) methanol, 3,6-bis (hydroxyl 21 methyl)-2-methyl-4H-pyran-4-one, 4-chloro-3,5-dimethoxybenzoic acid, 5-hydroxy-6-(1hydroxyethyl) isobenzofuran-1(3H)-one, and erinacine (Song et al., 2020). Some other small molecules that have been isolated from North American mushrooms include pentadecanoic acid, hexadecanoic acid, octadecadienoic acid, octadecanoic acid and β-sitosterol from Pleurotus djamor (Pineda-Alegria et al., 2017), and orellanine from Cortinarius armillatus (Shao et al., 2016). Four new compounds belonging to the butenolide groups called ramariolides A-D were isolated from the coral mushroom Ramaria cystidiophora collected in southwestern British Columbia (Centko et al., 2012). Ramariolides A was found to have antimicrobial activity against Mycobacterium smegmatis and Mycobacterium tuberculosis (Centko et al., 2012). 1.5 Overview of strategies used for natural products discovery Bioactive compounds can be categorized based on their molecular size and pharmaceutical importance as large molecular weight (LMW) compounds and small molecules. LMW compounds do not have a definite molecular weight as they are a population of molecules (polymeric) that display a distribution of molecular weights (Zhu et al., 1998) compared to the small molecules which are discrete and have a definite structure with exact molecular weight. LMW compounds usually have molecular weight more than 600-700 Da and include polysaccharides, proteins, polysaccharide conjugated with proteins and nucleic acids whereas small molecules have a molecular size less than 600 Da and comprise of steroids, lanostanoids, terpenes and others as described later in this section. LMW compounds are important from a biopharmaceutical perspective while small molecules are a more attractive target for formulating pharmaceutical drugs. The purpose of defining these two categories is to set a basis for conducting my current research with a focus on designing methodologies that will specifically isolate either of these compounds. Fig. 1.2 shows a comparative methodology for 22 characterization of small and large molecular weight compounds. Table 1.4 and 1.5 illustrate specific methodologies used by researchers to characterize small and large molecules from mushrooms. Fig. 1. 2. Research methodology towards the discovery of bioactive compound(s) from natural products. 23 Table 1. 4. Methodologies for purification and characterization of large molecules Mushroom (Compound) Lentinus edodes Extraction Purification Aqueous extraction Alcohol precipitation, HPGPC G. lucidum Alkaline (Polysaccharide) extraction (0.1N NaOH) G. lucidum Aqueous (Polysaccharide) extraction G. lucidum Aqueous (Polysaccharide) extraction Tricholoma matsutake (Polysaccharide TMP-A) Aqueous extraction G. tsugae (Glycans) 85% ethanol, hot water, ammonium oxalate, 5% NaOH. G. tsugae (Polysaccharideprotein complexes) Phosphate buffer, water and NaOH Anion exchange chromatography (Diaion– WA30 column), Gel filtration chromatography (Sephacryl S-500 and TSK HW-75 column) Trichloroacetic acid treatment, DEAE–cellulose column, Sephacryl S-300 HR and Sephadex G-10 SEC Deproteination (trichloroacetic acid), DEAE–cellulose column, Sephacryl S-200 HR and Sephadex G-10 SEC Sevag method, Ion exchange chromatography (DEAE-cellulose column), Sephadex (G-100 column) SEC Ion-exchange chromatography (DEAEcellulose column), gel filtration (Toyopearl HW65F column), Affinity chromatography (Con AAF-Formyl Toyopearl 6S0M column) Defatted with acetone and ethyl ester, treated with ethanol, Sevag method, Size exclusion chromatography 24 Chemical characterization Phenol-sulfuric acid method, FTIR, GC-MS, Congo Red test Phenol-sulfuric acid method Reference RI detection, NMR, GC-MS, ESI-MS Bao et al., 2002 Phenol-sulfuric acid method, NMR, TLC, GLC, HPSEC (Ultrahydrogel 1000 column), Congo Red Phenol sulfuric acid method, FTIR, GC–MS, NMR Bao et al., 2001 Phenol-sulfuric acid method, Total protein by Lowry method, Mol wt by gel filtration (Toyo pearl HW-65F column), GC, FTIR, Amino acid analysis, NMR Protein content assay, FTIR, Monosaccharide analysis (HPLC with RI detector Wang et al., 1993 Wang et al., 2013 Chen et al., 2004 Ding et al., 2010 Peng et al., 2003 and Shodex Sugar C1011 column), GC, NMR *FTIR (Fourier Transform Infrared); GC (Gas chromatography); NMR (Nuclear magnetic resonance); High performance gel permeation chromatography (HPGPC); MS (Mass spectrometry); TLC (Thin layer chromatography); RI (Refractive Index). Table 1. 5. Methodologies for purification and characterization of small molecules Mushroom (Compound) G. tsugae (steroids) G. tsugae (Ganodone) G. tsugae (Lanostanoidstsugaric acids) Piptoporus betulinus (Hydroquinone) G. pfeifferi (farnesylhydroquin one, ganomycin K) G. lucidum (Ganodermenonol, ganodermadiol, and ganodermatriol G. lucidum (Ganoderic acid ∑) Extraction Purification Extract with CHCl3 Extracted with ethanol Silica gel column chromatography Partitioning, Dry column vacuum chromatography (DCVC) and flash chromatography, HPLC Silica gel flash column chromatography Structure elucidation FTIR, NMR, EIMS FTIR, LCMS, X-ray crystallograp hy, NMR FTIR, NMR, EIMS Reference Lin et al., 1997b La Clair et al., 2011 Extracted Su et al., with 2000 methanol Extracted Partitioning with CHCl3 and FTIR, EIMS, Kawagishi with ethanol EtOAc, Silica gel flash column NMR et al., 2002 chromatography, RP-HPLC DCM Sephadex LH-20 column, LCMS, NMR Niedermey extract TLC, silica gel flash column er et al., chromatography, HPLC 2013 MeOH Partitioning with different FTIR, NMR, Arisawa et extract solvents, silica gel column MS al., 1986 chromatography (EtOAc/hexane) Extracted Partitioning with hexanes, RP- NMR, ESIMurata et with H2O, HPLC HRMS al., 2016 EtOH and ionic liquids G. lucidum Ethanol Flash chromatography, HPLC ESI-MS, Ruan et al., (Triterpenoids) extract NMR 2014 G. lucidum Refluxed Flash chromatography, HPLC ESI-MS, Ruan et al., (Triterpenoids) with NMR 2015 ethanol * RP-HPLC (Reversed-phase high-performance liquid chromatography); NMR (Nuclear magnetic resonance); LC-IT-TOFMS (Liquid chromatography ion trap-time of flight mass spectrometry); ESI-MS (Electrospray ionization-mass spectrometry) 25 1.6 Mushrooms from North America as a source for drug discovery As shown in Table 1.1, to date 75 species of mushrooms native to North America have been investigated and exhibited medicinal properties. Out of these, 47 species (63%) have bioactivities that have not been previously reported (Table 1.6). These include A. augusta with strong antiproliferative activity, C. dryophila with anti-inflammatory activity, G. esculenta and H. coralloides with strong immuno-stimulatory activities (Deo et al., 2019; Smith et al., 2017). This also includes C. tomentosus, G. helvelloides, L. conifericola, L. connata, T. abietinum with potent activity in anti-proliferation, immuno-stimulation and anti-inflammation (Deo et al., 2019; Smith et al., 2017). There were two specimens that could only be identified to the genus level, Inocybe sp. and Hydnellum sp., suggesting that they are poorly known or undescribed species. Inocybe sp. showed strong anti-proliferative, immuno-stimulatory and anti-inflammatory activities (Deo et al., 2019), while Hydnellum sp. exhibited strong anti-proliferative activity (Smith et al., 2017). There were 13 species that had been studied elsewhere, but their new bioactivities were discovered in the species collected in North America (Table 1.6). This includes Haploporus odorus with antimicrobial activity; Clavulina cinerea, Hydnum repandum, Hygrophoropsis aurantiaca, Hypholoma fasciculare, Phellinus nigricans, and Pleurotus ostreatus with anti-inflammatory activity; Ramaria cystidiophora, Russula paludosa, Tricholomopsis rutilans, and Tyromyces chioneus with anti-proliferative and anti-inflammatory activities. In addition to the reported studies described above, we have recently collected additional mushroom specimens in the northern interior of BC. Out of the 49 species collected, we found 21 species (43%) and 34 species (69%) that exhibited potent anti-proliferative and antiinflammatory activities respectively, and these have not been previously reported (Lee, 2020, 26 pers. comm.). We also found 25 species (51%) exhibiting potent immuno-stimulatory activity that has not been previously reported (Lee, 2020, pers. comm.). As shown in Table 1.3, there has been very limited number of studies exploring bioactive compounds from North American mushrooms. Despite the limited number of studies, 7 new small molecules, 8 new polysaccharides and a new protein with bioactivity have been discovered (Table 1.6). Such observations and the fact that a large number of mushrooms native to North America having bioactivities that have never been previously described, strongly suggests that North American mushrooms are indeed an excellent source for drug discovery. Table 1. 6. Medicinal properties of North American mushrooms Species1 New bioactivity described for the species Known activity2 Amanita augusta None Astraeus pteridis Barrsia oregonensis Chroogomphus tomentosus Clavulina cinerea Anti-proliferative, immunostimulatory, anti-inflammatory Antituberculosis Antituberculosis Anti-proliferative, immunostimulatory, anti-inflammatory Anti-inflammatory Collybia dryophila Coprinellus sp. Coprinus comatus Anti-inflammatory Anti-proliferative Anti-proliferative Elaphomyces muricatus Gautieria monticola Geopora clausa Guepinia helvelloides Gyromitra esculenta Haploporus odorus Anti-inflammatory, antioxidant, antituberculosis Antioxidant Antioxidant, anti-proliferative Anti-proliferative, immunostimulatory, anti-inflammatory Anti-proliferative, immunostimulatory Antimicrobial Hericium corraloides Immuno-stimulatory, antiinflammatory 27 None None None Anti-proliferative (Njue et al., 2017) None None Antioxidant (Li et al., 2010), Hypoglycemic (Han et al., 2006) None None None None None Anticancer (Zmitrovich et al., 2019) None Hydnellum sp. Hydnum repandum Anti-proliferative, immunostimulatory Anti-inflammatory Hygrophoropsis aurantiaca Hymenogaster subalpinus Hymenopellis furfuracea Hypholoma fasciculare Anti-inflammatory Inocybe sp. Anti-proliferative, immunostimulatory, anti-inflammatory Anti-proliferative, immunostimulatory, anti-inflammatory Antimicrobial Laetiporus conifericola Lentinellus subaustralis Leucocybe connata Leucogaster rubescens Melanogaster tuberiformis Phellinopsis conchata Phellinus conchatus Phellodon atratus Anti-inflammatory, antituberculosis Antimicrobial None Anti-proliferative (Takahashi et al., 1992; Vasdekis et al., 2018), antioxidant, antimicrobial (Ozen et al., 2011) Anti-proliferative (Nowak et al., 2016) None None Anti-inflammatory Anti-proliferative (Beattie et al., 2011), antioxidant (Barros et al., 2008), antimicrobial (Millar et al., 2019; Pereira et al., 2013) None None None Anti-proliferative, immunostimulatory, anti-inflammatory Antioxidant None Antituberculosis, antiinflammatory, antioxidant Antimicrobial None Antimicrobial Anti-proliferative (Ren et al., 2006) None Phellinus nigricans Anti-proliferative, immunostimulatory, anti-inflammatory Anti-inflammatory Pholiota terrestris Pleurotus ostreatus Antimicrobial Anti-inflammatory Polyporus badius Pyrofomes demidoffi Ramaria cystidiophora Antimicrobial Antimicrobial Anti-proliferative, antiinflammatory 28 None None Anticancer (Li et al., 2008), antioxidant (Wang et al., 2014), immuno-stimulatory (Li et al., 2008; Wang et al., 2014) None Immuno-stimulatory (Ooi & Liu, 2000), anti-proliferative, antioxidant (Wong et al., 2020) None None Antimicrobial (Centko et al., 2012) Rhizopogon couchii Rhizopogon nigrescens Rhizopogon pedicellus Rhizopogon subareolatus Rhizopogon subaustralis Rhizopogon subgelatinosus Russula paludosa Anti-inflammatory, antioxidant, antituberculosis Anti-inflammatory, antioxidant None Antioxidant, antituberculosis None Antimalarial None Anti-inflammatory, antioxidant None None Anti-inflammatory, antiNone proliferative Anti-proliferative, antiAnti-HIV (Wang et al., 2007) inflammatory Scleroderma laeve Anti-inflammatory, antioxidant, None antituberculosis Trichaptum Anti-proliferative, immunoNone abietinum stimulatory, anti-inflammatory Tricholomopsis Anti-proliferative, antiAntioxidant (Ribeiro et al., 2006) rutilans inflammatory Tyromyces chioneus Anti-proliferative, antiAnti-HIV (Liu et al., 2007) inflammatory 1 Species from Table 1.1 that has novel bioactivity compared to elsewhere but has remained unstudied. 2Bioactivities reported for mushrooms found elsewhere. North American mushrooms offer a wide variety of medicinal benefits and some have been appreciated for their exquisite flavors. While some are unique and edible, others are toxic for human consumption. North American medicinal mushrooms that are considered toxic or nonedible for general human consumption can be tailored as drugs after sufficient toxicity testing and targeted dosage from development. Some of the features that make mushrooms inedible are the presence of toxic metabolites in the whole mushroom, its taste and toughness. E. tinctorium is a hard inedible conk. P. atratus and J. hirtus are also tough mushrooms and apparently J. hirtus has a bitter taste. P. involutus is considered poisonous for human use. E. granulatus is also non-edible. C. armillatus is considered toxic at higher doses due to the presence of orellanine, a potent nephrotoxin (Shao et al., 2016). 29 1.7 Conclusions Having extensively reviewed the literature on the bioactivities and compounds isolated from mushrooms native to North America, the questions posed at the beginning of this review are answered. (i) Do species found in North America similar to those in Asia or Europe exhibit similar bioactivities and produce similar group of bioactive compounds? Based on subjective approach of bioactivity-guided investigations on selective species, similar species found in North America and Asia or Europe indeed produce similar bioactivities. For example, this is true for the commonly studied I. obliquus and P. ostreatus. Furthermore, compounds such as grifolin, neogrifolin and confluentin produced in North American A. flettii, are also made by other Albatrellus species found elsewhere (Liu et al., 2010; Yaqoob et al., 2020). However, a more definitive answer can only be obtained by using more advanced and objective methods such as Quadrupole Time-of-Flight Mass Spectrometry to globally examine the metabolites produced and using multiple biological screening assays to simultaneously monitor different bioactivities. (ii) Do similar species found in North America and elsewhere exhibit distinct bioactivity and produce distinct compounds? Again, the answer to this question will come from more advanced approaches described above. (iii) Do new species found in North America produce new compound(s)? Due to the limited number of studies on mushrooms native to North America, it is currently unknown whether new species found in North America produce new compound(s). However, based on very limited study (Centko et al., 2012), it is highly likely new medicinal compounds are produced by new species found in North America. (iv) Based on the answers to (i) to (iii) above, is it worth the efforts to explore mushrooms native to North America for medicinal properties and for new compounds? The answer to this question is a definite yes. This is based on the fact that there are many species, including new species, in North America whose 30 bioactivities have never been previously reported (Table 1.6) (Lee, 2020). Furthermore, novel compounds have been isolated from North American mushrooms despite the very limited number of studies. In summary, to date only 75 mushroom species in North America have been studied and reported to possess medicinal properties. Of these, 47 species (63%) exhibited bioactivities that have never been previously reported. To date, only 15 mushroom species in North America have been subjected to bioactivity-guided compound isolation studies. Out of this limited number of studies, already 7 new small molecules, 8 new polysaccharides and a new protein with medicinal properties have been discovered. In conclusion, mushrooms native to North America are indeed an excellent source for drug discovery. Further exploration of new species as well as known species that are found elsewhere are therefore warranted. Since most of the studies conducted to determine the medicinal effects have so far involved in-vitro cell lines, it would be of interest to see if the in-vitro cell studies can be translated to in-vivo studies performed using relevant animal models. 1.8 Echinodontium tinctorium Echinodontium tinctorium, known as Indian body paint fungus, is a tree-dwelling conk with a distinct orange to red color. Due to its distinct color, it was commonly used by Native American tribes as a war paint (Ye et al., 1996). E. tinctorium is an Agaricomycete fungus that belongs to genus Echinodontium of the Echinodontiaceae family. Based on morphological similarities, Gross (1964) classified six species of Echinodontiaceae which included E. tinctorium, E. tsugicola, E. japonicum, E. ballouii, E. taxodii and E. sulcatum. However, Liu et al. (2017) revised the taxa based on molecular techniques where E. tinctorium, E. tsugicola and a new specie E. ryvardenii were included in Echinodontium. In addition to this, E. japonicum was 31 placed in a new genus Echinodontiellum due to its different hardwood host (Quercus). The remaining species; Lauriliella taxodii and Laurilia sulcata, classified by Gross (1964) in Echinodontium were placed in different genera of family Bondarzewiaceae. E. ballouii being a rare specie was not included in the analysis. Phylogenetic studies have also been conducted (Tabata et al., 2000) to indicate the placement of E. tinctorium with other related species in the same group. Studies regarding the bioactivity of this mushroom remain limited and the literature available deals mostly with its taxonomy, phylogeny and symbiotic relationships (Aho et al., 1987; Larsen et al., 1987, Liu et al., 2017). Bond et al. (1966) have characterized a triterpene echinodol from E. tinctorium (Bond et al., 1966). Ye et al. (1996) have also isolated, purified and characterized a small molecule echinotinctone from E. tinctorium. Echinotinctone was isolated from a methanolic extract of E. tinctorium and was referred to as a first natural orange pigment that structurally resembles xanthene dyes fluorescein and eosin. One of the studies cross-referenced by Ye et al. (1996), based on a private communication by Dr. D. H. French (Reed College, Portland, Oregon), stated that Oregon natives used this mushroom for antibacterial activity and also that the extracts possess antitumor properties (Ye et al., 1996). 1.9 Research Hypothesis At the outset of this literature review, I hypothesized that immuno-stimulatory and growthinhibitory large and small molecules are present in BC wild mushrooms. Besides the description of only a few small molecules with unknown bioactivity (Ye et al., 1996) and anti-inflammatory polysaccharide (Javed et al., 2019) from E. tinctorium, this species has not been fully explored for its therapeutic potential. Therefore, the goal of my PhD project is to uncover the therapeutic potential of E. tinctorium. I will isolate, purify and characterize the responsible bioactive compounds which are currently unexplored. 32 1.10 Research Objectives To test my hypothesis, this project was divided into 3 experimental chapters. 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International journal of medicinal mushrooms, 21(8), 783-791. 44 Chapter 2: Structural elucidation and immuno-stimulatory activity of a novel polysaccharide containing glucuronic acid from the fungus Echinodontium tinctorium ABSTRACT An immuno-stimulatory polysaccharide (EtISPFa) was purified from water extract of the fungus Echinodontium tinctorium. EtISPFa has an estimated weight average molecular weight (Mw) of 1354 kDa and is composed of glucose (66.2 %), glucuronic acid (10.1 %), mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), rhamnose (3.1 %), fucose (1.8 %), and arabinose (0.2 %). It has multiple glycosidic linkages, with 3-Glcp (19.8 %), 4-GlcpA (10.8 %), 6-Glcp (10.7 %), and 3,6-Glcp (8.7 %) being the most prominent. NMR analysis showed that EtISPFa has a backbone containing mostly 3-substituted β-glucopyranose with 4-substituted glucopyranosyl uronic acid. Short side chains consisting of an average of two β-glycopyranose residues, connected through 1→6 linkages, are attached to the 6-position of about every 4th or 5th backbone glucose residue. EtISPFa is a novel glucuronic acid-containing β-glucan capable of significantly inducing the production of cytokines IL-17, IL-16, MIP-2, G-CSF, GM-CSF, LIF, MIP-1α, MIP-1β, and RANTES in vitro. EtISPFa should be further explored for its immunostimulatory activity in vivo. 2.1. Introduction Recent success in immunotherapy and immune checkpoint blockade has led to a renewed interest in finding compounds with immuno-modulatory effects (Havel, Chowell, & Chan, 2019). Many immuno-modulatory compounds isolated from mushrooms are polysaccharides (El Enshasy & Hatti-Kaul, 2013; Lull, Wichers, & Savelkoul, 2005; Rowan et al., 2003). Given the limited information on the therapeutic potential of E. tinctorium, crude extracts of the fungus were screened for three major bioactivities related to cancer treatment, including immuno- 45 stimulatory activity. At the outset of this research, I hypothesized that E. tinctorium would yield immuno-stimulatory polysaccharide(s) and it was documented that its water extract possessed strong immuno-stimulatory activity (Javed et al., 2019). To assess for immuno-stimulatory activity, activation of Raw 264.7 murine macrophage cells was monitored to produce the proinflammatory cytokine tumor necrosis factor (TNF)-α. Activation of macrophages to produce TNF-α is one of the critical innate immune responses and has proven to be a gold standard in monitoring for immuno-stimulation (El Enshasy & Hatti-Kaul, 2013; Lull et al., 2005). The aim of the present study was to purify and characterize an immuno-stimulatory polysaccharide from E. tinctorium. The water extract from E. tinctorium was subjected to multiple chromatographic steps (Sephadex LH-20, DEAE-Sephadex, Sephacryl S-500 HR, and HPLC BioSEC-5 chromatography) for purification of the immuno-stimulatory compound. Following this, structural analyses were performed. This included gas chromatography-mass spectrometry (GC–MS) to determine monosaccharide content and glycosidic linkages, Fourier transform infrared (FTIR) spectroscopy for identification of functional groups, and finally, 2-D NMR analyses for determination of the constitution and monosaccharide sequence of the EtISPFa polysaccharide. 2.2. Materials and methods 2.2.1. Materials and reagents All the reagents used were of analytical grade. Dulbecco’s Modified Eagle Medium (DMEM) from LONZA (Walkersville, Maryland, USA), and fetal bovine serum (FBS) from Life Technologies Inc. (Waltham, Massachusetts, USA) were used. SephadexTM LH-20, DEAESephadex, and HiPrep 26/60 SephacrylTM S-500 HR pre-packed columns were obtained from GE Healthcare (Chicago, IL, USA). Dextran standards (T1, T5, T12, T25, T50, T80, T150, 46 T270, T410) were purchased from Sigma- Aldrich (Oakville, ON, Canada), and HPLC BioSEC5 column and guard column were purchased from Agilent (Santa Clara, CA, USA). 2.2.2. Collection and extraction of the mushroom E. tinctorium conks were collected from hemlock trees in Terrace (CL103) and Smithers (CL37), BC, Canada, in August 2014 and 2015, respectively. Voucher specimens for these collections were deposited at the University of Northern British Columbia, Canada. The specimens, previously confirmed using morphological and molecular techniques (Javed et al., 2019), were dried in a hot air oven (55 ◦C, 24− 48 h), cut into smaller pieces using a saw machine, and ground to fine powder using a hammer mill. Powdered mushrooms (300 g) were sequentially extracted with 80 % ethanol (1.5 L, 65 ◦C, 3 h). The extract was vacuum filtered through Whatman filter paper No. 3 and the filtrate was designated 1A. The residue was further extracted with 50 % methanol (1.5 L, 65 ◦C, 3 h). The methanol extract was filtered and the filtrate was designated 1B. The residue was further extracted with water (1.5 L, 65 ◦C, 6 h) and the filtrate was named 1C. Crude extract 1C was concentrated, lyophilized, and filter sterilized before assessment for immuno-stimulatory activity. 2.2.3. Immuno-stimulatory assay Raw 264.7 murine macrophage cell line was used to conduct immuno-stimulatory assays essentially as described previously (Smith et al., 2017). After 16−18 h of plating at a density of 1 × 105 cells/well in 96-well plates in 200 μL of serum-free DMEM, cells were treated with 1C at doses ranging from 0.1−1 mg/mL and incubated for 6 h. Water was used as a negative control, whereas lipopolysaccharide (LPS) at 500 ng/uL was used as a positive control in most experiments. After 6 h incubation, 100 μL of cell supernatants were collected to determine the levels of TNF-α as well as other cytokines produced upon stimulation by test agents. TNF-α 47 stimulation was determined by sandwich ELISA as previously described (Smith et al., 2017). Samples were analyzed at 450 nm and 550 nm using Synergy-2 multiplate reader (BioTek®, VT, USA). Other cytokines and chemokines were measured by Eve Technologies (Calgary, AB, Canada) using a mouse cytokine array/chemokine array 32-plex. 2.2.4. Purification of immuno-stimulatory compound(s) from E. tinctorium The crude water extract 1C was subjected to purification on a 400 mL Sephadex LH-20 designated column-2 (C26/100 by GE Healthcare), previously equilibrated with 2–3 column volumes (C.V.) of degassed water. 1C was reconstituted in water to a concentration of 30 mg/mL, spun at 1000 × g for 5 min, before loading onto the column. Sample injection volume was 2% of the total bed volume. Degassed water was allowed to run through the column at a flow rate of 1 mL/min and 10 mL fractions were collected over 2 C.V. The eluted fractions were assessed for carbohydrate content using phenol sulfuric acid method (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956) and protein content using PIERCE BCA protein assay (Waltham, MA, USA). The pooled fractions from column-2 were concentrated to yield bioactive fraction 2A (100 mg) and subjected to anion exchange chromatography using DEAE-Sephadex A50 resin (designated column-3). Multiple buffers were assessed on small scale (10 mL) DEAE gravity drip column as shown in Fig. S1 to get the optimized conditions (L-Histidine buffer, pH 6.2, 1 mL/min). A XK-50 size column-3 (50 mm x 100 cm, GE Healthcare) was used. Flow-through and eluent (using 1 M NaCl) were concentrated, dialyzed (MWCO 3500 Da), lyophilized, resuspended (1 mg/mL), and assessed for immuno-stimulatory activity. Bioactive elution 3A was then purified using a Sephacryl S-500 HR (column-4) (150 mM NaCl, 1.3 mL/min, 1.5 C.V., 10 mL fractions) connected to an AKTA Pure system. Sample (2 mL at 50 mg/mL) was loaded onto 48 the column using a 2 mL sample loop. Eluted fractions were analyzed for TNF-α stimulation by immuno-stimulatory assay as described in Section 2.2.3. 2.2.5. Molecular size distribution and ultrapurification using HPLC HPLC SEC was used to further purify the bioactive compound, and for estimation of purity as well as molecular size distribution. HPLC SEC was performed on Agilent HPLC 1200 series using an Agilent BioSEC-5 column-5 (5 μm, 500 A, 4.6 × 300 mm), equipped with a guard column (5 μm, 500 A, 4.6 × 50 mm). Refractive index detector (Agilent 1200 Infinity II series, G7162A RID) with standard optical unit was used for detection purposes. The temperature was kept at 28 ± 0.2 ◦C. The peak maxima molecular weight (Mp) of EtISPFa was estimated by calibrating with T-series Dextran standards (25–2000 kDa). The column was equilibrated with mobile phase to get a stable baseline. Active fraction 4A (10 μL at 10 mg/mL) was injected through an autosampler (Agilent 1200 series G1329A) and water was used as a mobile phase (flow rate = 0.4 mL/min). Once the HPLC profile was obtained, the respective peaks were purified by fraction collection. Fraction collected peaks were re-injected to confirm sample purity. The peaks were assessed for immuno-stimulatory activity as described in Section 2.2.3. Fractions containing the bioactive peak were lyophilized and subjected to structural elucidation methods as described below. 2.2.6. Enzyme digestion and heat denaturation of polysaccharides from E. tinctorium To further delineate the role of polysaccharides from E. tinctorium in contributing to the immuno-stimulatory activity, both EtISPFa and EtISPFb were digested with several enzymes capable of cleaving glycosidic bonds. Two mg/mL of EtISPFa and EtISPFb were treated with cellulase (10 and 30 units) for 2 h at 37 ºC followed by heat deactivation at 80 ºC for 20 min. In the case for the treatment with fucosidase (0.01 unit) and galactosidase (0.1 unit), both 49 polysaccharides were incubated at 37 ºC for 24 h and heat deactivated as described above. The reaction mixtures were kept at -20 ºC until further analysis for immuno-stimulatory activity as described above. 2.2.7. Monosaccharide composition analysis Monosaccharide content of HPLC-purified sample was determined by GC–MS. EtISPFa (360 μg) and internal standard inositol (20 μg) were subjected to acid methanolysis by heating with 1 M methanolic HCl in a sealed screw capped glass tube (18 h, 80 ◦C). The samples were dissolved under nitrogen stream and treated with methanol, pyridine, and acetic anhydride for 30 min. Excess solvent was removed and samples were subjected to derivatization with Tri-Sil HTP (80 ◦C, 30 min). The resulting per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides were analyzed by GC–MS (Agilent 7890A GC, 5975C MSD) on a Supelco Equity-1 fused silica column (30 m × 0.25 mm ID). 2.2.8. Methylation and linkage analysis Glycosyl linkage analysis was conducted by methylating 1 mg of EtISPFa with dimsyl potassium (15 min) and methyl iodide (15 min) in a screw-capped glass tube. After extracting with dichloromethane and reducing with LiAlD4 (lithium aluminum deuteride) in THF for 4 h at 100 ◦C, the sample was permethylated with NaOH (15 min) and methyl iodide (45 min), depolymerized with 400 μL of 2 M TFA (trifluoro acetic acid) in a sealed tube (2 h, 121 ◦C), reduced with NaBD4 (sodium borodeuteride), and acetylated with a mixture of acetic anhydride and TFA (25 min, 50 ◦ C), resulting in partially methylated alditol acetates (PMAAs). The undermethylated polysaccharides after permethylation using NaOH and methyl iodide were permethylated again. This fraction was then depolymerized, reduced, acetylated, and combined with the fraction permethylated in the first run. The combined collection was subjected to GC– 50 MS analysis using a 30 m Supelco SP-2331 fused silica column on a GC–MS instrument (Agilent 7890A GC, Agilent 5975C MSD with electron impact ionization source). 2.2.9. Structural elucidation by spectral analysis The presence of proteins and nucleic acids was determined through UV nanodrop scan on a UV spectrophotometer with a detection range of 200-400 nm. Sample was analyzed at a wavelength of 280 nm for proteins and 260 nm for nucleic acids. FTIR spectroscopy was conducted to analyze functional groups of EtISPFa using Bruker ATR-FTIR spectrophotometer (Billerica, MA, USA), with a detection frequency from 4000-400 cm-1. For FTIR, a small amount of EtISPFa was placed on a diamond window and analyzed by OPUS software. Twenty two scans were performed to obtain a representative FTIR spectrum. For further detailed structural characterization, NMR analysis was conducted. EtISPFa (5.4 mg) was dissolved in 99.8 % of deuterium oxide (D2O, Sigma), lyophilized and redissolved in 320 μL of 99.96 % D2O (Cambridge Isotope Laboratories). D2O exchanged EtISPFa was then subjected to 1D proton NMR (1H-NMR) and 2D NMR including 1H-1H- correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), 1H-13C NMR heteronuclear single quantum correlation spectroscopy (HSQC), HSQC-TOCSY, and heteronuclear multiple bond correlation (HMBC) on an Agilent Inova 600 MHz NMR, equipped with a cryoprobe, using standard pulse sequences. Acetone was used as an internal standard (δH = 2.218 ppm, δC = 33.0 ppm). NMR analysis was carried out at 65◦C. 2.2.10. Estimation of glucan content in EtISPFa The α- and β-glucan contents were determined using a Megazyme kit (Wicklow, Ireland), based on manufacturer guidelines. Using this kit, EtISPFa was subjected to multiple steps of acid 51 hydrolysis and enzyme cleavage to yield D-glucose molecules which were then detected by measuring absorbance at 510 nm using Synergy 2 multiplate reader. Yeast β-glucan was used as a standard. 2.3. Results and discussion 2.3.1. Extraction and assessment of E3 water extract for immuno-stimulatory activity We have previously shown that the water extract from E. tinctorium possesses strong immuno-stimulatory activity (Javed et al., 2019). Here, we took the initiative to purify, identify and characterize the immuno-stimulatory compound(s) from E. tinctorium. The powdered mushroom was sequentially extracted, as illustrated in Fig. 2.1A. The sequential extractions with ethanol and methanol were expected to remove any small molecules, thereby retaining large molecules (polysaccharides, proteins or complexes of both) in the water extract. Extraction of powdered E. tinctorium (300 g) generated water extract 1C which was concentrated by freeze drying to yield a dried extract (4 g). Crude extract 1C was reconstituted in water to give a concentration of 20 mg/mL and filter sterilized. A dose-dependent immuno-stimulatory assay was then performed. As shown in Fig. 2.1B, E3 was found to induce TNF-α in Raw 264.7 cells at concentrations ranging from 0.1-1 mg/mL. 52 Fig. 2. 1. Chemical extraction of E. tinctorium and assessment of the water extract E3 for immuno-stimulatory activity on macrophage cells (A) Chemical extraction scheme to obtain water extract E3 from E. tinctorium. (B) Dose-dependent immuno-stimulatory assay shows induction of TNF-α production in Raw 264.7 macrophage cells by E3 extract from 0.1-1 mg/mL. Error bars represent S.D. Results shown are representative from three biological replicates. 2.3.2. Purification of immuno-stimulatory polysaccharide from E. tinctorium The overall scheme adopted for the purification of the immuno-stimulatory polysaccharide from E. tinctorium (EtISPFa) is shown in Fig. 2.2A and the amounts obtained at each step are given in Table S2. The 1C extract was first subjected to Sephadex LH-20 size-exclusion chromatography column-2. As shown in Fig. 2.3A, the major immuno-stimulatory activity was found at elution volume 30-48 mL (0.5-0.7 C.V.) (fractions 27-39), suggesting that the immunostimulatory compound has a relatively large molecular weight. The fractions collected from column-2 were also assessed for carbohydrate (CHO) and protein content. Results in Fig. 2.2B show that the major immuno-stimulatory activity correlated strongly with carbohydrate content. The activity also correlated with the first protein content peak (Fig. 2.2C). This result suggests 53 that the major immuno-stimulatory activity eluted at volume 30-48 mL contains both carbohydrate and protein components. Fig. 2. 2. Purification of the immuno-stimulatory polysaccharide (EtISPFa) from E. tinctorium. (A) Summary of the purification scheme used. 1C extract from E. tinctorium was purified using Sephadex LH-20 size-exclusion chromatography (column-2). Fractions collected from column-2 were assessed for immuno-stimulatory activity (solid line in B and C), carbohydrate content (dotted line in B) and protein content (dotted line in C). Error bars represent S.D. Results shown are representative from three biological replicates. The immuno-stimulatory fractions 10-16 from column-2 (Fig. 2.3A) were then pooled, concentrated, lyophilized, and resuspended in water. It was then subjected to column-3, DEAE- 54 Sephadex anion exchange chromatography using L-Histidine (pH 6.2) as the buffer of choice. The flow-through and eluent from column-3 were concentrated, dialyzed, lyophilized, filter sterilized, and assessed for immuno-stimulatory activity. As shown in Fig. 2.3B, the eluent referred to as 3A (but not the flow-through from DEAE-Sephadex) contains the immunostimulatory activity (Fig. 2.3B). This result suggested the presence of acidic groups on the immuno-stimulatory polysaccharide that were effectively associated with positively charged DEAE-Sephadex resin and were eluted upon addition of salt to the mobile phase. Fig. 2. 3. Purification of the immuno-stimulatory polysaccharide from E. tinctorium (EtISPFa) (A) Purification using Sephadex LH-20 size exclusion chromatography (column-2). Fractions collected were assessed for immuno-stimulatory activity. (B) Purification using DEAE-Sephadex anion exchange chromatography (column-3). Pre-load, flow-through and eluent were assessed for immuno-stimulatory activity. Medium and water were used as negative controls. LPS was used as a positive control. (C) Sephacryl S-500 HR SEC (column-4) elution profile correlating TNF-α stimulation with relative size of bioactive fractions. Raw 264.7 macrophage cells were 55 used to assess TNF-α production as an indicator of immuno-stimulatory activity. Error bars represent S.D. Bioactive elution collected from column-3 (Fig. 2.3B) was concentrated, lyophilized, filter sterilized, and subjected to column-4, Sephacryl S-500 high resolution size-exclusion chromatography. As shown in Figs. 2.3C & S2, 4A (fractions 14-18) and 4B (fractions 30-32) recovered from the elution of column-4 at retention volumes 150-190 mL and 300-320 mL retained bioactivity. T-series Dextran standards were used to estimate the relative molecular size of bioactive compounds on column-4. As shown in Fig. 2.3C, 4A had a predicted molecular size of 670-2000 kDa or greater, whereas 4B was 200-300 kDa in size. The two sets of bioactive fractions were pooled separately, concentrated, dialyzed, and lyophilized. 56 Fig. 2. 4. HPLC BioSEC-5 full elution profile of (A) 4A and (B) 4B. The collected two peaks in (A) and (B) were assessed for immuno-stimulatory activity shown in (C). Bioactive fractions 4A and 4B were further purified by HPLC using Agilent Bio SEC-5 column-5. The HPLC profiles of 4A and 4B (Fig. 2.4) show the presence of two significant peaks eluting at retention times 6.66 min and 12.44 min for 4A (Fig. 2.4A), and 8.48 min and 12.31 min for 4B (Fig. 2.4B), respectively. The respective peaks were fraction collected and 57 assessed for immuno-stimulatory activity. Peak 1 in both 4A and 4B at retention times 6.66 min and 8.48 min showed maximum TNF-α stimulation, compared to their respective peak 2 (Fig. 2.4C). The fractions collected containing the bioactive peaks were re-assessed for purity using column-5 (Fig. 2.5). The bioactive peak 1 (4A5A; EtISPFa) from 4A was retained at 5.72 min while bioactive peak 1 (4B5A) from 4B was eluting at retention time of 5.70 min. Since these retention times are quite similar, both 4A5A (referred to as EtISPFa) and 4B5A are most likely similar compounds or they share common chemical characteristics. Fig. 2. 5. (A) HPLC BioSEC-5 profile of purified Peak 1 EtISPFa. (B) HPLC BioSEC-5 purified Peak 1 EtISPFb. Based on the assumption that both are similar compounds, EtISPFa was chosen for further structural elucidation studies as described below. To more accurately estimate the size of EtISPFa, T-series dextrans with molecular weight ranging 25-2000 kDa were used on BioSEC-5 (Fig. S3). Based on the results, the peak maxima molecular weight (Mp) of EtISPFa was 58 estimated to be 1302 kDa. We further performed calculations to determine the number (Mn) and weight average molecular weight (Mw) of EtISPFa. As shown in Table S1, the Mn and Mw of EtISPFa was calculated to be 1302 kDa and 1354 kDa respectively. The polydispersity index was calculated to be 1.04. 2.3.3. Cytokine and chemokine secretion in Raw264.7 cells induced by 4A Besides TNF-α, secretion of cytokines such as colony stimulating factor (CSF), interleukins (ILs), chemokines, and interferons (IFNs) by macrophages is also important in regulating the immune system. A mouse cytokine/chemokine array 32 plex was used to determine the cytokine and chemokine secretion profile of Raw 264.7 cells induced by a 6 h treatment with bioactive fraction 4A. The array covers both pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, GM-CSF) and chemokines (RANTES, MCP-1, MIP-1α, MIP-1β) as well as anti-inflammatory cytokines (IL-4, IL-10, IL-11, G-CSF) that are commonly used to monitor the capacity of mushroomderived compounds to elicit an immune response (El Enshasy & Hatti-Kaul, 2013; Lull et al., 2005). The results are summarized in Table 2.1. Treatment with 4A markedly enhanced the secretion of cytokines, which ranged from an increase of 2.1-683 times higher than the control. The cytokines that were most significantly induced (> 10-fold increase) included interleukin-17 (IL-17), interleukin-6 (IL-6), macrophage inflammatory protein-2 (MIP-2), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), and RANTES (regulated on activation, normal T cell expressed and secreted). The cytokines that were moderately induced (2 to 10-fold increase) included interleukin-10 (IL-10), interferon gamma-induced protein 10 (IP-10), eotaxin, 59 interleukin-1α (IL-1α), monocyte chemo attractant protein-1 (MCP-1), and interleukin-12 (IL12). Table 2. 1. Cytokines induction by EtincFa in Raw 264.7 murine macrophage cells Fold + S.Da Cytokine a IFNγ Interferon gamma 1.22 + 0.32 TNF-α Tumor necrosis factor-α 58.04 + 6.83 IL-2 Interleukin-2 0.87b IL-10 Interleukin-10 4.47 + 1.42 IL-12 p70 Interleukin-12 1.46b IL-4 Interleukin-4 1.48 + 0.33 IL-5 Interleukin-5 1.0 IL-9 Interleukin-9 0.97 + 0.05 IL-17 Interleukin-17 12.95 + 1.06 IL-6 Interleukin-6 65.05 + 7.22 IP-10 Interferon gamma-induced protein-10 3.63b MIP-2 Macrophage inflammatory protein-2 24.55b CCL11 Eotaxin 2.11 + 1.56 G-CSF Granulocyte-colony stimulating factor 683.93 + 22.73 GM-CSF Granulocyte macrophage-colony stimulating factor 208.78 + 27.56 IL-1α Interleukin-1α 2.40 + 1.66 IL-1β Interleukin-1β 1.50 + 0.98 LIF Leukemia inhibitory factor 86.47b MCP-1 Monocyte chemo attractant protein-1 1.58b IL-15 Interleukin-15 1.50 + 0.27 MIP-1α Macrophage inflammatory protein-1α 21.7 + 6.87 MIP-1β Macrophage inflammatory protein-1β 22.34 + 7.57 RANTES (CCL5) Regulated on activation, normal T cell expressed and secreted [Chemokine (C-C motif) ligand 5] 5.19b The fold changes indicated in the table are relative to the control (H2O). The mean fold change ± standard deviation (S.D) is taken for data set n = 2. 60 These results confirmed the immuno-stimulatory effect of 4A on murine macrophage cells in vitro. It is important to point out that this in vitro study cannot distinguish whether our purified polysaccharide exerts immuno-stimulatory effect and/or induce inflammation. Such distinction can only be determined using an animal model. 2.3.4. Glucan content analysis The α-glucan and β-glucan contents were estimated in Sephacryl S-500-purified 4A and 4B. Yeast β-glucan was used as a positive control. β-glucan content in 4A and 4B fractions was estimated to be 89.9 % (10.16 % w/w) and 82.4 % (9.9 % w/w). However, α-glucan in 4A and 4B was estimated to be 10.1 (1.14 % w/w) and 17.6 % (2.11 % w/w), respectively. In summary, both 4A and 4B predominantly contain β-glucan and this was further studied using GC–MS and NMR analyses as described below. 2.3.5. Role of polysaccharide component in contributing to the immuno-stimulatory activity Several enzymes capable of cleaving glycosidic bonds were used to digest the Sephacryl S500-purified 4A and 4B (Fig. 2.6-2.9). EtISPFa and EtISPFb were subjected to enzymes digestion to assess the role of a particular glycosidic linkage in contributing to the immunostimulatory activity. As shown in Fig. 2.6, the immune-stimulatory activity of 4A and 4B was not affected by cellulase digestion. Cellulase are specific in hydrolyzing the β-1,4 linkage. Interestingly, from the chemical characterization data (Table 2.4), it was revealed that EtISPFa indeed has a β-1,4 linked GlcA. The unexpected negative results suggest that β-1,4 linkage may not be easily accessible to cellulase due to the complex structure of EtISPFa. Alternatively, it could be that celuulase hydrolyzes β-1,4 linkage between Glc molecules whereas EtISPFa has β1,4 linkage between Glc and GlcA. 61 Fig. 2. 6. Effect of cellulase on immuno-stimulatory activity of EtISPFa (A) and EtISPFb (B) from E. tinctorium. One-way ANOVA (Tukey’s test) was used for statistical analysis. Error bars represent S.D. ns represents non-significant. Fig. 2. 7. Effect of fucosidase (A) and galactosidase (B) on immuno-stimulatory activity of EtISPFa from E. tinctorium. One-way ANOVA (Tukey’s test) was used for statistical analysis. Error bars represent S.D. ns represents non-significant (p > 0.05). 62 In addition to this, α-fucosidase (0.01 units) and α-galactosidase (0.1 units) had no effect on the immuno-stimulatory activity of EtISPFa (Fig. 2.7). EtISPFa does not contain any α-linked fucose or galactose (Table 2.4), which explains the unaffected immuno-stimulatory activity. ns Fig. 2. 8. Effect of heat denaturation on immuno-stimulatory activity of EtISPFa from E. tinctorium. One-way ANOVA (Tukey’s test) was used for statistical analysis. ns represents nonsignificant (p > 0.05). Error bars represent S.D. Another important point to draw from these studies is that EtISPFa is a very stable polysaccharide. As shown in Fig. 2.8, that the immuno-stimulatory activity of EtISPFa was not abolished at high temperature. 2.3.6. Monosaccharide composition analysis The monosaccharide analysis by TMS derivatization using GC–MS revealed that EtISPFa consisted of predominantly glucose (Glc). Relatively large amounts of glucuronic acid (GlcA), mannose (Man), and galactose (Gal) were also found in EtISPFa (Fig. S4 and Table 2.2). Some other glycosyl residues were also found in small amounts including xylose (Xyl), rhamnose (Rha), fucose (Fuc) and arabinose (Ara). These glycosyl residues accounted for 49.2 % 63 of the total weight of carbohydrate. It is likely that this somewhat underestimates the actual carbohydrate due to the resistance of GlcA to acid-catalyzed depolymerization. Table 2. 2. Monosaccharide composition of EtISPFa Monosaccharide residue Mass (µg) Mol % Glucose (Glc) 118 66.2 Glucuronic acid (GlcA) 19.4 10.1 Mannose (Man) 11.9 6.7 Galactose (Gal) 11.4 6.4 Xylose (Xyl) 8.3 5.6 Rhamnose (Rha) 5.0 3.1 Fucose (Fuc) 2.9 1.8 Arabinose (Ara) 0.4 0.2 Total = 177.2 100 *Analysis performed by Complex Carbohydrate Research Center (CCRC), University of Georgia, US. 2.3.7. Glycosyl linkage analysis by GC–MS Consistent with the composition analysis, the glycosyl linkage analysis showed a majority of glucose linkages (61.8 %) in EtISPFa (Fig. S5 and Table 2.3). The main glucose linkages were 3substituted, terminal, 6-substituted and 3,6-disubstituted. The linkage analysis also showed a greater proportion of glucuronic acid residues than what the composition analysis detected. The Glc:GlcA ratio was 6:1 in the composition analysis, but 3:1 in the linkage analysis. This difference is explained by the known resistance of uronic acids to acid hydrolysis or methanolysis (De Ruiter, Schols, Voragen, & Rombouts, 1992), which is required for the composition analysis. This resistance is not a factor in the linkage analysis because before hydrolysis, the glucuronic acids are reduced to glucose, which is readily hydrolyzed. According 64 to the composition analysis, about 10 % of the total carbohydrate is GlcA, whereas that number is slightly above 20 % in the linkage analysis. We noted the presence of fully-branched glucose (2,3,4,6-Glcp) and this is most likely due to undermethylation during the derivation. The proportion of this fully-branched glucose is only 2% and is considered insignificant. The presence of significant amounts of glucuronic acid in EtISPFa distinguishes it from other fungal polysaccharides, which are mostly devoid of or low in GlcA content (Wang, Wang, Xu, & Ding, 2017). Minor glycosidic linkages were from mannose, galactose, fucose, xylose, and rhamnose residues. These results suggested that EtISPFa is similar to a (1→3) (1→6)-β-glucan with 3linked backbone and 6-linked side chains. However, a significant portion of the backbone 3substituted Glc residues are replaced by 4-substituted GlcA residues. Considering only the abundances of major backbone residues in the linkage analysis, 3-Glc, 3,6-Glc, and 4-GlcA, the ratio of straight-chain to branched residues is about 4:5, indicating the presence of side chains on every 4th or 5th backbone residue. Many studies have reported the presence of β-1à3 and β1à6 in immuno-stimulatory polysaccharides isolated from mushrooms. Some examples include polysaccharides from Lentinula edodes, Sclerotium rolfsii, Dictyophora indusiata and Pleurotus sajor-caju (Ferreira et al., 2015). These studies relate to the presence of β-1à3 and β-1à6 as a structural feature that contributes to the immuno-stimulatory activity of EtISPFa. Table 2. 3. Glycosyl linkage analysis* of EtISPFa by partially methylated alditol acetates PMAA 1,5-Di-O-acetyl-1-deuterio-6-deoxy-2,3,4-tri-O-methylgalactitol 1,2,5-Tri-O-acetyl-1-deuterio-6-deoxy-3,4-di-O-methylmannitol 1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methylmannitol 1,3,5-Tri-O-acetyl-1-deuterio-6-deoxy-2,4-di-O-methylmannitol 1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methylglucitol 1,5-Di-O-acetyl-1,6,6’-trideuterio-2,3,4,6-tetra-O-methylglucitol 1,3,5-Tri-O-acetyl-1-deuterio-6-deoxy-2,4-di-O-methylgalactitol 1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methylgalactitol 65 Linkage t-Fucp 2-Rhap t-Manp 3-Rhap t-Glcp t-GlcpA 3-Fucp t-Galp Mol%a 1.1 1.4 1.1 1.8 12.8 3.5 1.1 1.5 1,3,5-Tri-O-acetyl-1-deuterio-2,4,6-tri-O-methylglucitol 1,3,5- Tri-O-acetyl-1,6,6’-trideuterio-2,4,6-tri-O-methylglucitol 1,2,5- Tri-O-acetyl-1-deuterio-3,4,6-tri-O-methylmannitol and 1,3,5- Tri-O-acetyl-1-deuterio-2,4,6-tri-O-methylmannitol 1,5,6- Tri-O-acetyl-1-deuterio-2,3,4-tri-O-methylmannitol 1,5,6- Tri-O-acetyl-1-deuterio-2,3,4-tri-O-methylglucitol 1,4,5- Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methylglucitol 1,4,5- Tri-O-acetyl-1,6,6’-trideuterio-2,3,6-tri-O-methylglucitol 1,5,6- Tri-O-acetyl-1-deuterio-2,3,4-tri-O-methylgalactitol 1,3,5,6- Tetra-O-acetyl-1-deuterio-2,4-di-O-methylglucitol 1,2,3,4,5-Penta-O-acetyl-1-deuterioxylitol 1,2,3,4,5,6- Hexa-O-acetyl-1-deuterioglucitol 3-Glcp 3-GlcpA 2-Manp and 3-Manp 6-Manp 6-Glcp 4-Glcp 4-GlcpA 6-Galp 3,6-Glcp 2,3,4-Xylp 2,3,4,6Glcp 19.8 6.0 3.1 1.4 10.7 7.6 10.8 2.6 8.7 2.7 2.2 *Analysis performed by CCRC, University of Georgia, US. 2.3.8. Structural analysis by FTIR Functional group analysis was conducted using FTIR within the frequency range of 4000-400 cm-1 (Fig. 2.9). The stretches found in EtISPFa are typical for fungal polysaccharides, except for glucuronic acid which was more abundant in EtISPFa. The FTIR spectrum of EtISPFa showed a broad OH stretch at 3285 cm-1, sp3 C–H stretches at 2892 cm-1, and 1036 cm-1 for C-O from pyranose ring. The band at 1608 cm-1 indicated a carbonyl C-O that might be due to glucuronic acid identified in high amounts by GC-MS analysis (Tables 2.2 and 2.3) of EtISPFa. There were some weak stretches at 875-890 cm-1 indicative of the presence of β-glucan that was confirmed by 2D-NMR. 66 Fig. 2. 9. FTIR spectrum of EtISPFa. 2.3.9. NMR analyses of EtISPFa For further structural analysis, 1D and 2D NMR was performed. The H-NMR (Fig. 2.10) shows the structural characteristics of a large molecular weight polysaccharide. The negative signal at 4.36 ppm originated from pre-saturation suppression of residual water. The signal at 2.218 ppm corresponded to acetone as internal reference. The peak pattern in the anomeric region between 4.5 and 5.5ppm was complicated and suggested that EtISPFa has a complex and heterogeneous structure. The majority of anomeric signals resonated below 5 ppm, suggesting that most residues were in the β-anomeric configuration. The signal around 1.3 ppm likely originated from Rha and Fuc, small amounts of which were detected in composition and linkage analysis. 67 Fig. 2. 10. 1H-NMR spectrum of EtISPFa. In order to get further details on the complexity of this large polysaccharide, 2D NMR was carried out. The 2D NMR results (Figs. 2.11-2.13) were consistent with a complex, highmolecular weight β-glucan polysaccharide containing a significant amount of glucuronic acid. Several different β-glucose residues were found, mostly in 3-, 6, and 3,6-linkages and as terminal residues, as well as terminal and 4-linked β-glucuronic acid residues (Table 2.4). Although significant signal overlap and broad signals due to high molecular weight precluded definitive sequence determination, we were able to assign several monosaccharide residues and their linkages. The HSQC spectrum (Fig. 2.11) showed two major clusters of overlapping anomeric signals. Both of these were in the β-anomeric region and were further resolved in the COSY spectrum (Fig. 2.12) into about 8 residues each. The H-1 and H-2 chemical shifts indicated that all of these spin systems belonged to Glc or GlcA residues (Hu, Jiang, Huang, & Sun, 2016; Skelton, Cherniak, Poppe, & van Halbeek, 1991). Cluster I had H-1 chemical shifts between 4.8 and 4.7 ppm, and Cluster II had H-1 chemical shifts between 4.6 and 4.5 ppm. The HMBC spectrum (Fig. 2.13) clearly showed 3-bond correlations from Cluster I to carbons between 86 and 88 ppm. This downfield carbon chemical shift is characteristic of C-3 of 3-substituted Glc (Barbosa, Steluti, Dekker, Cardoso, & Da Silva, 2003), demonstrating that the residues whose anomeric protons belonged to Cluster I were attached to O-3 of their neighbors. The HMBC 68 spectrum also showed a strong cross peak correlating the Cluster II protons with carbons resonating between 71 and 72 ppm. The corresponding signals in the multiplicity-edited HSQC spectrum at this carbon chemical shift were from methylene groups, as attested by their negative intensity. This indicated that they originated from the secondary alcohols in the 6-position of 6substituted Glc (Barbosa et al., 2003), and further demonstrated that most of the residues belonging to Cluster II were attached to O-6 of their neighboring Glc residues. However, there was also a weaker HMBC cross peak around 84 ppm, which is indicative of C-4 of 4-substituted Glc or GlcA (Katzenellenbogen et al., 1994). Hence at least one of the residues in Cluster II was attached to O-4 of its neighbor. We were able to assign several of the residues in each of the clusters, based on the correlations found in the COSY and TOCSY spectra, along with the carbon chemical shifts read from the HSQC and HMBC spectra (Table 2.4). Thus, in Cluster I, Residue A was identified as 3,6-disubstituted β-Glc by the downfield displacement of its C-3 (87.7 ppm) and C-6 (71.7 ppm) chemical shifts (Barbosa et al., 2003). Residue B also had a downfield C-3 chemical shift, but its C-6 resonated at 63.7 ppm, showing that Residue B was 3-monosubstituted β-Glc. Residue C was found to have a downfield C-4 (83.9 ppm) and a downfield H-5 (3.90 ppm) that was correlated in HMBC to a C-6 at 177.5 ppm, consistent with 4-substituted β-GlcA (Katzenellenbogen et al., 1994). Residue D was characterized by a relatively downfield H-5 (3.75 ppm) that was correlated in HMBC to a C-6 at 177.8 ppm, indicating unsubstituted β-GlcA (Heiss, Klutts, Wang, Doering, & Azadi, 2009). The final residue we were able to assign in Cluster I was unsubstituted β-Glc (Residue E). As mentioned above, all the residues in Cluster I were attached to O-3 of their neighboring residues. Analysis was performed by CCRC. 69 Table 2. 4. NMR chemical shift assignments* for the residues found in EtISPFa. No. Residue Chemical shift (ppm) 1 2 3 4 5 A 4.80 3.53 3.79 3.53 3.65 3,6-b-Glcp-1®3 105.1 75.6 87.7 71.2 77.7 B 4.80 3.53 3.74 3.53 3.51 3-b-Glcp-1®3 105.1 75.6 87.7 71.2 78.5 C 3.46 3.68 3.71 3.90 4-b-GlcpA-1®3 4.78 105.3 75.8 77.5 83.9 78.2 D 4.75 3.44 3.54 3.64 3.75 b-GlcpA-1®3 105.5 75.9 78.5 73.1 78.7 E 4.72 3.36 3.54 3.39 3.49 b-Glcp-1®3 105.8 76.0 78.6 72.5 78.7 F 4.54 3.54 3.80 3.63 3.51 3-b-Glcp-1®4 105.3 75.8 86.4 72.3 78.5 G 4.52 3.38 3.63 3.45 3.63 6-b-Glcp-1®6 105.5 76.4 77.4 72.6 77.7 H 4.51 3.34 3.48 3.42 3.45 b-Glcp-1®6 105.6 76.0 78.5 72.6 78.7 *Analysis performed by CCRC, University of Georgia, US. 70 HMBC 6 4.20/3.87 71.7 3.91/3.74 63.7 177.5 177.8 3.91/3.74 63.7 3.91/3.74 63.7 4.20/3.87 71.7 3.91/3.74 63.7 A/B-3 A/B-3 A/B-3 A/B-3 A/B-3 C-4 A-6 G-6 Fig. 2. 11. Partial multiplicity-edited 1H-13C-HSQC NMR spectrum of EtISPFa, showing anomeric Clusters I and II and peak assignment detailed in Table 2.4. Red signals are positive and correspond to CH groups, and blue signals are negative and correspond to CH2 groups. 71 Fig. 2. 12. Partial 1H-1H COSY spectrum of EtISPFa. 72 Fig. 2. 13. Partial 1H-13C HMBC spectrum of EtISPFa. The inset represents part of the anomeric region of the HSQC spectrum, showing Clusters I and II. The residues in Cluster II were identified as follows: Residue F clearly showed downfield displacement of C-3 and an HMBC correlation between its C-4 at 71.2 ppm the pair of methylene protons at 3.91 and 3.74 ppm, which resonated in HSQC at 63.7 ppm, proving that Residue F was 3-substituted β-Glc. The HMBC correlation between its H-1 and a signal at 83.9 ppm showed that it was attached to O-4 of Residue D, a 4- substituted GlcA residue. Residue G showed an upfield H-3 chemical shift, indicating that it was not 3-O-substituted. It was not possible to directly ascertain that this residue was 6-O-substituted, but the presence of a cross peak in the HSQC-TOCSY spectrum at 4.54 ppm and 77.7 ppm, which belonged to C-5 next to a glycosylated C-6 (Lundborg & Widmalm, 2011), indicated that at least part of Cluster II 73 consisted of 6-substituted Glc residues. Residue H on the other hand, had a cross peak in HSQCTOCSY at 78.7 ppm, belonging to C-5 next to unglycosylated C6, and thus Residue H was identified as terminal β-Glc residue substituting O-6 of its neighbor and concluded to be the terminal residue of the side chain. In addition to the anomeric signals belonging to Clusters I and II, we observed minor anomeric peaks whose chemical shifts were consistent with α-mannose, in agreement with the presence of terminal and 6- substituted Man in the linkage analysis. However, because of their low abundance, they were not assigned further. Fig. 2. 14. Proposed representative structure of the immuno-stimulatory polysaccharide EtISPFa. The labels A-G correspond to those in Table 2.4. Based on the sugar composition, methylation analysis and NMR spectroscopy, a representative structure incorporating the chemical and spectroscopic data of EtISPFa is proposed in Fig. 2.14. This is an average structure, and the exact sequence of Glc and GlcA residues is presently unknown. For example, linkages such as 3-GlcA and 4-Glc are not accounted for in the proposed structure because we believe that it is part of the heterogeneity of the polysaccharide. To confirm the composition and linkages for a more definite structure or other possible structures, in addition to the already available data more vigorous techniques need to be used. Some of these techniques include capillary electrophoresis, matrix assisted laser 74 desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and HPLCfluorometric/UV detection. In capillary electrophoresis, derivatized monosaccharides are separated based on their electrophoretic mobility (Guo et al., 2013; Volpi et al., 2008). For HPLC analysis, polysaccharides are hydrolyzed, derivatized with 1-phenyl-3-methyl-5pyrazolone (PMP), separated and analyzed with fluorometric or UV detection (Harazono et al., 2011). For analysis on MALDI-TOF-MS, complex polysaccharides are converted to smaller oligosaccharide fragments by partial acidic or enzymatic hydrolysis. The fragments obtained after hydrolysis are separated and subjected to analysis (Xing et al., 2015). In addition to this, since EtISPFa contains more glucuronic acid, it is recommended that uronic acids be reduced with sodium borodeuteride first into neutral monosaccharide before carrying out methylation during GC-MS analysis. To the best of our knowledge, the β–glucan structure of EtISPFa with a high amount of glucuronic acid (10–20 %) is unique. Mushroom polysaccharides containing such high amounts of glucuronic acid are rare (Ruthes, Smiderle, & Iacomini, 2016; Wang et al., 2017). There are two known mushroom polysaccharides that contain high amounts of glucuronic acid are PUP80S1 and PUP60S2, isolated from Polyporus umbellatus, a widely used mushroom in traditional Chinese medicine. PUP80S1 is an 8.8 kDa β–glucan containing about 8.5 % uronic acid (He, Zhang, Wang et al., 2016; He, Zhang, Zhang, Linhardt, & Sun, 2016), while PUP60S2 is a 14.4 kDa β–glucan containing about 22.3 % uronic acid (He, Zhang, Wang et al., 2016; He, Zhang, Zhang et al., 2016). Polysaccharides containing uronic acid, rhamnose and mannose are known to display significant anti-oxidant activity (He, Zhang, Wang et al., 2016; He, Zhang, Zhang et al., 2016; Wang et al., 2017). Therefore, it would be of interest to determine whether EtISPFa possesses such biological activity. 75 2.4. Conclusion This is the first description on the isolation of an immuno-stimulatory polysaccharide from E. tinctorium. Using Sephadex LH-20, DEAE-Sephadex, Sephacryl S-500 HR, and HPLC BioSEC5, an immuno-stimulatory polysaccharide (EtISPFa) was successfully isolated with weight average molecular weight (Mw) of 1354 kDa from the water extract of E. tinctorium. EtISPFa is a complex and unique β-glucan polysaccharide rich in glucuronic acid (10–20 %). Besides glucose and glucuronic acid, the minor sugars present include mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), rhamnose (3.1 %), fucose (1.8 %) and arabinose (0.2 %). The combined results from GC–MS and NMR analyses reveal that EtISPFa is made up mostly of a backbone consisting of β-(1→3)-Glcp residues and β-(1→4)-GlcpA residues, with branching points at β(1→3, 6)-Glcp to which are attached short β-(1→6)-linked glucooligosaccharide side chains. EtISPFa also has terminal xylose, fucose, rhamnose, and arabinofuranose. Besides TNF-α, EtISPFa was found to significantly enhance IL-6, MIP-2, G-CSF, GM-CSF, LIF, MCP-1, MIP1α, MIP-1β and RANTES in macrophage cells. Further biological characterization of EtISPFa is required to determine its mechanism of action and whether it exerts immuno-stimulatory activity in animals. Appendix A. Supplementary data Supplementary material related to this chapter can be found in Appendix A. 2.5. References Barbosa, A. M., Steluti, R. M., Dekker, R. F., Cardoso, M. S., & Da Silva, M. C. (2003). Structural characterization of Botryosphaeran: a (1→ 3; 1→ 6)-β-d-glucan produced by the ascomycetous fungus, Botryosphaeria sp. Carbohydrate research, 338(16), 1691–1698. De Ruiter, G. A., Schols, H. A., Voragen, A. G., & Rombouts, F. M. (1992). Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high- performance anion-exchange chromatography using methanolysis combinaed with TFA hydrolysis is superior to four other methods. 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Structure elucidation and antioxidant activity of a novel polysaccharide from Polyporus umbellatus sclerotia. International journal of biological macromolecules, 82, 411–417. He, P., Zhang, A., Zhang, F., Linhardt, R. J., & Sun, P. (2016). Structure and bioactivity of a polysaccharide containing uronic acid from Polyporus umbellatus sclerotia. Carbohydrate polymers, 152, 222–230. Heiss, C., Klutts, J. S., Wang, Z., Doering, T. L., & Azadi, P. (2009). The structure of Cryptococcus neoformans galactoxylomannan contains β-D-glucuronic acid. Carbohydrate research, 344(7), 915–920. Hu, T., Jiang, C., Huang, Q., & Sun, F. (2016). A comb-like branched β-d-glucan produced by a Cordyceps sinensis fungus and its protective effect against cyclophosphamide- induced immunosuppression in mice. Carbohydrate polymers, 142, 259–267. Javed, S., Li, W. M., Zeb, M., Yaqoob, A., Tackaberry, L. E., Massicotte, H. B., et al. (2019). 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Mushroom heteropolysaccharides: A review on their sources, structure and biological effects. Carbohydrate polymers, 136, 358– 375. Skelton, M. A., Cherniak, R., Poppe, L., & van Halbeek, H. (1991). Structure of the De-Oacetylated glucuronoxylomannan from Cryptococcus neoformans serotype D, as determined by 2D NMR spectroscopy. Magnetic resonance in chemistry, 29(8), 786–793. Smith, A., Javed, S., Barad, A., Myhre, V., Li, W. M., Reimer, K., et al. (2017). Growthinhibitory and immunomodulatory activities of wild mushrooms from north-central British Columbia (Canada). International journal of medicinal mushrooms, 19(6), 485–497. Volpi, N., Maccari, F., & Linhardt, R. J. (2008). Capillary electrophoresis of complex natural polysaccharides. Electrophoresis, 29(15), 3095–3106. Wang, Q., Wang, F., Xu, Z., & Ding, Z. (2017). Bioactive mushroom polysaccharides: A review on monosaccharide composition, biosynthesis and regulation. Molecules, 22, 955. Xing, X., Cui, S. W., Nie, S., Phillips, G. O., Goff, H. D., & Wang, Q. (2015). Study on Dendrobium officinale O-acetyl-glucomannan (Dendronan®): Part V. Fractionation and structural heterogeneity of different fractions. Bioactive carbohydrates and dietary fibre, 5(2), 106–115 78 Appendix A. Supplementary data DEAE-Sephadex Optimization Multiple buffers including piperazine (pH 5.3 and 9.73), bis-Tris (pH 6.4), propane (pH 6.8), Tris (pH 8.1), and L-Histidine (pH 6.2) were first assessed to determine the optimum buffer for use in the purification. For the optimum buffer, the bioactive compound should retain its activity whether it binds or does not bind to the resin. If it does not bind, it would be present in the flowthrough. On the other hand, if it binds to the resin, then it would be eluted (present in elution buffer) by an increasing concentration of salt. For most of the buffers except L-Histidine, the bioactivity was present in both the flow-through and eluent and, for some, the bioactivity was lower than the preload. When using L-Histidine (pH 6.2) as buffer as shown in Fig. 3B, the bioactive compound was bound to DEAE-Sephadex resin and was then eluted by high salt concentration (1 M NaCl). In addition, the bioactivity was fully retained in the eluent. Therefore, L-Histidine (pH 6.2) was selected as the buffer of choice. 79 Fig. S 1. Optimization of conditions for anion exchange chromatography using multiple buffers. TNF-α stimulation by 2A using bis-Tris at pH= 6.4 (A), Tris at pH= 8.1 (B), propane 1,2-Diamino buffer at pH= 6.8 (C), piperazine at pH= 5.33 (D), and piperazine at pH= 9.73 (E). Preload, flow through and elution were assessed for immuno-stimulatory activity. Medium and water were used as negative controls. Error bars represent S.D. 80 Fig. S 2. Purification of the immuno-stimulatory polysaccharide from E. tinctorium (EtISPFa) using Sephacryl S-500 size exclusion chromatography. Fractions collected were assessed for immuno-stimulatory activity. Medium and water were used as negative controls. LPS was used as positive control. 81 Fig. S 3. Estimating the peak maxima molecular weight (Mp) of EtISPFa using HPLC BioSEC5. (A) Overlay spectra of dextran standards (25-2000 kDa) with EtISPFa. Samples were run in water at a flowrate of 0.4 mL/min. RID detector was used. (B) Retention times (min) of dextran standards were plotted against their molecular weight (kDa). 82 Fig. S 4. The GC-MS chromatograms from glycosyl composition analysis using TMS derivatization. Fig. S 5. GC-MS chromatogram resulting from glycosyl linkage analysis of neutral and uronic acid residues. Glycosyl linkage analysis was performed by partially methylated alditol acetates (PMAAs). 83 Table S 1. Calculating the number (Mn) and weight average molecular weight (Mw) of EtISPFa Concentrati on (g) Rt (min) MW (kDa) 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 0.00002 4.5 4.7 4.9 5.1 5.3 5.5 5.7 5.9 6.1 6.3 6.5 6.7 6.9 1737.397 1664.833 1592.269 1519.706 1447.142 1374.578 1302.015 1229.451 1156.887 1084.324 1011.760 939.196 866.633 MW or Mi (g/mol) 1737397.2 1664833.5 1592269.8 1519706.1 1447142.5 1374578.8 1302015.1 1229451.4 1156887.7 1084324.0 1011760.4 939196.7 866633.0 Moles ni niMi wi wiMi 1.1511E-11 1.2013E-11 1.2561E-11 1.316E-11 1.382E-11 1.455E-11 1.5361E-11 1.6267E-11 1.7288E-11 1.8445E-11 1.9768E-11 2.1295E-11 2.3078E-11 6.93221E+12 7.23436E+12 7.56404E+12 7.92522E+12 8.32261E+12 8.76196E+12 9.25028E+12 8.76196E+12 8.32261E+12 7.92522E+12 7.56404E+12 7.23436E+12 6.93221E+12 ∑=1.02731E+ 14 1.2044E+19 1.2044E+19 1.2044E+19 1.2044E+19 1.2044E+19 1.2044E+19 1.2044E+19 1.07724E+19 9.62832E+18 8.5935E+18 7.653E+18 6.79448E+18 6.00768E+18 1.33757E+20 0.090043621 0.090043621 0.090043621 0.090043621 0.090043621 0.090043621 0.090043621 0.080536859 0.071983488 0.064246937 0.057215534 0.050797075 0.044914759 156441.5393 149907.6425 143373.7457 136839.849 130305.9522 123772.0554 117238.1586 99016.15954 83276.81712 69664.50198 57888.41242 47708.44643 38924.61416 1354357.894 Molecular weight peak maxima (Mp) = 1302015 g/mol. Weight average molecular weight (Mw) = 1354357 g/mol. Number average molecular weight (Mn) = 1302015 g/mol. (Mn = ΣniMi/Σni) Polydispersity index = 1.04 (PDI = Mw/Mn) 84 Table S 2. Quantitative estimation of material recovered from each purification step Processing step Aqueous extract (E3) Sephadex LH-20 SEC 25 mL column 85 mL column 400-500 mL column Sephadex DEAE AEC 70 mL column 100 mL column 150 mL column 400 mL column 800-1000 mL column Sephacryl S500 HR SEC (5x) HPLC BioSEC-5 (100-120) Starting material 300 g Amount obtained 3.7 g % Yield 1.2 % 8.8 mg 63 mg 250 mg 4 mg 12.8 mg 50 mg 45 % 20 % 20 % 18 mg 27 mg 40 mg 100 mg 400 mg 500 mg 650 mg 100 mg 60-70 mg 6.8 mg 8 mg 13.7 mg 30 mg 120 mg 150 mg 171 mg 12 mg 3 mg 37 % 29 % 34 % 30 % 30 % 30 % 26 % 12 % 5% 85 Chapter 3: Structural elucidation of an anti-proliferative polysaccharide from the fungus Echinodontium tinctorium ABSTRACT An anti-proliferative polysaccharide (EtGIPL1a) was isolated from methanol extract of the fungus Echinodontium tinctorium. EtGIPL1a has an estimated weight average molecular weight of 275 kDa and is composed of glucose (54.3%), galactose (19.6%), mannose (11.1%), fucose (10.3%), glucuronic acid (4%) and rhamnose (0.6%). It has multiple glycosidic linkages, with 3Glcp (28.9 %), 6-Glcp (18.3 %), 3,6-Glcp (13 %), 4-GlcpA (9.2 %), 6-Galp (3.9 %), 2,6-Galp (2.6 %), 3-Fucp (2.5 %), 6-Manp (2.4 %) being the most prominent, and unsubstituted glucose (15.3 %), mannose (1.3 %) and fucose (0.9 %) as major terminal sugars. NMR analysis showed that EtGIPL1a has a backbone containing mostly 3-substituted b-glucopyranose with 4substituted glucopyranosyluronic acid. EtGIPL1a showed anti-proliferative activity against multiple cancer cell lines with IC50 ranging from 50.6-1446 nM. EtGIPL1a induced apoptosis in U251 glioblastoma cells and caused subG0 phase arrest with significant DNA fragmentation. 3.1. Introduction It is believed that structural complexity of polysaccharides contributes to its bioactive potential. Medicinal mushrooms have demonstrated anti-proliferative effects from multiple studies (Barad et al., 2018; Blagodatski et al., 2018; Shnyreva, Shnyreva, Espinoza, Padrón, & Trigos, 2018; Panda et al., 2020; Souilem et al., 2017; Yaqoob et al., 2020; Zeb & Lee, 2021). Fungal polysaccharides have been shown to possess diverse bioactivities and have been studied extensively, especially from known medicinal and edible mushrooms (Ferreira et al., 2015; He et al., 2020; Venturella, Ferraro, Cirlincione, & Gargano, 2021). Examples include lentinan from Lentinula edodes (Dubey et al., 2019), schizophyllan from Schizophyllum commune (Zhong et 86 al., 2015), Maitake D-fraction from Maitake (Zhuang, & Wasser, 2004), polysaccharide K (PSK or Krestin) from Coriolus versicolor (Blagodatski et al., 2018), ganoderan from Ganoderma lucidum (Wang, Gou, Xue, & Liu, 2019), and pleuran from Pleurotus species (Urbancikova et al., 2020); all have been shown to possess anti-cancer activity. To this end, extensive investigations have been conducted on the chemistry of fungal polysaccharides in relation to their bioactivities (Wang, Wang, Xu, & Ding, 2017). Studies have shown that fungal polysaccharides are structurally complex molecules due to their monosaccharide distribution, degree of branching, large molecular size and complex linkages. Based on their monosaccharide content, they can be homo- or heteropolysaccharide with respective bioactivities. For example, homopolysaccharide from Agaricus bisporus (Pires et al., 2017), Lentinula edodes (Ya, 2017), Pleurotus eryngii (Ma et al. 2014), and Ganoderma lucidum (Yang, Yang, Zhuang, Qian, & Shen, 2016) have shown anti-proliferative effects. Heteropolysaccharide from Lentinula edodes (Wang et al., 2017), Pleurotus eryngii (Ren, Wang, Guo, Yuan, & Yang, 2016), and Flammulina velutipes (Chen et al., 2018) have also shown anti-proliferative potential. An anti-inflammatory and immuno-stimulatory polysaccharide from E. tinctorium has already been isolated (Javed et al., 2019; Zeb et al., 2021), but its potential anti-proliferative role against cancer cells has yet to be explored. The aim of the present study was to purify and characterize an anti-proliferative polysaccharide from E. tinctorium. As in previous studies, E. tinctorium was sequentially extracted using 80% ethanol followed by 50% methanol. The methanol extract was phase separated and assessed for anti-proliferative activity using the cytotoxic MTT assay. Our results indeed showed that the methanol extract of E. tinctorium has anti-proliferative activity against HeLa human cervical cancer cells. The methanol extract from E. tinctorium was subjected to 87 multiple chromatographic steps (Sephadex LH-20, DEAE-Sephadex, Sephacryl S-500 HR, and HPLC BioSEC-3 chromatography) for purification of the anti-proliferative compound. Structural analyses were performed which included gas chromatography-flame ionization detector (GCFID) to determine monosaccharides content, gas chromatography-mass spectrometry (GC-MS) to determine the glycosidic linkages, Fourier transform infrared (FTIR) spectroscopy for identification of functional groups, and finally, NMR analyses to determine the constitution and monosaccharide sequence of the EtGIPL1a polysaccharide. Growth-inhibitory effect of EtGIPL1a was assessed on multiple cancer cell lines and details on mechanism of induction of apoptosis in U251 glioblastoma cells was explored. 3.2. Materials and methods 3.2.1. Materials, reagents and cell lines All the reagents were of analytical grade. Eagle’s Minimal Essential Medium was from LONZA (Walkersville, Maryland, USA). 3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO) and the dextran standards (T1, T5, T12, T25, T50, T80, T150, T270, T410) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Life Technologies Inc. (Waltham, Massachusetts, USA). Sephadex™ LH-20 resin, DEAE-Sephadex, and HiPrep 26/60 Sephacryl™ S-500 HR pre-packed columns were purchased from GE Healthcare (Chicago, IL, USA). HPLC BioSEC-3 column and guard column were purchased from Agilent (Santa Clara, CA, USA). All cell lines were obtained from American Type Culture Collection. All cells were maintained in Eagle’s Minimal Essential Medium except Panc-1 which was maintained in Dulbecco’s Modified Eagle Medium (LONZA). 88 3.2.2. Collection and extraction of the mushroom E. tinctorium conks were collected from hemlock trees (Tsuga heterophylla) in Terrace (CL103) and Smithers (CL37), BC, Canada, in August 2014 and 2015, respectively. Voucher specimens for these collections were deposited at the University of Northern British Columbia, Canada. The specimens, previously confirmed using morphological and molecular techniques (Javed et al., 2019), were dried in a hot air oven (55 ºC, 24-48 h), cut into smaller pieces using a saw machine, and ground to fine powder using a hammer mill. Powdered mushrooms (300 g) were sequentially extracted with 80% ethanol (1.5 L, 65 ºC, 3 h). The extract was vacuum filtered through Whatman filter paper No. 3 and the filtrate was designated 1A. The residue was further extracted with 50% methanol (1.5 L, 65 ºC, 3 h). The methanol extract was filtered and the filtrate was designated 1B. Crude extract 1B was concentrated, lyophilized, and filter sterilized before assessment for anti-proliferative activity. 3.2.3. Anti-proliferative assay Anti-proliferative activity of 1B was assessed by % cell viability using the cytotoxic MTT assay. HeLa cells were plated (96 well, 1.5 x 104 cells/well), and after 22-24 h, cells were treated with crude methanol extract (1B) and phase separated aqueous layer (L1) for 48 h at concentrations ranging from 0.1-1 mg/mL. Cells were observed for morphological changes under a microscope and incubated with 50 µL of MTT solution (3 h, 37°C). Medium was then removed from the wells and 150 µl DMSO was added and incubated for another 5 min. Different purple color intensities of formazan were observed, indicative of dose-dependent anti-proliferative response. Formazan purple color was quantified by determining the absorbance of samples at 570 nm using Bio-Tek’s Synergy-2 multi-plate reader. The purified polysaccharide EtGIPL1a was assessed against multiple cancer cell lines including HeLa, SW-480, U87, U251, DU145, 89 HCT116, Panc-1, MD-MB-231, MCF-7, and SKOV-3. Cell viability was assessed using MTT assay as described above. 3.2.4. Purification of anti-proliferative polysaccharide from E. tinctorium Phase separation was performed by dissolving 500 mg of 1B in water and partitioning with chloroform, that resulted in two distinct layers: aqueous (L1) and organic (L2). L1 was then subjected to a 100 mL Sephadex LH-20 designated column-1 (80 mg L1,1 mL/min, 3 mL fraction size, 2 CV). Collected fractions containing anti-proliferative activity were pooled, lyophilized and subjected to DEAE-Sephadex (designated column-2) using L-Histidine as the running buffer (pH = 5.7-6.4). The column was first equilibrated (L-Histidine buffer, 2 CV, 1 mL/min), followed by sample application (500 mg). Initially, L-Histidine buffer (2 CV) was allowed to run through the column to obtain flow-through (FT). Elution buffer (1 M NaCl in LHistidine buffer, 2.5 CV) was added and eluent was collected. The FT and eluent were concentrated, dialyzed (MWCO 3500 kDa), lyophilized, filter sterilized and tested for antiproliferative activity. The bioactive eluent 2A from DEAE-Sephadex was then subjected to Sephacryl S-500 HR SEC designated as column-3. Column-3 was equilibrated (4 CV, 150 mM NaCl, 1.3 mL/min) and injected with 100 mg 2A (2 mL sample loop). Fractions collected (10 ml fraction size, 2.5 CV) were assessed for anti-proliferative activity, carbohydrate (phenol-sulfuric acid method) and protein contents (BCA protein assay, Waltham, MA, USA). The bioactive fractions from column-3 were pooled, dialyzed, lyophilized and designated as L1a. 3.2.5. Molecular size distribution and ultrapurification using HPLC L1a was subjected to HPLC size-exclusion chromatography for purification and molecular size estimation. The molecular size was estimated by running standard T-series Dextrans. Initially, the HPLC BioSEC-3 column (designated column-4) (Agilent BioSEC-3, 3 µm, 100 A, 90 7.8 × 300 mm, Guard column Agilent BioSEC-3, 3 µm, 100 A, 7.8 × 50 mm) was equilibrated and then L1a was injected (5-10 uL, 1.2 mL/min, Water) through an autosampler. A Refractive Index Detector (RID) was used for analysis. The HPLC profile for L1a showed two peaks retained at 5.835 and 7.941 mins. These peaks were fraction collected (300-400 runs), lyophilized, and assessed for anti-proliferative activity. The bioactive Peak 1 was subsequently named EtGIPL1a. 3.2.6. Monosaccharide composition analysis Monosaccharide content of EtGIPL1a was determined by GC-MS. EtGIPFa (330 µg) and internal standard inositol (20 µg) were hydrolyzed, acetylated and derivatized using the same approach as previously described (Zeb et al., 2021). The TMS methyl glycoside derivatives were analyzed by GC-MS (Agilent 7890A GC, 5975C MSD) using a Supelco Equity-1 fused silica capillary column (30 m ´ 0.25 mm ID). 3.2.7. Methylation and linkage analysis Glycosyl linkage analysis was conducted as previously described (Zeb et al., 2021). One mg of EtGIPL1a was suspended in 200 µL of DMSO, stirred, permethylated, hydrolyzed, reduced and acetylated to yield partially methylated alditol acetates (PMAAs). The PMAAs were then analyzed on an Agilent 7890A GC connected to a 5975C MSD (EI ionization source) using a 30 m Supelco SP-2331 bonded phase fused silica capillary column. 3.2.8. Structural elucidation by spectral analysis EtGIPL1a was subjected to functional group analysis by FTIR (Bruker ATR-FTIR spectrophotometer, Billerica, MA, USA) with a detection wave range of 4000-400 cm−1. Twenty two scans were obtained and an IR spectrum was generated using OPUS software. 91 For further structural analysis, NMR was conducted as previously described (Zeb et al., 2021). 1D proton NMR (H1-NMR) and 2D NMR including 1H-1H-correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), 1H-13C-NMR heteronuclear single quantum correlation spectroscopy (HSQC), HSQC-TOCSY, and heteronuclear multiple bond correlation (HMBC) were carried out on an Agilent Inova 600 MHz NMR. 3.2.9. Growth-inhibition in U251 glioblastoma cells EtGIPL1a was tested for growth inhibition in U251 cells through MTT assay as mentioned in section 2.3. Morphological changes induced by EtGIPL1a were observed at different doses and time intervals under a microscope. In order to further confirm whether the changes were due to differentiation, western blot was carried out and the expression levels of glial fibrillary acidic protein (GFAP) differentiation marker as well as vimentin were determined. For first two biological replicates, cells were plated at a density of 30 x 104 cells/well and 50 x 104 cells/well for biological replicate 3, incubated overnight and treated at 12.5 µg/mL and 6.25 µg/mL of EtGIPL1a for 48 h. Cell lysates were collected and protein content was estimated. For immunoblot analysis, 15 ug of protein (21 ug for biological replicate 3) was loaded and resolved on 13.3 % of SDS-PAGE gel (200 V, 15 min) and transferred to a nitrocellulose membrane (100 V, 1 h, 4ºC). The protein was blocked with skim milk (5% w/v, 1 h, RT), incubated overnight with primary antibody (1:1000), washed, and incubated with secondary antibody (1:2000, 1 h, RT); it was then imaged with Pico ECL solution and visualized with the FluorChem Q system (ProteinSimple, CA, USA) alongwith AlphaView Q software (ProteinSimple). The primary antibodies that were used include vimentin (1:1000), GFAP (1:1000), LC3B (1:1000), and cleave 92 caspase 3 (1:1000) whereas the secondary antibodes included thioredoxin (1:2000) and GAPDH (1:2000). 3.2.10. Flow cytometry analysis Flow cytometry analysis was conducted on SW480 and U251 cells. SW480 cells were plated at a density of 5 x 105 cells/well in a 6-well plate; after 24 h, cells were treated with 0.4 mg/mL of filter-sterilized EtGIPL1a for 48 h. In contrast, U251 cells were plated at a density of 20 x 104 cells/well and treated with 27nM of EtGIPL1a. Water was used as a negative control. After 48 h of treatment, cells were observed for any changes in morphology. Each well treated with sample and control was trypsinized, centrifuged, and washed with PBS twice. Live cells were then double stained with PE Annexin-V and 7-AAD supplied with the Apoptosis detection kit I (BD Pharmingen), kept in dark and filtered into a sieve cap glass test tube. Stained cells were then analyzed by BD FACSMelody cell sorter flow cytometer (BD Biosciences) using BD FACSChorus software (V 1.0). For apoptosis, a total of 10,000 events were recorded for each sample. The percentage of viable and apoptotic cells was calculated from FACS Chorus version 1.0 (Becton Dickinson & Company, USA). For cell cycle analysis, a total of 40,000 events were recorded for each sample. The percentage of cells residing in each G1, S and G2/M phase of cell cycle were calculated from Flowjo software version 10.7.2 (Becton Dickinson & Company, USA). 3.3. Results and Discussion 3.3.1. Extraction and assessment of methanolic extract from E. tinctorium for antiproliferative activity The powdered E. tinctorium was sequentially extracted using the following solvents: 80 % ethanol, 50 % methanol, water and 5 % sodium hydroxide. We found that only the 50 % 93 methanol extracts exhibited anti-proliferative activity against HeLa human cervical cancer cells (Smith, 2017). Therefore, I aimed to purify, identify and characterize the anti-proliferative compound(s) from E. tinctorium. Fig. 3.1A summarizes the scheme taken to obtain the methanolic extract 1B which was then subjected to chloroform extraction. The aqueous phase L1 obtained, as well as the crude methanol extract 1B were lyophilized and assessed for antiproliferative activity. As shown in Fig. 3.1B, both 1B and L1 displayed dose-dependent antiproliferative effect on HeLa cells. Fig. 3. 1. Chemical extraction of E. tinctorium and assessment of the ethanol extract 1B for antiproliferative activity against HeLa cells. (A) Chemical extraction scheme to obtain ethanol extract from E. tinctorium. (B) Dose-dependent anti-proliferative MTT assay shows inhibition in HeLa cells by 1B extract and L1 from 0.1-1 mg/mL. Error bars represent S.D. Results shown are representative from three biological replicates. 3.3.2. Purification of anti-proliferative polysaccharide (EtGIPL1a) from E. tinctorium The overall scheme adopted for the purification of the anti-proliferative polysaccharide from E. tinctorium (EtGIPL1a) is shown in Fig. 3.2A. L1 was first subjected to Sephadex LH-20 size- 94 exclusion chromatography designated as column-1. As shown in Fig. 3.2B, the major antiproliferative activity was found in fractions 1-7, suggesting that the anti-proliferative compound has a relatively large molecular weight; these fractions 1-7 from column-1 were then pooled, concentrated, lyophilized, and resuspended in water. It was then subjected to column-2, DEAESephadex anion exchange chromatography using L-Histidine (pH 6.2) as the buffer of choice. Optimized conditions for DEAE-Sephadex were obtained after analyzing the purification profiles with multiple buffers (Fig. S6). The flow-through and eluent from column-2 were concentrated, dialyzed, lyophilized, filter-sterilized, and assessed for anti-proliferative activity. As shown in Fig. 3.2C, the eluent referred to as 2A (but not the flow-through from DEAE-Sephadex) contains the anti-proliferative activity. This result suggested the presence of acidic groups on the compound that were effectively associated with positively charged DEAE-Sephadex resin and were eluted upon addition of salt to the mobile phase. Eluent 2A was then subjected to column-3, Sephacryl S-500 high resolution size-exclusion chromatography. As shown in Fig. 3.3A, the anti-proliferative activity was retained in fractions 25-28 (designated L1a) and 29-31 (designated L1b). The fractions collected from column-3 were also assessed for carbohydrate (CHO) and protein contents. Results in Fig. 3.3B show that the major anti-proliferative activity L1a correlated strongly with carbohydrate content but not with the protein content. In contrast, the L1b activity appears to correlate better with the protein content (Fig. 3.3C). 95 Fig. 3. 2. Purification of the anti-proliferative polysaccharide (EtGIPL1a) from E. tinctorium. (A) Summary of the purification scheme used. (B) 1B extract from E. tinctorium was purified using Sephadex LH-20 size-exclusion chromatography (column-1). Active fractions (F1-7) were pooled, lyophilized and ran through DEAE-Sephadex anion exchange chromatography (column2) as shown in (C). The eluent (2A) showed anti-proliferative activity while the flow-through (FT) had no activity. Error bars represent S.D. Results shown are representative from three biological replicates. 96 Fig. 3. 3. Purification of the anti-proliferative polysaccharide from E. tinctorium (EtGIPL1a) using Sephacryl S-500 (column-3). Collected fractions were assessed for cell viability (A), carbohydrate (B) and protein content (C). Error bars represent S.D. and results are representative from three separate experiments. 97 3.3.3. Estimation of molecular size and purification by HPLC The major bioactive fraction L1a was further purified by HPLC using Agilent BioSEC-3 designated as column-4. As shown in Fig. 3.4A, the HPLC BioSEC-3 profile of L1a shows two peaks referred to as Peak1 and Peak 2. These were subjected to further purification by fraction collection. The collected Peak 1 and Peak 2 exhibited purity as shown in Fig. 3.4B and 3.4C respectively. Peak 1, designated as EtGIPL1a, has a retention time of 5.633 mins while Peak 2 has a retention time of 7.068 mins. Fig. 3.4D shows that both EtGIPL1a and Peak 2 dosedependently inhibited proliferation of HeLa cells, with notable stronger effect by EtGIPL1a. Using dextran standards on BioSEC-3 column, a standard curve was obtained (Fig. S7). Based on the standard curve, the peak maxima molecular weight (Mp) of EtGIPL1a was estimated to be 216271 g/mol (Da) or 216 kDa (Fig. S7 and Table S3). Further calculations were performed to determine the number (Mn) and weight average molecular weight (Mw) of EtGIPL1a. As shown in Table S3, the Mn and Mw of EtGIPL1a was calculated to be 232 kDa and 275 kDa respectively. The polydispersity index was calculated to be 1.18. 98 Fig. 3. 4. HPLC BioSEC-3 full elution profile of (A) L1a (B) Peak 1 (EtGIPL1a), and (C) Peak 2. The collected two peaks in (B) and (C) were assessed for anti-proliferative activity as shown in (D). 99 3.3.4. Assessing EtGIPL1a against a panel of human cancer cell lines To determine whether EtGIPL1a has a broader anti-proliferative effect, we assessed it at various concentrations against a panel of human cancer cell lines. Results from the dosedependent experiments summarized in Table 3.1 show that EtGPLL1a indeed exhibited strong anti-proliferative effect on the panel of human cancer cell lines. All the experiments shown in Table 3.1 were conducted by Dr. Lee. The purified polysaccharide was most effective against DU145 prostate cancer (IC50 = 50.6 nM), HCT116 colon cancer (IC50 = 122.2 nM) and the two glioblastoma cancer cells, U87 (IC50 = 136 nM) and U251 (IC50 = 193.2 nM). It was also effective against HeLa cervical cancer (IC50 = 287.9 nM), Panc-1 pancreatic (IC50 = 343.2 nM), MD-MB-231 breast cancer (IC50 = 514.3 nM), SKOV3 ovarian cancer (IC50 = 634.2 nM), and MCF-7 breast cancer cells (IC50 = 839.5 nM). EtGIPL1a is anti-proliferative on SW480 human colorectal cancer cells with an IC50 of 1446 nM, which is 10-fold lower than on HCT116 colon cancer cells. While both HCT116 (KRASG13D) and SW480 (KRASG12V) cells carry mutated KRAS, they have different activated cellular pathways that are related to proliferation. For example, HCT116 cells had high basal level mTORC1 activity while SW480 cells displayed low basal level mTORC1 activity (Thomas et al., 2014). SW480 cells showed hyperactivated mTORCs and AKT pathways leading to increased proliferation upon autophagy inhibition which was not observed in HCT116 cells (Lauzier et al., 2019). Whether the differential antiproliferative effects of EtGIPL1a are due to these differentially active pathways in the cell lines will need further investigations. 100 Table 3. 1. IC50 of EtGIPL1a against human cancer cell lines Cell lines Types *IC50 (nM) DU145 Human prostate cancer 50.6 HCT116 Human colon cancer 122.2 U87 Human glioblastoma 136 U251 Human glioblastoma 193.2 HeLa Human cervical cancer 287.9 Panc-1 Human pancreatic cancer 343.2 MD-MB-231 Human breast cancer 514.3 SKOV-3 Human ovarian cancer 634.2 MCF-7 Human breast cancer 839.5 SW480 Human colon cancer 1446 *IC50: the data shown is an average taken from two independent experiments. 3.3.5. Growth inhibition in U251 glioblastoma cells 3.3.5.1. Morphological changes in glioblastoma cell lines (U251) EtGIPL1a suppressed the growth of U251 cells in-vitro in a dose-dependent manner and induced morphological changes at lower doses. U251 cell morphology transformed from flat oval shape to more stellate shape with thin long processes extending from the cells as shown in Fig. 3.5. The experiments shown in Fig. 3.5 were conducted by Dr. Lee. 101 Fig. 3. 5. Morphological changes induced by EtGIPL1a after treatment at 13.7-220 nM for 48 h in U251 cells. 3.3.5.2. Flow cytometry analysis of EtGIPL1a EtGIPL1a induced apoptosis in U251 cells at a concentration of 27 nM for 24-48 h (as shown in Fig. 3.6B & 3.7). A significant increase in number of cells was observed after treatment with EtGIPL1a as compared to water (negative control) and resveratrol (positive control) (Fig. 3.7). The apoptosis was not significant in SW480 cells even at very high dose (1.76 µM) when compared to U251 cells, suggesting that EtGIPL1a might be causing growth-inhibition in SW480 cells through mechanisms other than apoptosis. 102 Fig. 3. 6. Flow cytometry analysis for apoptosis induced by EtGIPL1a at 1.76 µM for 48 h in SW480 cells (A) and at 27 nM for 24 h in U251 cells (B). Error bars represent standard deviation and results shown are representative from three biological replicates. One-way ANOVA was used for statistical analysis. * shows p = 0.0046. EtGIPL1a showed antiproliferative effects in multiple cancer cells including U251 glioblastoma cells. Glioblastomas (GB) are generally considered very aggressive brain tumors with higher malignancy. Although quite rare, due to its malignancy, GB have been categorized as grade IV astrocytic tumor by World Health Organization (Louis et al., 2007). There are quite a few treatment options for the management of GB including surgery, radiotherapy and chemotherapy with Temozolomide. 103 Fig. 3. 7. Apoptosis induced by treatment with 27 nM EtGIPL1a compared to 40 µM resveratrol for 48 h in U251 cells. Error bars represent standard deviation. The plots are representative of n=3. One-way ANOVA was used for statistical analysis. ** represents p = 0.0011 and * shows p= 0.0102. The development of tumors is associated with dedifferentiation and uncontrolled growth of cells (Linskey & Gilbert, 1995), whereas normal cells are well differentiated and have a controlled differentiation, which helps us understand their morphology. EtGIPL1a caused growth-inhibition and induced differentiation in U251 glioblastoma cells. The morphological changes induced by EtGIPL1a suggested a well differentiated astrocyte-like morphology. It is known from the literature (Bovolenta et al., 1984) that well differentiated astrocytes appear stellate shape with long processes, similar to what was observed with EtGIPL1a-treated U251 cells. It is believed that if cancer cells are well differentiated, the prognosis for cancer treatment is better. Astrocytes have an increased content of glial fibrillary protein (GFAP), which is exclusively expressed in mature astrocytes (Eng, 1985). A strategy proposed by researchers for limiting glial tumors is by inducing differentiation (Linskey & Gilbert, 1995). 104 Fig. 3. 8. Cell cycle analysis of EtGIPL1a at 27 nM for 48 h on U251 cells (A & B) and % cell population in G1, S, G2/M and subG0 phases of cell cycle (C). Error bars represent S.D. Results shown are representative from three biological replicates. One-way ANOVA was used for statistical analysis. * indicates p < 0.0001. Cell cycle analysis of EtGIPL1a showed a significant increase in the sub G0 population (Fig. 3.8) which is indicative of DNA fragments. The presence of high cell fractions in subG0 is indicative of cell death via apoptosis. There are few compounds from natural products that have shown cytotoxic potential against GB. A polysaccharide peptide (GL-PP) from Ganoderma lucidum has shown growth inhibition in U251 cells through G0/G1 cell cycle arrest and apoptosis; GL-PP has shown to induce the expression of caspase-3 in U251 cells (Wang et al., 2018) which is considered as an apoptotic marker. Another polyphenolic compound (hispolon) isolated from Phellinus linteus has been shown to induce apoptosis and G2/M phase cell cycle arrest in U87MG glioblastoma cells (Arcella et al., 2017). Cytotoxic triterpenoids from Antrodia camphorata (Li et al., 2020), polysaccharides from medicinal plants like Angelica sinensis (APs) (Zhand et al., 2017), Cyclocarya paliurus (CPP) (Du et al., 2020) and Aconitum coreanum (ACP1) (Sun et al., 2018) have also shown to induce apoptosis in U87MG and U251 glioblastoma cell lines. 105 3.3.5.3. Molecular markers involved in differentiation and apoptosis Molecular markers for differentiation as well as apoptosis in U251 cells were assessed. Vimentin is a cytoskeleton dedifferentiation marker whereas GFAP indicates differentiation in astrocytes. Both protein markers were inconclusive due to the fact that the primary antibodies underwent non-specific interactions. LC3B was also assessed to determine the possibility of autophagy leading to apoptosis. Unfortunately, LC3B was not expressed to a greater extent. Another marker, cleaved caspase 3, one of the important markers for apoptosis was found to be highly expressed in U251 cells after treatment with EtGIPL1a (Fig. 3.9). The western blot analysis was performed by a former student Mr. Victor Liu. Fig. 3. 9. Western blot analysis of EtGIPL1a on expression of cleaved caspase 3 in U251 cells. The three bold bands represent three batches of EtGIL1a at 12.5 µg/mL treatment for 48 h. 3.3.6. Monosaccharide composition analysis The monosaccharide analysis by TMS derivatization using GC-MS revealed that EtGIPL1a consisted of predominantly glucose (Glc) (54.3 %). Relatively large amounts of galactose (Gal) (19.6 %), mannose (Man) (11.1 %), and fucose (Fuc) (10.3 %) were also found in EtGIPL1a (Fig. S8 and Table 3.2). EtGIPL1a also contains glucuronic acid (GlcA) (4.0 %) and traces of rhamnose (Rha) (0.6 %). An immuno-stimulatory polysaccharide EtISPFa also isolated from E. tinctorium contains a higher amount of glucuronic acid (10.1 %) (Zeb et al., 2021). GC-MS was performed and analyzed by CCRC, USA. 106 3.3.7. Glycosyl linkage analysis by GC-MS Consistent with the composition analysis, the glycosyl linkage analysis showed a majority of glucose linkages (%) in EtGIPL1a (Fig. S9 and Table 3.2). The main glucose linkages were 3substituted, 6-substituted, and terminal. A small amount of 4-substituted glucose was also present. Galactose was found in 6- and 2,6- linkages, and GlcA was 4-substituted. Beside these, there were small amounts of terminal mannose (1.3%) as well as 3-substituted (2.5%) and terminal (0.9%) fucose. Compared with the polysaccharide EtISPFa reported previously (Zeb et al., 2021), 3-Fucp and 6-Galp were elevated, and 2,6-Galp was not detected at all in the EtISPFa polysaccharide. Table 3. 2. Monosaccharide composition of EtGIPL1a Monosaccharide residue Mass (µg) Mol % Glucose (Glc) 90.8 54.3 Galactose (Gal) 32.7 19.6 Mannose (Man) 18.6 11.1 Fucose (Fuc) 15.7 10.3 Glucuronic acid (GlcA) 7.3 4.0 Rhamnose (Rha) 0.9 0.6 Total = 165.9 100 The polysaccharide composition is known to link with bioactivity. Glucose, mannose, and galactose are amongst the commonly studied monosaccharide components of mushrooms whereas glucuronic acid, galacturonic acid, fructose, N-acetylglucosamine, N-acetyl galactosamine, and ribose are the least studied (Wang, Wang, Xu, & Ding, 2017). According to Chen et. (2005), fucose, glucose and mannose were considered essential for the anti-proliferative properties of two fungi: Antrodia xantha and Rigidoporus ulmarius; the bioactivity of these fungi 107 was compared with the inactive A. cinnamomea and A. malicola, which lacked the aforementioned monosaccharides in their structure (Chen, Lu, Cheng, & Wang, 2005). Another study showed the anti-proliferative effect of a fucose-containing highly branched 1,3-βmannoglucan isolated from the fungus Poria cocos, a well-known Chinese medicine. This compound was found to inhibit lung cancer by down-regulating TGFβR signaling pathway, leading to inhibition of the migration of human metastatic lung cancer cells CL1-5 (Lin, Lu, & Chang, 2020). An anti-proliferative heterogenous polysaccharide (GIPinv) isolated from Paxillus involutus also contained fucose as terminal sugar (Barad et al., 2018). A heterogenous high molecular weight fucose-rich polysaccharide fraction FMS with fucose attached at the terminals was isolated from Ganoderma lucidum. FMS was found to induce production of IgM antibodies against tumor-specific glycans in Lewis lung cancer cells. FMS mediated antibody response and suppressed monocyte chemoattractant protein-1 which is an inflammatory mediator associated with cancer. Moreover, FMS suppressed Globo H, a carbohydrate antigen only found on the surface of cancer cells. This immunogenic ability of FMS was believed to be due to the presence of terminal fucose in its structure, which is capable of interacting with the surface antigens on tumor cells (Liao et al., 2013). Since EtGIPL1a also contains reasonable percentage of fucose at the terminals, it will be important to investigate whether EtGIPL1a has anti-cancer activity in animal model and whether it exhibits in vivo properties similar to FMS. The overall linkage analysis of EtGIPL1a showed (1à3)-linked Glc, (1à6)-linked Glc and (1à6)-linked Gal to be the major monomers, indicating that these components are the main chain of EtGIPL1a structure. β-glucans isolated from Grifola frondosa (Fang et al., 2012) and Schizophyllum commune (Klaus et al., 2011) share the same structural features, as their main chain is composed of (1à3)-linked Glc with branching at (1à6)-linked Glc. According to 108 several studies conducted on fungal polysaccharides, β-1à3 linkage in the major backbone with β-1à6 branching points is required for the antiproliferative activity (Wasser, 2002). Table 3. 3. Glycosyl linkage analysis of EtGIPL1a by partially methylated alditol acetates a PMAA Linkage 1,5-Di-O-acetyl-1-deuterio-6-deoxy-2,3,4-tri-O-methylgalactitol t-Fucp Peak Area % 0.9 1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methylmannitol t-Manp 1.3 1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methylglucitol t-Glcp 15.3 1,3,5-Tri-O-acetyl-1-deuterio-6-deoxy-2,4-di-O-methylgalactitol 3-Fucp 2.5 1,3,5-Tri-O-acetyl-1-deuterio-2,4,6-tri-O-methylglucitol 3-Glcp 28.9 1,5,6- Tri-O-acetyl-1-deuterio-2,3,4-tri-O-methylmannitol 6-Manp 2.4 1,5,6- Tri-O-acetyl-1-deuterio-2,3,4-tri-O-methylglucitol 6-Glcp 18.3 1,4,5- Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methylglucitol 4-Glcp 1.7 1,4,5- Tri-O-acetyl-1,6,6’-trideuterio-2,3,6-tri-O-methylglucitol 4-GlcpA 9.2 1,5,6- Tri-O-acetyl-1-deuterio-2,3,4-tri-O-methylgalactitol 6-Galp 3.9 1,3,5,6- Tetra-O-acetyl-1-deuterio-2,4-di-O-methylglucitol 3,6-Glcp 13.0 1,2,5,6- Tetra-O-acetyl-1-deuterio-3,4-di-O-methylgalactitol 2,6-Galp 2.6 For clarity, residues found at < 1% were omitted. Polysaccharides isolated from basidiomycetes are known to have more complex heteroglucan structural traits as compared to ascomycetes (He et al., 2017; Zhang, Nie, Yin, Wang, & Xie, 2014). For instance, polysaccharides from ascomycetes contain a systematic monosaccharide chain unit such as those found in Cordyceps species (Lu, Gu, Hao, Jin, & Wang, 2016; Smiderle, Sassaki, Griensven, & Iacomini, 2013). In contrast, monosaccharides in polysaccharides isolated from basidiomycetes are very complex and do not have a systematic monosaccharide chain unit. 109 Diverse monosaccharide composition contributes to the structural complexity of polysaccharides of basidiomycetes which applies to EtGIPL1a and EtISPFa which was previously isolated immuno-stimulatory polysaccharide from E. tinctorium (Zeb et al., 2021). Despite the reported studies where β-1à3 and β-1à6 has been linked to immuno-stimulatory activity, which is true for EtISPFa but does not apply to growth-inhibitory polysaccharide EtGIPL1a. Although EtGIPL1a does have β-1à3 Glc backbone with branching at β-1à6 linked Glc, it does not possess immuno-stimulatory activity. The growth-inhibitory activity of EtGIPL1a is likely to be due to the variability in the monosaccharide composition and linkages from EtISPFa. The presence of acidic group was also confirmed by GC-MS analysis. EtGIPL1a is an acidic polysaccharide that has a considerable amount of glucuronic acid (4 %), which is not commonly found in fungal polysaccharides. Interestingly, we also found high content of glucuronic acid (10 %) in another bioactive polysaccharide isolated from E. tinctorium (Zeb et al., 2021). Other fungi that contain acidic polysaccharides are Pleurotus abalonus (Shi, Zhao, Jiao, Shi, & Yang, 2013), Fusarium and Gibberella species (Ahrazem et al., 2000), Plectosphaerella cucumerina, Verticillium dahliae, and V. albo-atrum (Ahrazem et al., 2006). 3.3.8. Structural analysis by FTIR FTIR showed the presence of characteristic absorption peaks for a polysaccharide (Cui, 2005) (Fig. 3.10): a broad band at 3283 cm-1 that referred to free hydroxyl group stretching and a weak signal at 2923 cm-1 for the C-H stretch. There were some other peak stretches as well: 1598 cm-1 for carboxyl C=O, 1383 cm-1 for C-H bending and 1039 cm-1 corresponding to pyranose ring stretching vibration. The observed functional groups present in EtGIPL1a are consistent with that of a typical polysaccharide due to the presence of free O-H groups, pyranose ring, alkanes and amine (Chen et al., 2012; Liu et al., 2015; Wang, Wang, Xu, & Ding, 2017). 110 Fig. 3. 10. FTIR spectrum of EtGIPL1a. 3.3.9. NMR analyses of EtGIPL1a To gain further structural insights, 1D and 2D NMR analyses were carried out and the data was analyzed by CCRC, USA. According to 1D proton NMR, there was a complex pattern of peaks in the 3.2-4.5 ppm region which referred to the non-anomeric proton region (H2 - H6) of carbohydrates (Fig. 3.11). Fig. 3. 11. 1H-NMR spectrum of EtGIPL1a. H1 protons resonating at 4.5-5 ppm were referred to as β-anomeric protons. Due to the complexity of overlapping non-anomeric proton peaks, 2D NMR was conducted. 111 Fig. 3. 12. Partial multiplicity-edited 1H-13C-HSQC NMR spectrum of EtGIPL1a with labels indicating signals that were of much lower intensity in the EtISPFa sample (see Table 3.4). Red signals are positive and correspond to CH groups, and blue signals are negative and correspond to CH2 groups. The H1 signals that resonate between 5-6 ppm refer to α-anomeric protons whereas those between 4-5 ppm are β-anomeric protons. The H1 signals for EtGIPL1a were in the 4.2-5 ppm region, suggesting the polysaccharide have a β-configuration of glycosidic bond. Polysaccharide isolated from Grifola frondosa (Fang et al., 2012) and Schizophyllum commune (Klaus et al., 2011) also showed a β-configured glucan structure with 1à3 linked Glc as the main chain with O-6 substitution. 112 Table 3. 4. NMR chemical shift assignments for the residues found in EtGIPL1a that were not found in EtISPFa No. Residue A 6-a-Galp- B 2,6-a-Galp- C 3-a-Fucp- 1 5.00 101.0 4.94 101.4 4.95 101.6 2 3.87 71.2 3.81 74.6 3.94 70.2 Chemical shift (ppm) 3 4 3.89 4.03 72.6 72.8 3.86 3.91 71.1 73.2 3.95 3.96 81.3 75.3 5 4.07 71.3 4.14 72.1 4.11 69.5 6 3.92/3.71 69.6 3.99/3.71 69.8 1.25 18.6 The spectra were similar to those reported for the EtISPFa polysaccharide (Zeb et al., 2021). The 2D NMR results were aligned with the complexity of signals seen in 1D NMR, concluding that EtGIPL1a is a complex large β-glucan with a 1à3 linked Glc backbone which is substituted at O-6. However, there were several additional signals indicating that the L1a polysaccharide was more complex than the EtISPFa polysaccharide sample (Chapter 2). Three additional residues were assigned (Table 3.4), confirming some of the differences observed in the linkage data in comparison with those of the EtISPFa polysaccharide. Thus, 6-substituted agalactopyranosyl, 2,6-disubstituted a-galactopyranosyl, and 3-substituted a-fucopyranosyl residues A, B, and C were identified, which were found in lower abundance in the EtISPFa polysaccharide than in EtGIPL1a. The anomeric signals of Residues A and B showed NOE contacts only with their own H2 and one H6 (Fig. 3.4 and Table 3.4). Unlike the H1-H2 correlation, the cross peak between H1 and H6 could only arise from an inter-residue contact, suggesting that both 6-Gal and 2,6-Gal may constitute the backbone of a separate galactan polysaccharide. No NOE signals were detected from H1 of Residue C, and thus, it is unknown whether this residue belonged to the glucan or the galactan. Although it was difficult to quantify the different residues in the NMR experiments because of extensive peak overlap, the intensities 113 of the signals belonging to 4-substituted glucuronic acids were somewhat smaller than in the EtISPFa sample (Zeb et al., 2021), thus corroborating the linkage data. Based on the chemical analyses that include monosaccharide composition, linkage analysis, FTIR, 1D and 2D NMR, a proposed backbone structure for the 275 kDa anti-proliferative EtGIPL1a (Fig. 3.13) is largely identical to that reported earlier for the 1354 kDa immunostimulatory EtISPFa polysaccharide (Zeb et al., 2021), with significantly lower proportion of GlcA. In addition to the main polysaccharide, there is a (1®6)-a-galactan with unknown side chains at O-2 of some of the Gal residues. Furthermore, there is a small amount of 3-substituted a-Fuc with an NOE correlation to a proton at 3.82 ppm (Table 3.4). This could be H3 of a b-Glc residue in the main backbone, but at the low abundance of Residue C, it is not possible to make this determination with certainty. In addition to having more 3-linked fucose (2.5 %), 6-linked mannose (2.4 %), 6-linked galactose (3.9%) and 2,6-linked galactose (2.6 %), in general. As mentioned in Chapter 2, this structure (Fig. 3.13) is an average structure with multiple possibilities. In addition to the current structure elucidation techniques, more evidence can be collected by other techniques which will confirm the monosaccharide components and linkages in EtGIPL1a. These techniques include hydrolysis of polysaccharide by acid or enzymes and then identification using MALDI-TOF-MS, capillary electrophoresis and analysis of derivatized monosaccharides on HPLC using fluorometry or UV detection. EtGIPL1a has approximately six times more fucose, three times more galactose and 2 times more mannose than EtISPFa. Therefore, it is tempting to speculate that rather than the backbone structure, the abundant fucose, mannose and galactose and their corresponding linkages distinguish EtGIPL1a from EtISPFa, and contributes to its anti-proliferative function. 114 Fig. 3. 13. Proposed representative structure of the anti-proliferative polysaccharide EtGIPL1a. 3.4. Conclusion This is the first study to describe isolation and characterization of an anti-proliferative polysaccharide from E. tinctorium. Using a phase separation method, Sephadex LH-20, DEAESephadex, Sephacryl S-500 HR, and HPLC Bio SEC-3, we successfully isolated an antiproliferative polysaccharide (EtGIPL1a) with weight average molecular weight (Mw) of 275 kDa from the methanol extract of E. tinctorium. EtGIPL1a is a complex and unique b-glucan polysaccharide rich in galactose (19.6 %), mannose (11.1 %), fucose (10.3 %) and glucuronic acid (4 %), along with trace amounts of rhamnose (0.6 %). The combined results from GC-MS and NMR analyses reveal that EtGIPL1a is made up mostly of a backbone consisting of β(1→4)-Glcp residues and β-(1→3)-GlcpA residues, with branching points at β-(1→6)-Glcp. The polysaccharide is anti-proliferative against a large panel of cancer cell lines. 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Medicinal value of culinary-medicinal Maitake mushroom Grifola frondosa (Dicks.: Fr.) SF Gray (Aphyllophoromycetideae). International journal of medicinal mushrooms, 6, 1-28. 120 Appendix B. Supplementary data DEAE-Sephadex Optimization The running conditions for anion exchange chromatography using Sephadex DEAE were optimized with buffers (Fig. S1) including Bis-Tris (pH = 6.48), L-Histidine (pH 6.2), Piperazine (pH 5.3 and 9.73), and Tris (pH 8.07). The same procedure was used to obtain the optimized buffer as mentioned in Appendix A of Chapter 2. The elution and flow through were assessed for growth-inhibitory activity via MTT. L-Histidine was used as final buffer for further purification. 121 Fig. S 6. Growth-inhibitory assay for optimization of Sephadex DEAE with buffers at different pH including Bis-Tris at pH 6.48 (A), L-Histidine at pH 6.2 (B), Piperazine at pH 5.3 (C) and 9.73 (D), and Tris at pH 8.07 (E). Medium and water were used as negative controls. Error bars represent S.D. 122 Fig. S 7. Estimating the peak maxima molecular weight (Mp) of EtGIPL1a using HPLC BioSEC-3. Dextran standards and EtGIPL1a were loaded onto BioSEC-3 at a flow rate of 1.2 mL/min. This was used to convert the retention time (Rt) to retention volume, and then to the Average Distribution Constant (Kav) as shown in Table S1. Closed circles represent the Dextran Standards used and open squares are all points of the interpolated molecular weight distribution of EtGIPL1a. 123 Fig. S 8. The GC-MS chromatograms from glycosyl composition analysis using TMS derivatization. Fig. S 9. GC-MS chromatogram resulting from glycosyl linkage analysis of neutral and uronic acid residues. Glycosyl linkage analysis was performed by partially methylated alditol acetates (PMAAs). In the interest of clarity, PMAAs below 1% are not labeled. 124 Table S 3. Calculating the number (Mn) and weight average molecular weight (Mw) of EtGIPL1a Rt time (min) 5 5.2 5.4 5.6 5.8 6 6.4 Rt volume (mL) 6 6.24 6.48 6.76 6.96 7.2 7.68 VeVo Kav=VeVo/Vc-Vo Mwt (log) MW or Mi (g/mol) 0.74 0.98 1.22 1.5 1.7 1.94 2.42 0.0814978 0.1079295 0.1343612 0.1651982 0.1872247 0.2136564 0.2665198 5.635 5.522 5.447 5.335 5.222 5.147 4.922 431519 332659 279898 216271a 166724 140281 83560 Moles ni (number of molecules) niMi wi wiMi 4.63479E-11 2.79107E+13 1.2044E+19 0.191902 82809.29 6.01216E-11 3.62052E+13 1.2044E+19 0.191902 63837.87 7.14546E-11 4.303E+13 1.2044E+19 0.191902 53712.94 9.24766E-11 5.56894E+13 1.2044E+19 0.191902 41502.8 1.19959E-10 4.303E+13 7.1741E+18 0.114308 19057.92 1.42571E-10 3.62052E+13 5.0789E+18 0.080924 11352.14 2.39349E-10 2.79107E+13 2.3322E+18 0.03716 3105.103 ∑= 2.69981E+14 6.2761E+19 275378.1b Twenty mg of EtGIPL1a was loaded onto BioSEC-3 at a flow rate of 1.2 mL/min. Vo is the void volume or Rt volume of Dextran Blue 2000 = 5.26 mL. Vc = 1 Column volume = 14.34 mL. Therefore, Vc-Vo = 9.08 mL. a Molecular weight peak maxima (Mp) = 216271 g/mol. b Weight average molecular weight (Mw) = 275378.1 g/mol. Number average molecular weight (Mn) = 232465.3 g/mol. (Mn = SniMi/Sni) Polydispersity index = 1.184 (PDI = Mw/Mn 125 Chapter 4: Lanostane triterpenoids, phenol and diphenylmethane derivatives from the fungus Echinodontium tinctorium ABSTRACT In our continuing search for bioactive metabolites from British Columbia wild mushrooms, bioassay-guided extraction, fractionation and chemical investigation of the organic extracts of the fruiting bodies from the fungus Echinodontium tinctorium resulted in the isolation of a new diphenylmethane derivative bis(2,4-dihydroxy-6-methylphenyl) methane (2), together with 5 known compounds (1, 3-6). The structures of 1-6 were determined by a combination of 1D and 2D NMR, ESI-MS and X-Ray crystallography. The full NMR data assignment of the known compound (4) and the crystal structures of (2), (4) and (5) are reported here for the first time. Furthermore, the biological activity of compounds (2), (4) and (5) are shown here for the first time; they exhibited antiproliferative activity against U251 human glioblastoma cells with an IC50 of 85 μM, 4.6 μM, and 5.45 μM respectively. Compound (2) and (4) were also effective against HeLa cervical cancer cells with an IC50 > 100 μM and 1.2 μM respectively. Additionally, (4) also showed growth inhibition in multiple cancer cell lines with IC50 ranging from 2-5 μM. Flow cytometry analysis showed that compounds (2) and (4) can induce apoptosis in U251 cells. 4.1. Introduction Glioblastoma (GB) is a highly aggressive and invasive cancer, most frequently diagnosed in adults in their mid 60’s (Omuro & DeAngelis, 2013); it arises from multiple cell types with neural stem cell-like properties. Despite scientific advances, GB remains incurable with a short life expectancy of approximately 18 months post-diagnosis (Davis, 2016). Temozolomide is the only standard chemotherapeutic drug approved for the treatment of GB, with a treatment regimen that includes surgical resection, followed by radiotherapy and chemotherapy (Lim et al., 2018). Due to its invasiveness, chance of tumor recurrence and unwanted treatment side effects, there is a strong therapeutic need to find more effective drugs and targets. A number of promising synthetic small molecules (dos Santos et al., 2019) and natural products against GB have recently been tested (Erices et al., 2018; Khan et al., 2020). 126 As mentioned in the previous chapters of this thesis, E. tinctorium has not been studied in detail for its potential bioactive compounds. To date, 12 small molecules have been isolated from other Echinodontium species, which included E. tsugicola and E. japonicum (Table 4.1). Ye et al. (1996) and Bond et al. (1996) identified two small molecules (echinodol and echinotinctone) from E. tinctorium, but no bioactivity studies were provided for these small molecules and, as described in Chapters 2 and 3, two immuno-stimulatory and growth-inhibitory polysaccharides have been isolated and characterized from E. tinctorium. Table 4.1. Small molecules isolated from E. tinctorium and related species Compounds m/z Source Activity Reference Echinotinctone 256 E. tinctorium None Ye et al., 1996 Echinodol 498 E. tinctorium None Bond et al., 1966 Tsugicolines A-E 267 (A) 351 (B) 269 (C) 249 (D) 284 (E) E. tsugicola A-active against Lepidium sativum Arnone et al., 1995 E. tsugicola E. tsugicola Antimicrobial None Echinodone 496 E. tsugicola None Arnone et al., 1998 Shiona et al., 2004, 2005 Kanematsu, 1972 DeacetylEchinotinctone 3-Epiechinodol 454 E. tsugicola None Kanematsu, 1972 E. tsugicola None Kanematsu, 1972 Deacetyl-3epiechinodol Echinolactone A Echinolactone B 3-Epi-illudol 440 E. tsugicola None Kanematsu, 1972 245 261 E. japonicum E. japonicum E. japonicum None None None Suzuki et al., 2005 Suzuki et al., 2005 Suzuki et al., 2005 Tsugicolines E-I Echinocidins A-D The goal of this study was to isolate and characterize small molecules from E. tinctorium and then assess them for growth-inhibitory activity, especially against human glioblastoma cells. To achieve this goal, multiple approaches were used which included two solvent phase separation, sequential phase separation and direct extraction of powdered mushroom. Sephadex LH-20 size exclusion chromatography (SEC), silica flash column chromatography (SFC) and high127 performance liquid chromatography (HPLC) were employed for purification. Structural elucidation was carried out with the help of electrospray ionization mass spectrometry (ESI-MS), Fourier transform infrared spectroscopy (FTIR), 1D and 2D nuclear magnetic resonance spectroscopy (NMR), and X-ray crystallography. For evaluation of biological activity, growthinhibitory assay MTT was performed and mechanism of growth-inhibition was confirmed by apoptosis and cell cycle analyses. Potential molecular targets of isolated small molecules were also identified using the MolTarPred software. 4.2. Materials and methods 4.2.1. Materials and reagents All reagents were of analytical grade. Eagle’s Minimal Essential Medium was from LONZA (Walkersville, Maryland, USA). 3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyltetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Life Technologies Inc. (Waltham, Massachusetts, USA). Sephadex™ LH-20 resin was purchased from GE Healthcare (Chicago, IL, USA). All cell lines were obtained from the American Type Culture Collection. HPLC analysis was performed on an Agilent 1260 Infinity Systems with DAD detector. Low resolution mass spectrometry was performed on an Agilent 6120 Single Quad MS. For high resolution electrospray ionization mass spectrometry (HRESIMS), an Agilent 1200 HPLC and a Bruker maXis Impact Ultra-High Resolution tandem TOF (UHR-Qq-TOF) mass spectrometer was used at the Mass Spectrometry Facility, Simon Fraser University (SFU). NMR analyses was conducted on a 600 MHz NMR spectrophotometer at University of British Columbia (UBC). HPLC grade solvents were used and were purchased from BDH (Mississauga, ON, Canada). 4.2.2. Collection and extraction of the mushroom E. tinctorium conks were collected as described in Chapters 2 and 3. The mushroom was sequenced to confirm its identity. For isolation of small molecules from E. tinctorium, three different extraction approaches were used: two solvent phase separation (approach 1), sequential phase separation (approach 2), and direct extraction (approach 3). 128 4.2.3. Approach 1: Two solvent phase separation 4.2.3.1. Extraction and phase separation Powdered E. tinctorium was sequentially extracted with 80 % ethanol (65 ºC, 3 h) followed by extraction with 50 % methanol (65 ºC, 3 h) to give crude extract E2. The crude extract E2 was reconstituted in water and partitioned with chloroform to yield two separate layers; top aqueous layer L1 and bottom organic layer L2. Both layers were dried; L1 was lyophilized whereas L2 due to high organic content was dried using a rotary evaporator. 4.2.3.2. Assessment of growth-inhibitory activity Dried L1 and L2 were reconstituted in methanol at 20 mg/mL, filter sterilized (2 µm) and tested for growth-inhibitory activity in HeLa cells using MTT assay as described in Chapter 3. L1 and L2 were tested on HeLa cells at a final concentration of 0.1-1 mg/mL. L2 was also tested on rat intestinal epithelial cells (RIE-1) to compare the results with colorectal adenocarcinoma cells (SW480). After confirmation of biological activity, L1 and L2 were subjected to Sephadex LH-20 SEC to estimate the relative size of bioactive compound(s). As discussed in Chapter 3, L1 contained relatively large molecular weight compound while low molecular weight compound with potential growth-inhibitory activity was expected to be in L2. Therefore, L2 was further studied here. 4.2.3.3. Purification by Sephadex LH-20 SEC Sephadex LH-20 resin was soaked in methanol for 48 h. Column was packed (2 mL/min, methanol) and equilibrated (2 CV, 1 mL/min, methanol). Initially, a 25 mL gravity drip column was used, which was then upscaled to a 80-100 mL column (mobile phase; methanol, 1 mL/min). Ten mL fractions were collected, concentrated to half the volume and tested for growth-inhibitory activity using MTT assay. 4.2.3.4. HPLC analysis and purification Post-Sephadex LH-20 purified bioactive fractions were pooled, concentrated and subjected to HPLC analysis. A solvent gradient (Fig. 4.1) of acetonitrile and 0.2 % formic acid in water was run at 0.7 mL/min. A 10 µL sample was injected via an autosampler onto the HPLC column 129 (Phenomenex synergy 4µ Hydro-RP 80 Å, 150 × 4.6 mm). Once the bioactive fractions were analyzed, the most abundant peak was fraction collected, purified and re-injected into HPLC to assess for % purity. Fig. 4. 1. HPLC solvent gradient for analysis and purification. B is % of acetonitrile and C is % of 0.2 % formic acid in water. 4.2.3.5. ESI-MS analysis HPLC-purified compound was subjected to ESI-MS (Agilent 6120 Single Quad MS) analysis using the same solvent gradient as in Fig. 4.1. Flow injection analysis (FIA) was conducted to optimize the MS conditions. The ESI-MS parameters optimized for analysis are shown in Table 4.2. Table 4. 2. Parameters for mass spectrometry analysis MSD signal parameters MSD spray chamber MSD status ESI MS mode = Positive mode Scan range = 100-1000 m/z Ion source = API-ES Fragmentor = 70-400 V (100 V optimized) Gain = 1 Threshold = 150 Step size = 0.10 Speed (µL/sec) = 867 Method spray chamber = API-ES Drying gas flow (L/min) = 12 Nebulizer pressure (psig) = 40 Drying gas temperature (°C) = 340 Capillary voltage VCap (V) = 3000 (+), 3000 (-) Quad temperature (°C) = 100 Capillary current (nA) = 6 Chamber current (µA) = 0.69 130 4.2.4. Approach 2: Sequential phase extraction 4.2.4.1. Sequential phase extraction and assessment for growth-inhibitory activity Crude methanol extract E2 was sequentially phase-separated with multiple solvents in order of increasing polarities (Fig. 4.8A). E2 was suspended in water at a concentration of 10 mg/mL extracted 3-5 times with hexane, followed by chloroform, and ethyl acetate (EA). It was assumed that water layer retained large molecules as seen in approach 1. The other three organic layers were expected to contain small molecules. All three layers were dried, resuspended in methanol and assessed for growth-inhibitory activity at 0.4 mg/mL for 48h in HeLa cells using MTT assay. 4.2.4.2. HPLC analysis of phase separated layers Once bioactivity was confirmed, each layer was subjected to HPLC analysis independently. HPLC method development involved optimization of column type, solvents, solvent gradients, and flow rate. HPLC analysis revealed that all the layers possess many peaks indicating the presence of many compounds. Due to many peaks in each layer, it was concluded that an additional purification step needs to be incorporated into the purification methodology. Based on the amount of material that each layer weighed, EA layer weighed the most, followed by chloroform layer and hexane layer, therefore, EA was considered first for further purification. 4.2.4.3. Approach 2a: Purification of EA layer by Sephadex LH-20 SEC The dried EA layer was subjected to purification by SEC using Sephadex LH-20 resin. Dried EA layer was resuspended in methanol (20 mg/mL), filtered and loaded onto the column. Methanol was used as a mobile phase and run at a flow rate of 1 mL/min. Fractions were collected (3 mL, 2-3 CV), concentrated, and tested for growth-inhibitory activity in HeLa cells using MTT assay. The Sephadex LH-20 column retained most of the compounds in EA layer as most of the preload was visible at the top of the column. 4.2.4.4. HPLC analysis of Sephadex LH-20 purified compounds Bioactive fractions were pooled and scanned using nanodrop for optimal wavelength. Sephadex LH-20 eluted fractions were further purified by HPLC. HPLC method development 131 was performed for optimized conditions. A 2 mg/mL preload was run through the column (Phenomenex Luna C18(2), 5µ, 250 × 4.6 mm) at a flow rate 0.7 mL/min using acetonitrile and water gradient. Due to the fact that most of the compounds interacted and were retained by the Sephadex LH-20 resin, it was concluded that perhaps an alternative strategy is needed to resolve a range of different compounds. 4.2.4.5. Approach 2b: Purification of EA layer by silica flash column chromatography Thin layer chromatography (TLC) was first used as a preliminary step to develop the solvent system for separating compounds in the EA layer. The optimized conditions were then used to run the column. 4.2.4.6. TLC method development Prior to running the silica flash column, method was developed on a TLC with individual and combination of solvents. A dilute solution of EA layer was made and spotted onto aluminum baked silica plate. The best results were obtained by running hexane first which removed very non-polar compounds. Apparently, no compounds were observed to migrate on the TLC plate with hexane. It could be that those compounds were not UV active. EA was then used to run through TLC which resulted in separation of compounds that appeared under UV. There were some compounds that appeared to be still present at the origin line, indicating that a more polar solvent can be used to mobilize them from the baseline. Therefore, methanol was used to mobilize the compounds retained at the origin line. Methanol was successful in achieving further separation of more polar compounds. 4.2.4.7. Purification by Silica Flash Column (SFC) Packing SFC: Silica was used as a stationary phase with hexane initially, followed by EA, methanol and finally chloroform were used as mobile phase. Initially, a 50-60 mL SFC was used and was later on up-scaled to a larger sized column. For stationary phase, silica powder was suspended in hexane to make a slurry. Silica slurry was poured by sliding through the side of the column to avoid entrapment of air bubbles. The column was packed by passing hexane and applying air pressure through an adaptor connected to the air hose. 132 Running SFC: A concentrated sample of EA layer was then applied to the column with a Pasteur pipette. Initially, hexane was allowed to run through the column. For each solvent elution, 20 fractions of 1 mL fraction size were collected and analyzed. A quadrant test was performed where each collected fraction was simultaneously spotted onto TLC plate and visualized under short and long wave UV. The hexane-eluted fractions were not visible under UV whereas the EA eluted fractions 4 to 9 were visible under UV. For methanol solvent elution, fractions 6 to 8 were visible. All the fractions from hexane, EA, and methanol were dried and resuspended in methanol to test for growth-inhibitory activity against HeLa cells using MTT assay. 4.2.4.8. Additional purification of Post SFC fractions The bioactive fractions from SFC were subjected to additional purification by the following approaches: (i) SFC with solvent gradient in the elution order of 20% methanol à 60% methanol à 80% methanol à 100% methanol à 100 % acetonitrile à IPA (ii) SFC with solvent gradient in the elution order 100 % methanol à 80 % methanol à 60 % methanol à40 % methanol à 1 % Acetic acid in water à Chloroform à EA (iii) SFC with solvent gradient as chloroform à EA à Acetonitrile à Methanol à 1 % Acetic acid in water à Acetone à Chloroform à EA (iv) Sephadex LH-20 using methanol as mobile phase. Since approach (iv) gave best results therefore, it was used for further purification of compounds from EA layer. 4.2.4.9. HPLC analysis and purification of Post SFC fractions After confirmation of growth-inhibitory activity, the bioactive fractions were subjected to HPLC analysis (Phenomenex Luna C18(2), 5µ, 250 × 4.6 mm), using acetonitrile, water, and methanol gradient (0.5-1 mL/min, λ = 260 nm) as in Fig. 4.2. The most abundant peaks were fraction collected in 6 mL vials. After collection, the peaks were concentrated and reinjected into HPLC to determine % purity. These pure peaks were designated as (1) and (2). 133 Fig. 4. 2. HPLC solvent gradient for analysis and purification. B, C and D refers to acetonitrile, water and methanol. Additionally, all the EA eluted fractions from large SFC were analyzed by HPLC. The analysis was performed on Phenomenex Luna C18(2) column at 1 mL/min using three solvent gradient (acetonitrile: water: methanol, 25.5:49:25.5 to 26:48:26 in 8 min, 100:0:0 from 10-15 min, 60:30:10 from 15-18 min, 25.5:49:25.5 from 20-25 min). Three abundant peaks containing compounds 1-3 were fraction collected and purified. 4.2.4.10. Structural elucidation of compounds from EA layer Structural elucidation of compounds (1-3) purified from EA layer was performed using ESIMS, FTIR and NMR. ESI-MS and FTIR were performed as mentioned earlier. A high resolution ESI-MS in positive scan mode (m/z 100 – 1500 Da), was performed on compound (3). Acetonitrile/water (0.1 % formic acid) was used as mobile phase. For FTIR, twenty-two scans were collected to generate an IR spectrum. Further structural details of the HPLC-purified compounds (1-3) were obtained from NMR. 1D proton (1H) and carbon (13C) NMR and 2D NMR including 1H-13C HSQC, 1H-13C HMBC, 1H-1H COSY, and 1H-1H NOESY, were carried out using a 600 MHz NMR spectrophotometer. 4.2.4.11. Crystallization of compound (2) For crystal formation of (2), vapor diffusion and slow evaporation methods with multiple solvent combinations were used. Vapor diffusion method A saturated solution of (2) was made in methanol and kept in a 1.5 mL glass vial. The 1.5 mL vial was placed in a 25 mL glass vial containing hexane as the surrounding medium. The vial was capped and kept for 2 weeks to allow slow vapor diffusion of hexane into the sample vial. 134 Slow evaporation Three solvent systems were used in an effort to crystallize (2): a. Saturated solution of (2) was made in methanol/acetonitrile and kept for slow evaporation at room temperature for 2 days in a small beaker. b. Saturated solution of (2) was made in methanol/acetone and kept for slow evaporation at -20 °C and monitored at multiple time points. c. Saturated solution of (2) was made in methanol/hexane and kept at -20 °C for slow evaporation overnight. 4.2.4.12. Crystallization of compound (3) Slow evaporation For crystal formation of (3), slow evaporation with different solvents was experimented. The solvents included acetonitrile/methanol, acetone/methanol, and methanol alone. About 2 mg of (4) was dissolved in 100 µL of solvents to make a saturated solution and kept in a 1.5 mL glass vial at room temperature to allow slow evaporation. Vapor diffusion A 2 mg of (3) was dissolved in methanol in a 300 µL narrow glass insert which was then placed in a small beaker containing antisolvent hexane. The beaker was covered with a plastic wrap. The antisolvent was allowed to vaporize and mix with the methanol dissolved (3) for 2 days as well as slow evaporation of both the solvents through the plastic wrap. Recrystallization A saturated solution of (3) was made by dissolving 2 mg in minimal amount of acetonitrile. The solution was kept in a 1.5 mL glass vial and heated in a heat block (55 ºC, 5-7 min) until all dissolved. The solution was cooled on ice bath and then kept undisturbed. 4.2.4.13. Purification and HPLC analysis of phase separated hexane layer Hexane layer was subjected to HPLC analysis on a Phenomenex Luna C18 HPLC column using acetonitrile/water gradient (20 % to 90 % acetonitrile in 10 min) at 0.8 mL/min. The absorption wavelength was set at 250 nm for analysis. 135 Due to the crude nature of hexane extract that contained many compounds, a small-scale gravity drip SFC was performed to purify compounds in the hexane layer. A 95:5 ratio of hexane: IPA was used as mobile phase. The solvent was optimized by method development on TLC. Twelve fractions of 1 mL each were collected. The eluted fractions from SFC were spotted onto TLC to run a quadrant test. The quadrant test confirmed the UV active compounds. The UV active fractions were then subjected to HPLC analysis (Isocratic Hexane/IPA 95/5%, 0.7 mL/min, 220 and 240 nm) on Phenomenex Luna C8(2) HPLC column. 4.2.4.14. Purification and HPLC analysis of phase-separated chloroform layer The phase-separated chloroform layer was analyzed by HPLC on Phenomenex Luna PhenylHexyl column (250 x 4.6 mm, 100 Å, 5µm) using three solvent gradient (acetonitrile: water: methanol 10:25:65 to for 10:10:80 in 10 min then 0:0:100 for an additional 5 min) at 1 mL/min flow rate. Further purification was carried out by Sephadex LH-20 SEC and SFC. The chloroform layer was run through a medium sized SEC column (100 mL) using methanol as mobile phase at 1 mL/min. A total of 30 fractions of 5 mL each was collected. The Sephadex LH-20 collected fractions were assessed for growth inhibition in HeLa cells. The bioactive fractions were then analyzed by HPLC. Based on similar HPLC profiles, the bioactive fractions 14-16 and fractions 17-20 were pooled separately. The peaks abundant in these fractions were collected and analyzed. Additionally, chloroform layer was also purified by another method using SFC. A solvent gradient approach was used to elute compounds starting with more non-polar hexane, followed by chloroform, acetone, ethyl acetate and finally more polar methanol. For each solvent elution 5-7 fractions of 4 mL each were collected, dried and injected into HPLC. The post SFC fractions were compared with the post-Sephadex LH-20 bioactive fractions. 136 4.2.5. Approach 3: Direct extraction method 4.2.5.1. Solid-liquid extraction and assessment for growth-inhibitory activity A solid-liquid extraction of E. tinctorium material was performed with organic solvents as shown in Fig. 4.60. Initially, 10 g of mushroom powder was extracted with hexane (200 mL, 3 h, RT). The flask was agitated several times and supernatant was collected. The procedure was repeated twice with same conditions and the final extract was soaked in hexane (100 mL, overnight, RT). All hexane supernatants were pooled, partitioned with water to remove any polar compounds and finally dried. The dried hexane layer was reconstituted in acetonitrile and subjected to HPLC analysis. The same procedure was repeated for diethyl ether (DEE) solidliquid extraction. The DEE dried extract was also reconstituted in acetonitrile and subjected to HPLC analysis. Moreover, hexane and DEE extracts were reconstituted in methanol at a concentration of 0.6 mg/mL and 0.7 mg/mL and assessed for growth-inhibitory activity in HeLa cells using MTT assay. 4.2.5.2. HPLC analysis and purification of hexane extract The hexane extract was subjected to HPLC analysis using reverse phase LC column (Phenomenex Luna C18(2) 5µm, 100 Aͦ, 250 × 4.6 mm). The following conditions were used to run the sample; mobile phase as acetonitrile: water 80:20 to 90:10 in 10 min, 90:10 from 10-20 min, 80:20 in 25 min and until 40 min, flow rate = 1 mL/min, DAD = 210, 230 nm. The purification method for hexane layer was upscaled to semi-preparative column (Agilent Zorbax Eclipse XDB C18 9.4 × 250mm 5µ) with isocratic solvent (Acetonitrile: Water 90:10) at a flow rate 2 mL/min for 40 min. 4.2.5.3. Structural elucidation by spectroscopy The MS analysis of compounds isolated from hexane extract was determined with positive mode ESI-MS using solvent gradient (Acetonitrile: Water 80:20 to 90:10 in 15 min, 90:10 from 15-22 min, 90:10 to 80:20 in 25 min) at 0.8 mL/min. Scan (100-1000 m/z) and SIM mode were used to confirm the mass. 137 The functional group analysis was performed by FTIR (Bruker ATR-FTIR spectrophotometer by Billerica, MA, USA) with a frequency range 4000-400 cm−1. An IR spectrum was generated using OPUS software. A total of twenty-two scans were obtained. For more detailed structural insights, 1D and 2D NMR analyses were carried out using a 600 MHz NMR spectrophotometer at UBC. 4.2.5.4. Crystallization of compound (4) Method 1: Compound (4) was dissolved in minimal volume of 90 % acetonitrile to yield a saturated solution. The solution was kept in a narrow glass insert with a 300 µL capacity and placed in a 1.5 mL glass vial with holes in the rubber cap. The vial was kept at room temperature for slow evaporation. Method 2: A saturated solution of compound (4) was made with acetonitrile in a 1.5 mL glass vial for recrystallization. The vial was heated at 55 °C in a heating block for 3-4 min until all dissolved. It was then kept on an ice bath for 10 min and kept at room temperature overnight. Method 3: Compound (4) saturated solution was made by adding 90 % acetonitrile and placed in a 6 mL glass vial. The solution was left for slow evaporation at room temperature. 4.2.5.5. Crystallization of compound (5) Method 1: Vapor diffusion method was used where a saturated solution of (5) was made in acetonitrile and kept in a 2 mL glass vial, which was then placed in a 10 mL glass vial (screw capped) with hexane anti-solvent. The vials were placed at 2-4 ºC for a few days. Method 2: (5) was dissolved in acetonitrile/methanol mix and kept for 4-5 days in a 6 mL wide glass vial (covered) for slow evaporation at room temperature. Method 3: A saturated solution of (5) was made with 90 % acetonitrile/water mix in a 2 mL glass vial. The sample vial was placed open in another 6 mL glass vial (covered) and kept overnight for slow evaporation at room temperature. 138 4.2.5.6. Melting point determination Melting point of compound (2) and (4) were determined using a melting point apparatus (DigiMelt MPA160, Stanford research systems, USA) with a ramp rate 2-5. Initial and final melting point range was recorded for each compound. 4.2.5.7. HPLC-MS analysis of Diethyl ether extract (DEE) Diethyl ether extract was reconstituted in acetonitrile and subjected to HPLC analysis on Phenomenex Luna C18(2) analytical column using gradient elution (Acetonitrile 60-100 % in 25 min, 100-60 % in 30 min) at 0.8 mL/min with DAD (210, 230 nm). The ESI-MS analysis was carried out by including 0.1 % formic acid in mobile phase. The fragmentor was set to 80 V with a gain of 1. 4.2.5.8. Growth-inhibitory activity and flow cytometry analysis of purified compounds The growth-inhibitory activity of purified compounds was first assessed on HeLa cells as well as on other cancer cell lines and normal immortalized cell lines. Percent cell viability was determined using MTT assay as described in Chapter 2. Flow cytometry analysis was carried out on U251 glioblastoma cells using Annexin V and PI double staining analysis. Cells were plated at a seeding density of 20 x 104 cells/mL in 6 well plates and incubated for 24 h after which they were treated with control and purified compounds at different concentrations. For compound (2), cells were treated with 50 µM and 100 µM for 2448 h and then assessed for apoptosis and cell cycle using flow cytometry. For compound (4), cells were treated with concentrations at 4.3 µM, 10 µM, 20 µM, and 40 µM for 24-48 h time intervals. After 24-48 h of treatment, cells were harvested, washed with DPBS, spin twice at 400 x g for 5 min at room temperature. Supernatant was discarded and cell pellets were resuspended in DPBS, cells were counted and spun again at 4 °C, after which the pellets were resuspended in binding buffer to achieve a cell density of 1.5 x 106 cells/mL. Cells were then stained with annexin V and PI and kept in the dark at room temperature for 15 min. DPBS was added and double stained cells were filtered and analyzed by flow cytometry. 139 4.2.5.9. Molecular target prediction of compounds (1-5) Initially, chemical structures were drawn in Chemdraw, which were then used to generate a chemical notation, using SMILE (Simplified Molecular Input Line Entry System). SMILE format was entered into the MolTarPred program and targets were predicted in different organisms with a reliability of prediction score. Given below are the SMILE format generated for purified compounds: Compound (1) OC1=CC(O)=CC(C)=C1 Compound (2) CC1=C(CC2=C(O)C=C(O)C+C2C)C(O)=CC(O)=C1 Compound (4) OC1CCC2(C)C(CCC3=C2CCC4(C)C3(C)CC5C4C(C)C(OC(C)=O) C(/C=C(C)/C)O5)C1(C)C Compound (5) O=C1CCC2(C)C(CCC3=C2CCC4(C)C3(C)CC5C4C(C)C(OC(C)=O) C(/C=C(C)/C)O5)C1(C)C 4.3.Results and Discussion 4.3.1. Approach 1-Two Solvent Phase Separation 4.3.1.1. Extraction and assessment of growth-inhibitory activity E2 was extracted from powdered E. tinctorium using manual extraction. L1 and L2 were obtained as a result of phase separation (Table 4.3). As shown in Fig. 4.3, L2 showed growthinhibitory activity on HeLa, RIE-1 and SW480 cells. Table 4. 3. Quantitative estimation of E2, L1 and L2 layers after phase separation Exp # E2 (mg) 1 2 3 20 20 30 L1 (H2O layer) (mg) 11 11.5 15 % Yield 55 57.5 50 140 L2 (Chloroform layer) (mg) 9 7.5 10.8 % Yield 45 37.5 36 RIE-1 SW480 Fig. 4. 3. Dose dependent growth-inhibitory effect (n=3) of L2 at 0.1-1 mg/mL for 48 h on HeLa cells (A), RIE-1 and SW480 cells (B). 4.3.1.2. Purification by Sephadex LH-20 and HPLC Bioactivity-guided purification (Fig. 4.4A) was done initially using Sephadex LH-20 and finally with HPLC. The Sephadex LH-20 elution profile (Fig. 4.4B) showed bioactive fractions from F17-F26, indicative of the presence of low molecular weight compounds contributing to the growth-inhibitory effects. An additional small scale liquid-liquid extraction was done and bioactive methanol layer was then subjected to HPLC analysis and purification. 141 Fig. 4. 4. A) Purification strategy using two solvent phase separation. B) Sephadex LH-20 SEC elution profile for growth-inhibitory small molecule(s). A representative data from three biological replicates. Data File D:\NALS HP...EENZEB NALS DATA\MEHREEN\F20 Sample Name: F20 Plh20 MeOH HydroRP Plh20 MeOH HydroRP2018-09-1711-42-23.D HPLC analysis of bioactive fractions on HydroRP column at 238 nm displayed one abundant ===================================================================== Acq. Operator : SYSTEM Sample Operator : SYSTEM Acq. Instrument : HPLC-MS-FC Location : 1 Injection Date : 9/17/2018 11:43:38 AM Inj Volume : 1.000 µl Acq. Method : C:\Chem32\1\Methods\MehreenMethods\E.tE2.L2s.Phl20.F20 .M Last changed : 9/17/2018 11:40:18 AM by SYSTEM Analysis Method : C:\ChemData\1\Methods\Nobu Method\P-Aurea-Feb 2021.M Last changed : 5/6/2021 9:49:24 AM by SYSTEM (modified after loading) Additional Info : Peak(s) manually integrated peak retained at 5.9 min with some shoulder peaks (Fig. 4.5A). Purification and fraction MeOH HydroRP (238nm) collection of this peak resulted in a pure peak (Fig. 4.5B). VW D1 A, W avelength=238 nm (D:\NALS HP...ZEB NALS DATA\MEHREEN\F20 Plh20 MeOH HydroRP2018-09-1711-42-23.D) 5.967 mAU 400 300 200 100 0 -100 0 2.5 5 7.5 10 12.5 15 17.5 min 20 ===================================================================== Area Percent Report ===================================================================== Sorted By Multiplier Dilution Sample Amount: Do not use Multiplier Signal 1: VWD1 A, : : : : & Signal 1.0000 1.0000 1.00000 [ng/ul] (not Dilution Factor with ISTDs Wavelength=238 used in calc.) nm Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] % ----|-------|----|-------|----------|----------|--------| 1 5.967 BV R 0.2329 1.13811e4 491.63745 100.0000 Totals LEE_HPLC : 5/6/2021 1.13811e4 9:58:10 AM 491.63745 Page SYSTEM 142 1 of 2 Acq. Method : Last changed Analysis Method Last changed : : : Additional : Info C:\Chem32\1\Methods\MehreenMethods\E.tE2.L2s.Phl20.F20 .M 9/17/2018 4:03:12 PM by SYSTEM C:\ChemData\1\Methods\Nobu Method\P-Aurea-Feb 2021.M 5/6/2021 10:06:43 AM by SYSTEM (modified after loading) Peak(s) manually integrated MeOH HydroRP (238nm) VW D1 A, W avelength=238 nm (D:\NALS HP...ZEB NALS DATA\MEHREEN\F20 Plh20 MeOH HydroRP2018-09-1716-30-32.D) 5.965 mAU 500 a Are :6 74 1.2 3 400 300 200 100 0 -100 0 2 4 6 8 10 min 12 ===================================================================== Area Percent Report ===================================================================== Fig. 4. 5. HPLC spectrum of Post Sephadex LH-20 purified F20 (A) and HPLC purified Sorted By Multiplier Dilution Sample Amount: Do not use Multiplier : : : : & Signal 1.0000 1.0000 1.00000 [ng/ul] (not Dilution Factor with ISTDs compound (B) at 238 nm. Signal 1: VWD1 A, Wavelength=238 used in calc.) nm 4.3.1.3. Mass spectrometric analysis of HPLC-purified compound Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] % ----|-------|----|-------|----------|----------|--------| 1 5.965 MM 0.2188 6741.23145 513.55762 100.0000 Flow injection analysis (FIA) showed the optimum ionization conditions at fragmentor Totals : 6741.23145 513.55762 voltage 100 and 130 V (Fig. 4.6), therefore 100 V was used for further MS analysis. MS scan LEE_HPLC 5/6/2021 10:10:24 AM Page SYSTEM 1 of 2 (Fig. 4.7A) showed the presence of three abundant MS ions at m/z 249, 251, and 293 [M + H] which were then analyzed in SIM (single ion mode). SIM (Fig. 4.7B) showed two equally abundant MS ions at m/z 249 and 293 [M + H] with a difference of -44. Such results suggest that a COO- group came off from 293 [M + H], giving rise to its fragment 249 [M + H]. ES-API, Pos, Sca 0.399 4.325 3.016 1.707 MSD1 TIC, MS File (C:\Chem32\1\Data\Mehreen\FIA 600-800 mass Fragmentor80-4002018-09-1916-34-12.D) 300000 5.631 250000 200000 6.942 150000 8.251 100000 70 r: nto gme Fra 100 r: nto gme Fra 130 r: nto gme Fra 160 r: nto gme Fra 190 r: nto gme Fra 220 r: nto gme Fra 250 r: nto gme Fra 280 9.558 r: nto gme Fra 50000 r: nto gme Fra 310 r: nto gme Fra 340 r: nto gme Fra 400 0 2 4 6 8 10 12 14 min 14 min 13.357 12.050 10.742 9.434 8.126 6.817 5.509 4.201 2.893 1.585 0.277 VWD1 A, Wavelength=238 nm (Mehreen\FIA 600-800 mass Fragmentor80-4002018-09-1916-34-12.D) mAU 60 50 40 30 20 10 0 0 2 4 6 8 10 12 Fig. 4. 6. ESI-MS FIA of HPLC purified compound at fragmentor voltage 40-400 V in UV (A) and MSD scan signal (B). 143 Fig. 4. 7. ESI-MS spectrum of F20 in scan mode (A) and SIM mode (B). 4.3.2. Approach 2: Sequential phase extraction 4.3.2.1. Sequential phase separation and assessment for growth-inhibitory activity E2 was subjected to sequential phase separation in order to extract small molecules of different polarities (Fig. 4.8A). As a result of phase separation, EA layer has the highest yield followed by chloroform and hexane layers (Table 4.4). When tested on HeLa cells, all three layers exhibited growth-inhibitory activity (Fig. 4.8B). 144 Fig. 4. 8. A) Purification methodology for sequential phase separation of E. tinctorium. B) Sequential phase separated layers treated at 0.4 mg/mL for 48 h were assessed for growthinhibitory activity in HeLa cells. A representative data from three biological replicates. Table 4. 4. Quantitative estimation of phase separated layers Type of extract Amount obtained (mg) % Yield Solubility Crude methanol extract E2 400 - Water Hexane layer 6.4 1.6 Methanol Chloroform layer 80 20 Methanol EA layer 150 37.5 Methanol 4.3.2.2. Purification of EA layer by Sephadex LH-20 SEC Bioactive compounds purified by Sephadex LH-20 column (Fig. 4.9) were present in fractions 7-9 (1 CV), suggesting that there are low molecular weight compounds contributing to the growth-inhibitory activity. 145 Fig. 4. 9. Sephadex LH-20 (70 mL column) elution profile of EA layer. Result shown is a representative from three biological replicates. 4.3.2.3. HPLC and ESI-MS analysis Bioactive fractions were then analyzed on HPLC where one prominent peak was retained at 8.6 min with several least abundant peaks (Fig. 4.10A). The ESI-MS analysis of this peak showed two major mass ions with m/z 296 and 317 [M + H], along with many other ions that were less abundant (Fig. 4.10B). Therefore, further purification was done to get cleaner HPLC and MS spectra. 146 Fig. 4. 10. A) HPLC spectrum of Post Sephadex LH-20 bioactive fractions, B) ESI-MS of peak retained at 8 min. 4.3.2.4. Purification of EA layer by SFC and HPLC TLC method was developed (Fig. 4.11) to isolate small molecules from the EA layer. The optimized solvent system was then used to run the silica column. TLC analysis showed 3 spots that were visible under short wave and long wave UV as well as with anisaldehyde stain. Short wave UV Long wave UV Anisaldehyde Ninhydrin KMnO4 Vanillin Fig. 4. 11. TLC visualization by UV and multiple stains. The TLC quadrant test (Fig. 4.12A) showed that EA-eluted fractions 5-9 and methanol-eluted fractions 6-8 from the silica column contained UV active compounds. The UV active EA-eluted fractions 6-9 also showed growth-inhibitory activity on HeLa cells (Fig. 4.12C). These fractions were therefore further analyzed using HPLC. 147 Fig. 4. 12. A) A TLC quadrant test of EA-and methanol-eluted fractions from silica column chromatography visualized under UV light, B) Full TLC on pooled UV visible fractions 4-9 visualized under UV light, C) Growth-inhibitory fractions of EA elution from silica column chromatography. Result shown is a representative of three biological replicates. The HPLC method was successfully developed on C18 reverse phase HPLC column where bioactive fractions 6-9 showed a well resolved spectrum with multiple potential peaks (Fig. 4.13A). The abundant peaks retained at 5.2, 6.4, and 11.08 min were fraction-collected and reinjected for purity check (Fig. 4.13B, C & D). 148 Fig. 4. 13. HPLC spectrum of SCC purified bioactive fractions 6-9 (A), and HPLC purity check of peak 1 (B), peak 3 (C) and peak 3 (D). Additional purification of F6-9 was achieved from Sephadex LH-20 SEC (approach (iv)) that resulted in cleaner and well-resolved spectrum (Fig. 4.14). The HPLC analysis showed that initial fractions F14-16 only had peak 1, F17-18 contained both peaks 1 and 2 and F19-22 contained predominantly peak 2. These peaks were fraction-collected and analyzed. Peak 1 was designated as compound (1) and peak 2 was designated as compound (2). Both compounds were then subjected to growth-inhibitory analysis and structural elucidation. 149 Fig. 4. 14. HPLC spectrum of approach (iv) fractions F14-16 (A), F17-18 (B), and F19-22 (C). 4.3.2.5. Chemical characterization of compound (1) Compound (1) was obtained as a colorless powder, with a molecular formula C7H8O2, confirmed from NMR data and ESI-MS ion peak at m/z 125 [M+H] as shown in Fig. 4.16. 4.3.2.6. HPLC and ESI-MS analysis of (1) The UV diode array detector (DAD) spectrum showed (1) was retained at 5 min with 100 % purity and absorption maxima at 220 and 280 nm (Fig. 4.15). 150 Fig. 4. 15. HPLC DAD spectrum of (1) at 220 (A) and 280 nm (B). The ESI-MS spectrum (Fig. 4.16) showed a molecular ion peak at m/z 125 [M+H] in both scan mode (m/z 100 – 1000) and SIM which confirmed the molecular formula C7H8O2 of orcinol. Fig. 4. 16. ESI-MS spectrum of (1) in scan mode (A) and SIM (B). 4.3.2.7. Structural elucidation of (1) by FTIR and NMR IR spectrum (Fig. 4.17) showed peak stretches for OH (3286cm-1) and C-H (2921 cm-1) functionalities. 151 Fig. 4. 17. FTIR spectrum of (1). 1D NMR data revealed that (1) constitutes seven carbons that include a 1, 3, 7 tri-substituted benzene ring with two hydroxy groups and a methyl group. 1H NMR (Fig. 4.18 & Table 4.5) showed presence of aromatic protons (δH 6.08, 1H), (δH 6.16, 1H), (δH 6.16, 1H) and methyl protons (δH 2.19, 3H). The C-1 (δC 157.9) and C-3 (δC 157.9) were de-shielded due to their OH substituents. 13C NMR (Fig. 4.19 & Table 4.5) showed three quaternary carbons (δC 157.9, 157.9, 140.4), three methine (δC 99.4, 107.5, 107.5), and a methyl group (δC 20.4). 13C and 1H signals were directly correlated in HSQC spectrum (Fig. 4.20). Fig. 4. 18. 1H NMR spectrum of (1). 152 Fig. 4. 19. 13C NMR spectrum of (1). Table 4. 5. 13C and 1H Chemical shifts of (1) Number 13 1 2 3 4/6 5 7 157.9 99.4 157.9 107.5 140.4 20.4 C Chemical shift δ ppm 1 H Chemical shift δ ppm (# H, multiplicity, J) 6.09 (1H, t, J = 2.27, 2.27) 6.17 (2H, d, J =2.18) 2.20 (3H, s) Fig. 4. 20. 13C-1H HSQC spectrum of (1). 153 Chemical group °4 C CH °4 C 2 CH °4 C CH3 The 13C-1H HMBC cross peaks (Figs. 4.21 & 4.23) were observed between C-1/C-3 (δC 157.9) and H-4/H-6 (δH 6.16) as well as H-2 (δH 6.08). C-2 (δC 99.4) had cross peak with H-4/H6 (δH 6.16), C-4 (δC 107.5) with H-6 (δH 6.16), H-2 (δH 6.08), and H-7 (δH 2.19), C-6 (δC 107.5) with H-4 (δH 6.16), H-2 (δH 6.08), and H-7 (δH 2.19), C-5 (δC 140.4) with H-7 (δH 2.19). 1H-1H COSY correlations (Figs. 4.22 & 4.23) were seen between H-7 (δH 2.19), H-4 (δH 6.16), and H-6 (δH 6.16). Fig. 4. 21. 13C-1H HMBC spectrum of (1). 154 Fig. 4. 22. 1H-1H COSY spectrum of (1). 6 1 7 5 4 2 3 1H-1H COSY HMBC Fig. 4. 23. 1H-1H COSY and 13C-1H HMBC structural correlations. 155 Fig. 4. 24. 1H-1H NOESY spectrum of (1). In summary, compound (1) was identified as orcinol, also called 5-methylresorcinol. Orcinol has been isolated from lichens including Parmelia subrudecta (Ivanova et al., 2010) and mold like Aspergillus niger (Sahasrabudhe et al., 1986). Some studies have described the role of orcinol as an important source of carbon and energy which is achieved by hydrolysis of benzene ring to acetate and pyruvate, which would possibly act as substrates for the energy cycles (Chapman & Ribbons, 1976, Sahasrabudhe et al., 1986). 4.3.2.8. Chemical characterization of compound (2) Compound (2) was obtained as yellow star-shaped crystals, with a molecular formula C15H16O4, determined from NMR data, X-ray crystal structure and ESI-MS spectrum. 4.3.2.9. HPLC and ESI-MS analysis of (2) The UV-DAD spectrum showed maximum absorption at 220, 260 and 280 nm (Fig. 4.25). ESI-MS (Fig. 4.26) molecular ion peak at m/z 261.1 [M+H]. 156 Fig. 4. 25. HPLC DAD spectrum of (2) at multiple wavelengths. Fig. 4. 26. ESI-MS spectrum of (2) in scan mode m/z = 100-1000 (A) and SIM mode (B). 157 4.3.2.10. Structural elucidation by FTIR and NMR spectroscopy The F TIR spectrum (Fig. 4.27) showed absorptions of hydroxy group (3233 cm-1) and aliphatic CH stretches (2854, 2922 cm-1) which were in agreement with the composition of compound (2). Fig. 4. 27. FTIR spectrum of (2). Data from 1D NMR revealed that (2) is a symmetric molecule with a total of 15 carbons (Fig. 4.28 & Table 4.6). Fourteen carbons are equally distributed on both sides of the molecule held together by another carbon. The carbons included one central methylene (δC 22.3), two methyl groups (δC 19.9), two methine (δC 100.7, 110.0), and four quaternary carbons (δC 156.0, 155.9, 155.97, 117.1). 1D NMR signals (Fig. 4.29 & Table 4.6) also revealed a substituted aromatic ring [δH 6.17 (4H, m), 3.78 (2H, s), 2.15 (6H, s); δC 100.7, 110.0, 22.3, and 19.9]. In addition to this, (2) also possessed four hydroxy groups which were confirmed from X-ray diffraction data (XRD). Moreover, the presence of one central methylene group was confirmed from 13C NMR DEPT135 (Distortionless Enhancement of Polarization Transfer) signals that were embedded in HSQC spectrum. 158 14 15 7 5 4 3 6 8 1 13 9 10 11 2 12 Fig. 4. 28. 13C NMR of (2). Fig. 4. 29. 1H NMR of (2). Table 4. 6. 13C and 1H NMR chemical shifts of (2) Number 1/13 2/12 3/11 4/10 5/9 13 C Chemical shift δ ppm 156.0 100.7 155.9 110.0 155.9 1 H Chemical shift δ ppm (# H, multiplicity, J) 6.17 (2H, d, J= 2.51) 6.18 (2H, d, J= 2.53) 159 Chemical group °4 C CH °4 C CH °4 C 6/8 7 14/15 117.1 22.3 19.9 3.78 (2H, s) 2.15 (6H, s) °4 C CH2 CH3 1D NMR peak assignments were confirmed from 2D correlations (Table 4.6 and Fig. 4.30). Cross peaks were observed between C-1/C-13 (δC 156.0) and H-4/H-10 (δH 6.18) and H-7 (δH 3.78). C-5/C-9 (δC 155.9) had cross peaks with H-2/H-12 ((δH 6.17) and H-7 (δH 3.78), C-3/C-11 (δC 155.9) with H-7 (δH 3.78) and H-14/H-15 (δH 2.15), C-6/C-8 (δC 117.1) with H-4/H-10 (δH 6.18), H-2/H-12 ((δH 6.17), H-7 (δH 3.77), and H-14/H-15 (δH 2.15), C-4/C-10 (δC 110.0) with H2/H-12 (δH 6.17), C-2/C-12 (δC 100.7) with H-4/H-10 (δH 6.18). C-7 (δC 22.3) was weakly correlated to H-4/H-10 (δH 6.18) and H-2/H-12 (δH 6.17) and finally C-14/C-15 (δC 19.9) had cross peaks with H-4/H-10 (δH 6.18). Fig. 4. 30. 13C-1H HSQC spectrum of compound (2). 160 Fig. 4. 31. 13C-1H HMBC spectrum of compound (2). The 1H-1H COSY spectrum (Fig. 4.32) showed proton couplings between H-4/H-10 (δH 6.18) and H-14/H-15 (δH 2.15). H-7 (δH 3.78) was strongly coupled to H-2/H-12 (δH 6.17) and weakly to H-14/H-15 (δH 2.15). NOE correlations (Figs. 4.33 & 4.34) were observed between H-4/H-10 (δH 6.18) and H-14/H-15 (δH 2.15), between H-7 (δH 3.78) and H-14/H-15 (δH 2.15) and H-4/H10 (δH 6.18). The long range 13C-1H multiple bond couplings are shown in Fig. 4.33 and Table 4.6. 161 Fig. 4. 32. 1H-1H COSY spectrum of compound (2). Fig. 4. 33. 2D NMR structural co-relations of compound (2). 162 Fig. 4. 34. 1H-1H NOESY spectrum of compound (2). Table 4. 7. 13C-1H multiple bond couplings (HMBC) # C (δ ppm) C-1/C-13 (156.0) C-5/C-9 (155.9) C-3/C-11 (155.0) C-6/C-8 (117.1) C-4/C-10 (110.0) C-2/C-12 (100.7) C-7 (22.3) C-14/C-15 (19.9) 4.3.2.11. # H (δ ppm) H-4/H-10 (6.18), H-7 (3.78) H-2/H-12 (6.17), H-7 (3.78) H-7 (3.78), H-14/H-15 (2.15) H-4/H-10 (6.18), H-2/H-12 (6.17), H-7 (3.78), H-14/ H-15 (2.15) H-2/H-12 (6.17) H-4/H-10 (6.18) H-4/H-10 (6.18), H-4/H-10 (6.18) Crystallization and X-ray crystallography of (2) The vapor diffusion method resulted in the formation of thin yellow irregular crystals whereas slow evaporation method with methanol/acetonitrile co-solvents generated star shaped yellow irregular crystals (Fig. 4.35A) which qualified for XRD analysis. None of the other methods were successful in achieving good quality crystals. Detailed crystal data can be found in supplementary data of appendix C. 163 Fig. 4. 35. A) Crystals of compound (2) under microscope, B) ORTEP style image of compound (2). Crystal Data: C15H16O4, triclinic, P-1 (No. 2), a=8.8847(5) Å, b=9.2916(5) Å, c= 9.4831(4) Å, a= 97.485(4)°, b= 105.756(4)°, g= 105.320(4)°, V= 709.25(7) Å3, T= 90(2) K, Z= 2, Z’= 1, µ(CuKa)= 0.838, 9454 reflections measured, 1829 unique (Rint = 0.0628) which were used in all calculations. The final wR2 was 0.0968 (all data) and R1 was 0.0378 (I > 2(I)). The XRD analysis was performed by Chemistry department, UBC. The crystal data of compound (2) was in agreement with the NMR data. The crystal structure of compound (2) is reported for the first time. 4.3.2.12. Assessment of growth-inhibitory activity of (2) Compound (2) showed growth-inhibitory activity on HeLa cells and U251 cells when treated for 48 h with an IC50 of 195 µM + 6.01 and 85.45 µM + 2.8 respectively (Fig. 4.36). To further explore the mechanism for growth inhibition by (2) on U251 cells, apoptosis and cell cycle analyses were performed. 164 % Cell Viability (% Control) %Cell Viability (%Control) 100 50 0 0 1 2 3 100 50 0 Concentration (logµM) 2 3 Concentration (logµM) Fig. 4. 36. Growth inhibition caused by (2) in HeLa cells (A) and U251 cells (B). Results shown are representative from three biological replicates. 4.3.2.13. Flow cytometry analysis of compound (2) As shown in Fig. 4.37, compound (2) induced significant apoptosis in U251 cells at 100 µM when cells were treated for 24 and 48 h. Twenty-four-hour treatment with 100 µM of (2) resulted in significant apoptosis (p = 0.04) with 42 % apoptotic cells as compared to 50 µM treatment and negative control methanol where there were 29 % and 26 % of apoptotic cells respectively. After 48 h treatment with 100 µM of (2), the percentage of apoptotic cells also increased significantly (p = 0.003) to 84 % as compared to 50 µM of (2) and methanol with 21 % and 28 % apoptotic cells respectively (Fig. 4.37). 165 Fig. 4. 37. Apoptosis induced by 50 µM and 100 µM of compound (2) in U251 cells (A), and % apoptotic cells at 24 h (B) and 48 h (C) time intervals; methanol as control. Results shown are representative from 3 separate experiments (n=3). For statistical analysis, one-way ANOVA was used. * represents p = 0.04 and ** represents p = 0.003. Cell cycle analysis of compound (2) showed a significant increase (p = 0.0038) in percentage of cells in G1 phase, suggesting that (2) induced G1 phase arrest in U251 cells (Fig. 4.38). 166 2June2021_U251Cellcycle_24h_Methanol control_24h.fcs Cell Cycle 47385 A 6.0K Methanol 4.0K Count G1= 43.8 % S= 45.5 % G2/M= 7.03 % C 2.0K 2June2021_U251Cellcycle_24h_PK3(260) 100uM_24h.fcs 0 Cell Cycle 0 50K 100K 150K 200K 250K 48491 B PI*-A :: PI*-A Compound (2) 8.0K RMSD : 13,4 %G1 : 43,8 %S : 45,5 G1= 77.8% %G2 : 7,03 S= 15.8% G1 Mean : 70358 G2 Mean : 138736 G2/M= 1.53% G1 CV : 2,64 G2 CV : 2,08 % less G1 : 3,54 % greater G2 : 0,33 Count 6.0K 4.0K 2.0K 0 0 50K 100K 150K 200K 250K PI*-A :: PI*-A Fig. 4. 38. A) Cell RMSD cycle :analysis of compound (2) at 100 µM for 48 h, B) % population of cells 19,4 %G1 : 77,8 %S : 15,8 %G2 : 1,53 G1 Mean : 72777 G2 Mean : 141765 G1 CV : 3,50 G2 CV : 2,85 % less G1 : 5,94 % greater G2 : -0,018 in G1, S and G2/M phase of cell cycle. Results shown are combined from three separate experiments. One-way ANOVA was performed for statistical analysis. Error bars represent standard deviation. ** and * indicates p = 0.0038 and 0.01. Matubara et al. (1998) indicates that diphenylmethane derivatives had been chemically synthesized from 5-alkyl resorcinol by reacting with para-formaldehyde, dissolved in formic acid. The chemical reaction resulted in formation of multiple types of molecules; Type A (orthoortho), Type B (para-para), Type C (ortho-para), and Type D (xanthenes). Compound (2) is structurally similar to one of the type A compounds named Bis (2, 4-dihydroxy-6-methylphenyl) methane. According to the same study, this compound was tested for anti-tyrosinase activity which turned out to be negative (Matubara et al., 1998). Besides the chemical synthesis described by Matsubara and coworkers, (2) has never been reported from any natural sources including mushrooms; therefore, this is the first description of its growth-inhibitory activity and X-ray crystal structure of (2). 167 4.3.2.14. HPLC-MS analysis of additional fractions from SFC HPLC analysis of fraction 9 obtained from large SFC resulted in three abundant peaks retained at 15, 16 and 18 mins (Fig. 4.39). All the peaks demonstrated maximum purity when analyzed at different wavelengths using DAD (Fig. 4.40). Fig. 4. 39. HPLC profile of fraction 9 of SFC. A B Fig. 4. 40. HPLC DAD spectrum of peaks at 16 min (A) and 18 min (B). ESI-MS analysis of peak at 16 min showed m/z ion peak 285 [M+H] as the most abundant peak in the spectrum with few least abundant peaks of m/z 303, 407 and 447 (Fig. 4.41A). The 168 aforementioned ions from the MS scan mode were re-run in the SIM mode which resulted in 285 being the most abundant one (Fig. 4.41B). Since the peaks at 16 min and 18 min were purified twice to achieve maximum purity, it was deemed practically impossible to generate more material to proceed with further structural elucidation studies. A 285.2 *M S D 1S P C , tim e = 1 0 .3 6 1o fC :\C h e m 3 2 \1 \D a ta \M e h re e n _ 2 0 2 1 \2 0 2 1 -0 2 -0 8 1 2 -2 5 -4 7 1 6 1 E 2 E A S C _ P k1 6 .8 .D E S -A P I, P o s, S ca n ,F ra g 1 0 0 M a x :2 1 9 2 6 4 8 0 6 0 444.4 447.3 267.2 124.1 2 0 407.2 286.2 303.3 4 0 0 1 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 285.0 B 2 0 0 m /z *M S D 2S P C ,tim e = 10.356o fC :\C he m 32\1\D a ta \M e hre e n_2021\2021-02-0812-25-47161E 2E A S C _P k16.8.D E S -A P I,P o s,S IM ,F ra g: 100 M a x :7 3 1 6 4 8 80 60 444.0 20 447.0 442.0 40 0 280 300 320 340 360 380 400 420 440 m /z Fig. 4. 41. ESI-MS scan mode (A) and SIM (B) of peak at 16 min. 4.3.2.15. Chemical characterization of compound (3) 4.3.2.16. HPLC, ESI-MS and FTIR analysis of (3) Further analysis was conducted on peak at 15 min, which was designated as compound (3). The HPLC DAD spectrum showed that compound (3) was retained at 15 min with a 100 % purity at three different wavelengths; 230, 240 and 260 nm (Fig. 4.42). 169 Fig. 4. 42. HPLC DAD spectrum of compound (3) at 230, 240 and 260 nm. A high-resolution electrospray ionization mass spectrum (HRESI-MS) scan from 100 – 1000 m/z revealed the parent ion peak at m/z 507.2731 [M+H] and another fragment at m/z 303.2326 [M+H]. The low resolution ESI-MS scan and SIM mode were in agreement with the HRESI-MS (Fig. 4.43). 170 A B C Fig. 4. 43. High resolution HRESI-MS (A) and low resolution ESI-MS (B) of compound (3) in scan mode, and SIM mode (C). The FTIR spectrum of compound (3) showed peak stretches at 3391 cm-1 and 2930 cm-1 for hydroxyl and C-H functionalities (Fig. 4.44). 171 95 % 90 T 3000 1728.47 1601.79 1453.70 1411.36 1372.96 1312.80 1276.78 1236.11 1174.67 1117.87 1043.20 1023.51 976.28 957.86 928.59 887.67 831.74 806.18 762.60 711.76 602.05 522.94 481.49 435.88 407.01 390.49 2930.39 2871.81 3391.67 85 3500 2500 W 2000 -1 1500 1000 500 Fig. 4. 44. FTIR spectrum of compound (3). C: U A 4.3.2.17. D B OPUS 8.0.19 DATA MEAS 161E2EAP 15.8 161E2EAP 15.8 ALPHA-II / D ATR 1/6/2021 Crystallization of compoundP (3)1/1 Amongst the three approaches selected (slow evaporation, vapor diffusion and recrystallization), the first two approaches successfully generated shiny white needle-shaped thin long crystals (Fig. 4.45). Methanol alone worked as a solvent for slow evaporation. Compared to slow evaporation, the crystal size was bigger with the vapor diffusion approach. Unfortunately, crystals obtained from both methods were unable to diffract light and therefore its crystal structure was unattainable. Fig. 4. 45. Crystals of compound (3) from vapor diffusion method (A) and slow evaporation (B). 4.3.2.18. HPLC analysis of hexane layer The HPLC analysis of phase-separated hexane layer showed a very complex spectrum (Fig. 4.46) with many peaks indicating the need for further purification. Therefore, a method was successfully developed on TLC with 95:5 hexane: IPA as mobile phase. The TLC quadrant test revealed the UV active compounds in the fractions 7 to 9 (Fig. 4.47). 172 161.E2.Hexane layer. SCC Fig. 4. 46. HPLC spectrum of phase separated hexane layer. Hexane: IPA 95:5 A SCC Short column Gravity drip) fractions (1mL each) UV Active B F7-9 were subjected to ysis: Phenomenex Luna C8(2) àHexane:IPA 95:5 mL/min 240nm Fig. 4. 47. TLC quadrant test (A) and post SFC collected fractions (B). 4.3.2.19. Purification and HPLC analysis of compounds from chloroform layer HPLC analysis of phase-separated chloroform layer displayed an unresolved spectrum of multiple peaks (Fig. 4.48). Therefore, further purification was carried out. Fig. 4. 48. HPLC profile of chloroform layer. 173 4.3.2.20. Purification by Sephadex LH-20 SEC The post-Sephadex LH-20 fractions 13-20 were found to contain growth-inhibitory compounds (Fig. 4.49). The bioactive fractions were analyzed by HPLC. Fractions 14-16 shared the same HPLC profile and were therefore combined for further purification (Fig. 4.50). Fig. 4. 49. Sephadex LH-20 SEC profile of chloroform layer. Result shown is a representative from three biological replicates. The fractions 4a, 4b, and 4c from chloroform elution of SFC also shared the same profile when injected into HPLC, therefore the fractions from both columns were combined and analyzed (Fig. 4.50). The peaks retained at 11.3 and 14.4 min were fraction collected and reinjected for purity check (Figs. 4.51 & 4.53). ESI-MS analysis was carried out in positive mode for both peaks. Peak at 11.3 min showed molecular ion peak with a m/z 507 Da [M + H] in both scan (Fig. 4.52A) and SIM mode (Fig. 4.52B). In contrast, the peak at 14.4 min had a molecular ion peak of m/z 439 Da [M + H] in SIM and scan mode (Fig. 4.54). 174 Analysis Method : C:\ChemData\1\Methods\Nobu Method\Mitac\Mitac.M Last changed : 5/21/2021 3:21:56 PM by SYSTEM (modified after loading) Additional Info : Peak(s) manually integrated DAD1 A, Sig=230,4 Ref=off (Mehreen_Dec2020\161E2Chcl3SCAc_EAF4a (1) 2021-01-06 11-59-31.D) mAU 1400 14.063 14.489 1200 1000 11.310 800 600 400 200 0 2.5 5 7.5 10 12.5 DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161E2Chcl3SCAc_EAF4a (1) 2021-01-06 11-59-31.D) 15 17.5 20 min 20 min 14.464 Data File C:\ChemDat...een_Dec2020\161E2Chcl3lh20e2F10-12_Pk11.4 (1) 2021-01-07 10-59-32.D mAU Sample Name: 161E2Chcl3lh20e2F10-12_Pk11.4 1400 1200 11.310 14.062 15.826 ===================================================================== 1000 Acq. Operator : SYSTEM 800 Sample Operator : SYSTEM 600 Acq. Instrument : LEE_HPLC Location : P1-A-01 400 Injection Date : 1/7/2021 11:00:26 AM Inj : 1 200 Inj Volume : 2.000 µl 0 Different Inj Volume from Sample Entry! Actual Inj Volume : 4.000 µl 5 7.5 10 12.5 15 17.5 Acq. Method :2.5 C:\ChemData\1\Methods\Mehreen_Dec2020\161E2ChCl3Lh20_PhenhexDec2020.M Last changed : 1/7/2021 10:59:21 AM by SYSTEM Analysis Method : C:\ChemData\1\Methods\Nobu Method\Mitac\Mitac.M ===================================================================== Last changed : 5/21/2021 3:28:50 PM by SYSTEM Area Percent Report (modified after loading) ===================================================================== Additional Info : Peak(s) manually integrated Fig. 4. 50. HPLC DAD spectrum of pooled fractions F14-16 and F4a-c. : Signal : 1.0000 : 1.0000 : 1.00000 [ng/ul] (not used in calc.) & Dilution Factor with ISTDs 11.532 DAD1 A, Sig=230,4 Ref=off (Mehreen_Dec2020\161E2Chcl3lh20e2F10-12_Pk11.4 (1) 2021-01-07 10-59-32.D) Sorted mAU By 600 Multiplier 500 Dilution Sample 400 Amount: Do not 300 use Multiplier 200 100 Signal 1: DAD1 A, Sig=230,4 Ref=off 0 2 4 6 8 10 12 DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161E2Chcl3lh20e2F10-12_Pk11.4 (1) 2021-01-07 10-59-32.D) 16 min 14 mAU 600 500 11.533 400 LEE_HPLC300 5/21/2021 3:22:38 PM SYSTEM Page 1 of 2 200 100 0 2 4 6 8 10 12 16 min 14 ===================================================================== Area Percent Report ===================================================================== Fig. 4. 51. UV DAD spectrum of peak at 11.5 min. Sorted By Multiplier Dilution Sample Amount: Do A not use Multiplier : Signal : 1.0000 : 1.0000 : 1.00000 [ng/ul] (not used in calc.) & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=230,4 Ref=off LEE_HPLC 5/21/2021 3:29:33 PM SYSTEM Page 1 of 2 B Fig. 4. 52. ESI-MS spectrum of peak at 11.3 min in scan mode (A) and SIM mode (B). 175 Fig. 4. 53. UV DAD spectrum of peak at 14.4 min. 439.4 *M S D 1S P C ,tim e = 8.710o fC :\C he m 32\1\D a ta \M e hre e n_2021\2021-02-0911-09-11161E 2C hcl3P k15.4_c18.D E S -A P I,P o s,S ca n,F 100 M a x :5 1 6 9 9 2 80 60 440.4 A 40 125.2 441.4 20 0 100 200 300 400 500 600 m /z 439.0 *M S D 2S P C ,tim e = 8.704o fC :\C he m 32\1\D a ta \M e hre e n_2021\2021-02-0911-09-11161E 2C hcl3P k15.4_c18.D E S -A P I,P o s,S IM ,F r 100 M a x :8 7 8 2 0 8 80 60 B 40 20 0 300 350 400 450 500 m /z Fig. 4. 54. ESI-MS ion chromatogram of peak at 14.4 min. In addition, HPLC analysis of the remaining bioactive fractions 17-20 from Sephadex LH-20 showed the same HPLC profile where there were two abundant peaks retained at 4.9 and 6.4 minutes (Fig. 4.55). 176 Last changed : 12/9/2020 12:12:57 PM by SYSTEM Analysis Method : C:\ChemData\1\Methods\Nobu Method\Mitac\Mitac.M Last changed : 5/17/2021 6:03:16 PM by SYSTEM Additional Info : Peak(s) manually integrated 4.981 6.439 DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161E2Chcl3Lh20e5_F17 (1) 2020-12-09 14-32-56.D) mAU 600 500 200 7.388 5.752 300 8.693 400 100 0 2.5 5 7.5 10 12.5 DAD1 D, Sig=230,4 Ref=off (Mehreen_Dec2020\161E2Chcl3Lh20e5_F17 (1) 2020-12-09 14-32-56.D) 17.5 20 min 15 17.5 20 min 4.981 mAU 15 1750 1500 1250 5.753 750 500 250 6.420 1000 0 2.5 5 7.5 10 12.5 ================================================================================== Fraction Information ================================================================================== No Fractions found. ================================================================================== ================================================================================== ===================================================================== Area Percent Report ===================================================================== Fig. 4. 55. HPLC spectrum of bioactive fractions 17-20 of Sephadex LH-20 column. The HPLC DAD spectrum for peak retained at 4.9 min at 210 and 230 nm was not pure (Fig. 4.56) and it was also reflected in the MS spectrum where there were multiple m/z ions, although the 271 Da peak being the dominant one (Fig. 4.57). Sorted By : Signal Multiplier : 1.0000 Dilution : 1.0000 Sample Amount: : 1.00000 [ng/ul] Use Multiplier & Dilution Factor with ISTDs (not used in calc.) LEE_HPLC 5/21/2021 3:06:17 PM SYSTEM Page Fig. 4. 56. HPLC DAD spectrum of peak at 4.9 min at 230 and 210 nm. 177 1 of 2 A B Fig. 4. 57. ESI-MS spectrum of peak at 4.9 min in scan mode (A) and SIM mode (B). Data File C:\ChemDat...reen_Dec2020\161E2Chcl3lh20Fa_Pk3(6.4min) (1) 2021-01-04 11-27-04.D Sample Name: 161E2Chcl3lh20Fa_Pk3(6.4min) The HPLC DAD spectrum of peak at 6.4 min displayed a pure peak (Fig. 4.58) at both 210 ===================================================================== Acq. Operator : SYSTEM Sample Operator : SYSTEM Acq. Instrument : LEE_HPLC Location : P1-A-01 Injection Date : 1/4/2021 11:27:58 AM Inj : 1 Inj Volume : 2.000 µl Different Inj Volume from Sample Entry! Actual Inj Volume : 5.000 µl Acq. Method : C:\ChemData\1\Methods\Mehreen_Dec2020\161E2ChCl3Lh20_PhenhexDec2020.M Last changed : 1/4/2021 10:36:41 AM by SYSTEM Analysis Method : C:\ChemData\1\Methods\Nobu Method\Mitac\Mitac.M Last changed : 5/21/2021 3:10:59 PM by SYSTEM (modified after loading) Additional Info : Peak(s) manually integrated and 230 nm. The ESI-MS showed ion peaks with m/z 543, 283 and 261 Da (Fig. 4.59). It is possible that the parent ion peak was 543 and that ion peaks 283 and 261 were its fragment ions as they both add up closely to the mass of parent ion peak. DAD1 A, Sig=230,4 Ref=off (Mehreen_Dec2020\161E2Chcl3lh20Fa_Pk3(6.4min) (1) 2021-01-04 11-27-04.D) 6.330 mAU 350 300 250 200 150 100 50 0 2 4 6 DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161E2Chcl3lh20Fa_Pk3(6.4min) (1) 2021-01-04 11-27-04.D) 8 10 min 8 10 min 6.340 mAU 1750 1500 1250 1000 750 500 250 0 2 4 6 ===================================================================== Area Percent Report ===================================================================== Fig. 4. 58. HPLC DAD spectrum of peak at 6.4 min. Sorted By Multiplier Dilution Sample Amount: Do not use Multiplier : Signal : 1.0000 : 1.0000 : 1.00000 [ng/ul] (not used in calc.) & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=230,4 Ref=off LEE_HPLC 5/21/2021 3:13:24 PM SYSTEM Page 178 1 of 2 A B Fig. 4. 59. ESI-MS spectrum of peak at 6.4 min in scan mode (A) and SIM mode (B). 4.3.3. Approach 3: Direct extraction method 4.3.3.1. Extraction and assessment of growth-inhibitory activity of extracts Hexane and DEE extracts were obtained from direct extraction of powdered mushroom (Fig. 4.60A) with yield of 1.8 % and 4 % respectively (Table 4.8). Both the extracts were tested for growth-inhibitory activity using MTT assay. Hexane and DEE extracts caused growth inhibition in HeLa cells with less than 20 % cell viability when treated at 0.6 mg/mL for 48 h (Fig. 4.60B). Fig. 4. 60. A) Methodology for direct extraction of small molecules from E. tinctorium, B) Hexane and DEE extracts at 0.6 mg/mL for 48 h were assessed for growth-inhibitory activity on HeLa cells. Result shown is a representative from three biological replicates. 179 Table 4. 8. Quantitative estimation of extracts and compounds from direct extraction method Sample Powdered mushroom Hexane extract Amount (mg) 5000 60 (Methanol soluble) 40 (DMSO soluble) % Yield 1.8 DEE extract Hexane extract for HPLC PK 2 PK 3 PK 4 PK5 (PK5a & PK5b) 200 50 4 - Solubility Water/methanol Hexane, Acetonitrile, Methanol, DMSO Acetonitrile Methanol 2.8*, 0.8** 5.6*, 1.6 Acetonitrile 0.3 0.6 Acetonitrile 3.6 7.2 Acetonitrile 1.1* (PK5a & PK5b) 2.2 Acetonitrile 0.5** (PK5a) 1 (PK5a) 0.4**(PK5b) 0.8 (PK5b) Data*indicates File C:\ChemData\1\Data\Mehreen_Dec2020\161Hex_isocraticC18 (1) 2021-03-19 single step purification on HPLC, **indicates two times purification12-09-54.D on HPLC Sample Name: 161Hex_isocraticC18 4.3.3.2. HPLC analysis and purification ===================================================================== Acq. Operator : SYSTEM Sample Operator : SYSTEM HPLC method was successfully developed using Phenomenex Luna C18 reverse phase Acq. Instrument : LEE_HPLC Location : P1-A-01 Injection Date : 3/19/2021 12:10:48 PM Inj : 1 Inj gradient. Volume : Some 7.000 compounds µl HPLC analytical column with acetonitrile and water had absorption Different Inj Volume from Sample Entry! Actual Inj Volume : 8.000 µl Acq. Method : C:\ChemData\1\Methods\Mehreen_Dec2020\161Hex_c18_Jan2021.M maxima at 210 nm while others were more Last changed : 3/19/2021 12:09:02 PM by visible SYSTEM at 230 nm. Therefore, HPLC analysis and Analysis Method : C:\ChemData\1\Methods\DEF_LC.M Last changed : 4/23/2021 10:10:19 AM by SYSTEM purification was done at bothafter wavelengths (modified loading)simultaneously (Fig. 4.61). Additional Info : Peak(s) manually integrated DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161Hex_isocraticC18 (1) 2021-03-19 12-09-54.D) 250 35.245 32.843 500 16.368 750 13.643 10.523 1000 23.974 1250 42.193 1500 40.165 28.608 mAU 1750 0 5 10 15 20 25 30 DAD1 D, Sig=230,4 Ref=off (Mehreen_Dec2020\161Hex_isocraticC18 (1) 2021-03-19 12-09-54.D) 40 45 min 40 45 min 35.246 23.982 mAU 1750 35 1500 1250 1000 500 28.610 16.372 750 250 0 5 10 15 20 25 30 35 ===================================================================== Fig. 4. 61. HPLC DAD profile of hexane layer from direct extraction method at 210 and 230 nm. Area Percent Report ===================================================================== After HPLC analysis on analytical column, the method was developed on C18 reverse phase Sorted By : Signal Multiplier : 1.0000 HPLC semi preparative: column with acetonitrile and water gradients (Fig. 4.62). The peaks Dilution 1.0000 Sample Amount: : 1.00000 [ng/ul] (not used in calc.) Do not use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 C, Sig=210,4 Ref=off 180 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] % ----|-------|----|-------|----------|----------|--------| eluting at retention times 19.3, 24.5, 26, 32 and 39.1 min were fraction collected and designated as PK1, 2, 3, 4 and 5 respectively. Fig. 4. 62. Semi preparative HPLC DAD profile of hexane layer. The collected peaks were reinjected (Figs. 4.63 & 4.65) into a C18 analytical column to check for purity at multiple wavelengths using DAD (210, 230 nm). The solvent gradient (Acetonitrile: Water 80:20-90:10 in 15min, 90:10 from 15-22min, 90:10-80:20 in 25min) was used at 0.8 mL/min. All the peaks were also subjected to ESI-MS analysis to obtain the molecular mass (Figs. 4.64 & 4.69). Fig. 4. 63. HPLC DAD profile of PK1. 181 Da a File C:\ChemDa ...F collec _Feb21 2021-03-01 14-22-50\001-P1-A1-161.He .Pk2 e if .D Sam le Name: 161.He .Pk2 e if ===================================================================== Ac . O e a o : SYSTEM Se . Line : 1 Sam le O e a o : SYSTEM Ac . In men : LEE_HPLC Loca ion : P1-A-01 Injec ion Da e : 3/1/2021 2:23:53 PM Inj : 1 Inj Vol me : 7.000 l Diffe en Inj Vol me f om Sam le En ! Ac al Inj Vol me : 50.000 l Me hod : C:\ChemDa a\1\Da a\Meh een_Dec2020\161He Pk2 e if F collec _Feb21 2021-03-01 14-22-50\161He _c18_Jan2021.M (Se ence Me hod) La changed : 3/1/2021 2:20:42 PM b SYSTEM Addi ional Info : Peak( ) man all in eg a ed ===================================================================== Fig. 4. 64. MSD spectrum of PK1 in scan (100-1000 m/z) and SIM mode. DAD1 C, S =210,4 Ref= ff (Me ee _De... ec _Feb21 2021-03-01 14-22-50\001-P1-A1-161.He .P 2 e f .D) 29.975 mAU 22.963 2000 1500 1 2 1000 33.308 27.536 23.924 500 0 5 10 15 20 25 30 35 ================================================================================== F ac ion Info ma ion ================================================================================== F ac ion li , ime calc la ed ing he dela of RID1 (fi de ec o ). ================================================================================== F ac Well Loca ion Vol me BeginTime EndTime Rea on Ma # # [ l] [min] [min] ----- ----- ---------- ---------- ---------- ---------- ------------- -----------1 1 1 880.00 21.500 22.600 Time 2 1 2 1240.00 28.300 29.850 Time ================================================================================== ===================================================================== A ea Pe cen Re o ===================================================================== Fig. 4. 65. HPLC DAD spectrum of PK2. When PK2 was kept over a week and reinjected again, it showed a desired peak at 29 min (named PK2b) and an unexpected peak (named PK2a) at 22 min, suggesting the unstable nature of PK2. Therefore, several attempts were taken to repurify the PK2, initially on Phenomenex So ed B M l i lie Dil ion Do no e M l i lie : : : & Dil Signal 1.0000 1.0000 ion Fac o Luna C18(2) (Fig. 4.66) and then on Agilent Poroshell C18 (Fig. 4.67) in order to improve the i h ISTD resolution of peaks (as the latter has small pore size) and shorten the time of analysis. Signal 1: DAD1 C, Sig=210,4 Ref=off Peak Re Time # [min] ---- ------1 22.963 2 23.924 T e Wid h A ea Heigh A ea [min] [mAU* ] [mAU] % ---- ------- ---------- ---------- -------BV R 0.4596 4.00330e4 1316.92395 29.7643 VV E 0.4090 3286.90381 120.96574 2.4438 LEE_HPLC 3/2/2021 9:57:57 AM SYSTEM Page 182 1 of 2 mi Acq. Method : 161Hex_c18_Jan2021.M Analysis Method : C:\ChemData\1\Methods\DEF_LC.M Last changed : 4/23/2021 10:05:09 AM by SYSTEM (modified after loading) Additional Info : Peak(s) manually integrated DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161HexPK2repurify_C18 (1) 2021-03-18 10-37-38.D) mAU 500 28.604 400 300 22.004 200 100 0 5 10 15 20 25 30 35 min 35 min 28.610 Data File C:\ChemData\1\Data\Mehreen_Dec2020\161HexPk2_PoroC18A (1) 2021-04-06 10-45-13.D DAD1 D, Sig=230,4 Ref=off (Mehreen_Dec2020\161HexPK2repurify_C18 (1) 2021-03-18 10-37-38.D) Sample Name: 161HexPk2_PoroC18A mAU 500 22.000 ===================================================================== 400 Acq. Operator : SYSTEM Sample 300 Operator : SYSTEM Acq. Instrument : LEE_HPLC Location : P1-A-01 200 Injection Date : 4/6/2021 10:46:07 AM Inj : 1 Inj Volume : 7.000 µl 100 Different Inj Volume from Sample Entry! Actual Inj Volume : 4.000 µl Acq. 0 Method : C:\ChemData\1\Methods\Mehreen_Dec2020\161HexPk_PoroC18A_March2021.M 5 10 20 25 30 Last changed : 4/6/2021 10:52:43 AM by 15 SYSTEM (modified after loading) ================================================================================== Analysis Method : C:\ChemData\1\Methods\DEF_LC.M Last changed : 4/23/2021Fraction 10:03:47Information AM by SYSTEM ================================================================================== (modified after loading) No FractionsInfo found. Additional : Peak(s) manually integrated DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161HexPk2_PoroC18A (1) 2021-04-06 10-45-13.D) ================================================================================== mAU ================================================================================== ===================================================================== 2000 Area Percent Report ===================================================================== 10.163 Fig. 4. 66. HPLC DAD spectrum of PK2 on Phenomenex Luna C18(2) column. 1500 7.946 Sorted : Signal 1000 By Multiplier : 1.0000 500 Dilution : 1.0000 Sample Amount: : 1.00000 [ng/ul] 0 Use Multiplier & Dilution Factor with ISTDs (not used in calc.) Signal 1: DAD1 C, Sig=210,4 Ref=off 25 min 20 10.163 5 10 15 DAD1 D, Sig=230,4 Ref=off (Mehreen_Dec2020\161HexPk2_PoroC18A (1) 2021-04-06 10-45-13.D) mAU 2000 7.946 Peak RetTime Type Width Area Height Area # 1500[min] [min] [mAU*s] [mAU] % ----|-------|----|-------|----------|----------|--------| 1000 1 22.004 BB 0.3396 295.20825 13.47512 3.7401 2 50028.604 BB 0.5240 7597.79932 216.19472 96.2599 0 LEE_HPLC 4/23/2021 10:06:25 AM SYSTEM 5 Page 10 15 1 of 2 25 min 20 ================================================================================== Fraction Information ================================================================================== No Fractions found. ================================================================================== ================================================================================== ===================================================================== Area Percent Report ===================================================================== Fig. 4. 67. HPLC DAD spectrum of PK2 repurification on Agilent Poroshell C18 column. Sorted By : Signal Multiplier : 1.0000 Dilution : 1.0000 Sample Amount: : 1.00000 [ng/ul] Use Multiplier & Dilution Factor with ISTDs (not used in calc.) Page LEE_HPLC 4/23/2021 10:04:17 AM SYSTEM 183 1 of 2 Ana l y s i s Me t hod : C : \ Chem32 \ 1 \ Me t hods \ Hoo i X i an \ I - Tomen t os a . M La s t changed : 4 / 20 / 2021 1 : 40 : 03 PM by SYSTEM ( mod i f i ed a f t e r l oad i ng ) Add i t i ona l I n f o : Pe a k ( s ) manua l l y i n t eg r a t ed ES-API, Pos, 14.403 MSD1 TIC, MS File (C:\Chem32\1\Data\Mehreen_2021\2021-03-1014-41-24161HxPk2brepurefresh2x_c18withMS.D) 1000000 800000 600000 400000 200000 0 16 ES-API, Pos, 18 min 16 18 min 16 18 min 14.407 0 2 4 6 8 10 12 14 MSD2 TIC, MS File (C:\Chem32\1\Data\Mehreen_2021\2021-03-1014-41-24161HxPk2brepurefresh2x_c18withMS.D) 2000000 1750000 1500000 1250000 1000000 750000 500000 250000 0 0 2 4 6 8 10 12 14 VW D1 A, W avelength=210 nm (Mehreen_2021\2021-03-1014-41-24161HxPk2brepurefresh2x_c18withMS.D) 14.349 mAU 120 100 80 60 40 20 0 0 2 4 6 8 10 12 14 ================================================================================== Fig. 4. 68. HPLC profile ofF r freshly collected PK2b for purity check at MSD1 scan mode, MSD2 a c t i on I n f o r ma t i on ================================================================================== No F r a c t i ons f ound . ================================================================================== ================================================================================== SIM mode and UV at 210 nm. A Page 6120 4 / 20 / 2021 1 : 40 : 29 PM SYSTEM 1 of 2 B Fig. 4. 69. MSD spectrum of PK2 in scan mode and SIM mode. PK3 was pure when reinjected into HPLC (Fig. 4.70). The ESI-MS analysis of PK3 revealed molecular ion peak with m/z 437 in both scan and SIM mode (Fig. 4.71). The same molecular ion peak was present in PK5b that was later identified as echinodone. PK3 had different retention time compared to PK5b which could indicate that PK3 is an isomer of PK5b. Due to 184 Injection Date : 1/27/2021 2:11:37 PM Inj : 1 Inj Volume : 7.000 µl Different Inj Volume from Sample Entry! Actual Inj Volume : 5.000 µl Acq. Method : C:\ChemData\1\Methods\Mehreen_Dec2020\161Hex_c18_Jan2021.M very low abundance of PK312:18:54 even after rounds of purification, there was not sufficient Last changed : 1/27/2021 PMmultiple by SYSTEM Analysis Method : C:\ChemData\1\Methods\DEF_LC.M Last changed 9:54:52 AM by SYSTEM amount of PK3: to4/26/2021 perform further structural elucidation studies. (modified after loading) Additional Info : Peak(s) manually integrated DAD1 A, Sig=254,4 Ref=off (Mehreen_Dec2020\161HexPk3_C18A (1) 2021-01-27 14-10-41.D) 20.660 mAU 20 15 10 5 0 2.5 5 7.5 10 12.5 15 DAD1 B, Sig=230,4 Ref=off (Mehreen_Dec2020\161HexPk3_C18A (1) 2021-01-27 14-10-41.D) 17.5 20 17.5 20 22.5 min 20.660 mAU 20 15 10 5 0 2.5 5 7.5 10 12.5 15 22.5 min ===================================================================== Fig. 4. 70. HPLC DAD spectrum of PK3. Area Percent Report ===================================================================== 437.3 *M S D 1S P C ,tim e=10.278of C :\C hem 32\1\D ata\M ehreen_2021\2021-03-2912-56-34161H exP k3_P oroc18A .D E S -A P I,P os,S can,Fr 100 Sorted By : Signal Multiplier : 1.0000 Dilution 80 : 1.0000 Sample Amount: : 1.00000 [ng/ul] (not used in calc.) Do not use 60 Multiplier & Dilution Factor with ISTDs 20 560.3 40 Signal 1: DAD1 A, Sig=254,4 Ref=off 519.4 438.3 A 354.97916 700 800 900 /z 1000 m 437.0 Totals : 479.3 419.3 439.4 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] % 0 ----|-------|----|-------|----------|----------|--------| 100 200 300 420.78864 00 500 1 20.660 BB 0.2632 354.97916 100.0000600 , Fra *M SD2SPC, tim e=10.288of C:\Chem 32\1\Data\M ehreen_2021\2021-03-2912-56-34161HexPk3_Poroc18A.D ES-API, Pos, SIM 100 Max: 127408 Max: 289856 20.78864 80 Page LEE_HPLC 4/26/2021 9:56:26 AM SYSTEM B 1 of 2 60 40 439.0 20 0 395 400 405 410 415 420 425 430 Fig. 4. 71. ESI-MS spectrum of PK3 in scan mode (A) and SIM mode (B). 185 435 440 m/z 4.3.3.3. Characterization of compound (4) PK4 was designated as compound (4): it was obtained as colorless needle shaped crystals, with a molecular formula of C32H50O4 which was obtained from NMR data, X-Ray crystallography and ESI-MS ion peaks. 4.3.3.4. HPLC and ESI-MS analysis of compound (4) The UV absorption maxima of compound (4) was obtained at 210 and 230 nm (Fig. 4.72). Fig. 4. 72. UV DAD profile of compound (4) at 210 and 230 nm. Compound (4) was subjected to mass spectral analysis using scan and SIM mode. The MS spectrum (Fig. 4.73) of compound (4) revealed MS ion peak of m/z 439 [M+H]. The m/z 439 from the ion chromatogram represented the base peak as a major deacetylated fragment of the parent compound. 186 Fig. 4. 73. MS spectrum of compound (4) in scan mode 100-1000 m/z (A) and SIM mode (B). 4.3.3.5. Structural elucidation by FTIR and NMR spectroscopy The IR spectrum of compound (4) (Fig. 4.74) showed peak stretches at 2937 cm-1 indicative 3500 3000 2500 W 2000 -1 C: U4. 74. A B OPUS 8.0.19 DATA MEAS Fig. FTIRD spectrum of compound (4).161H P 4E P .0 1500 161H P 4E 1000 ALPHA-II / D ATR 441.40 601.83 1099.38 1037.17 1014.94 932.33 911.10 1240.44 1369.05 1455.10 1738.51 1707.71 2937.13 3273.14 80 85 T 90 % 95 of sp3 C-H, a weak stretch at 3273 cm-1 for hydroxy group and at 1738 cm-1 for C=O group. 500 4/8/2021 1/1 Based on the 1D NMR data, compound (4) possessed 32 carbons (Fig. 4.76), that included seven methylenes (δC 35.2, 27.5, 18.0, 26.7, 19.9, 29.7, and 35.3), nine methyl groups (δC 15.9, 18.3, 14.5, 24.8, 17.6, 27.4, 15.0, 27.1, and 20.0), a carbonyl (δC 170.2), seven quaternary carbons (δC 38.6, 133.9, 135.1, 37.0, 48.9, 43.5, and 37.2), eight methine groups (δC 50.3, 82.6, 53.2, 38.8, 77.7, 77.8, and 78.1), and vinylic carbon (δC 123.3). The HSQC data showed 13C signals correlating well with the 1H NMR peaks (Table 4.9, 4.10 & Fig. 4.75). There were several methylene proton pairs (Ha and Hb) identified from HSQC signals including H-1a (δH 1.72, 1H) and 1-Hb (δH 1.22, 1H), overlapped H2-a/b (δH 1.57, 2H), 187 H-6a (δH 1.73, 1H) and H-6b (δH 1.56, 1H), H-7a (δH 2.17, peak hidden behind water peak) and H-7b (δH 2.02, 1H), overlapped H-11a/b (δH 2.08, 2H), H-12a (δH 1.84, 1H) and H-12b (δH 1.58, 1H), H-15a (δH 2.31, 1H) and H15b (δH 1.14, 1H). The H-24 (δH 5.03, 1H) attached to the unsaturated carbon was de-shielded the most. H-24 was cis-coupled to the H-26 (δH 1.70, 3H) and trans-coupled to H-27 (δH 1.69, 3H). The H-32 acetyl protons (δH 1.96, 3H) were overlapped by the CD3CN solvent peak. Additionally, there was also a peak for a hydroxy proton (δH 2.49, 1H). Fig. 4. 75. 1H NMR of compound (4). 188 Fig. 4. 76. 13C NMR of compound (4). Table 4. 9. 13C Chemical shift data of compound (4) 13 Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 C Chemical shift δ (ppm) 35.2 27.5 77.8 38.6 50.3 18.0 26.7 133.9 135.1 37.0 19.9 29.7 48.9 43.5 35.3 82.6 53.2 15.9 18.3 38.8 14.5 77.7 78.1 123.3 137.2 27.4 15.0 27.4 15.0 27.1 170.2 20.0 Functionality CH2 CH2 CH °4 C CH CH2 CH2 °4 C °4 C °4 C CH2 CH2 °4 C °4 C CH2 CH CH CH3 CH3 CH CH3 CH CH =CH °4 C CH3 CH3 CH3 CH3 CH3 C=O CH3 Table 4. 10. 1H Chemical shift data of compound (4) Number 1 1a 1b 2a/b 3 5 6a 6b H Chemical shift δ ppm (#, multiplicity) 1.63-1.56 (1H, m) 1.14-1.07 (1H, m) 1.57 (2H) 3.04-2.98 (1H, m) 1.04 (1H) 1.73 (1H, m) 1.56 (1H, m) 7a 2.17 (1H) J value (Hz) Comments Signal overlap with 26 and 27 Signal overlap H-2a and H-2b Signal overlap with H-30 H-6a overlap with H-1a, H-6b overlap with H-17, H-12b, and H2a/b H-7a behind water peak 189 7b 11a/b 2.02 (1H, m) 2.08 (2H, m) 12a 12b 15a 15b 16 1.84 (1H) 1.58 (1H) 2.31 (1H, dd) 1.14 (1H, dd) 3.83 (1H, ddd) 17 18 19 20 21 22 23 24 26 27 28 29 30 32 OH 1.60 (1H, m) 0.60 (3H, s) 1.01 (3H, s) 1.73 (1H, m) 0.78 (3H, d) 4.31 (1H, t) 3.97 (1H, t) 4.91 (1H, dq) Signal overlap between H-11a/b and H7b Signal overlap with H-20 14, 10.9 14, 4.8 10.8, 9.4, 4.8 Signal overlap with 2a/b, 12b and 6b Signal overlap with 12a 6.4 9.5 9.1 8.9, 1.4 1.63-1.56 (7H, m) 0.86 (3H, s) 0.67 (3H, s) 1.04 (3H, s) 1.9 (3H) 2.38 (1H, d) Signal overlap with 1a Signal behind CD3CN solvent peak 5.5 Fig. 4. 77. 13C-1H HSQC spectrum of compound (4). 190 Fig. 4. 78. 13C-1H HMBC spectrum of compound (4). Fig. 4. 79. 2D COSY and HMBC structure correlations of compound (4). 191 Fig. 4. 80. 1H-1H COSY spectrum of compound (4). Fig. 4. 81. 1H-1H NOESY spectrum of compound (4). 192 4.3.3.6. Crystallization and crystal data of compound (4) Compound (4) was crystallized using three different methods. Method 1 resulted in formation of thin and small fragments of needle-like crystals that were mixed with powder (Fig. 4.82A). Method 2 resulted in the formation of bunches of white shiny crystals (Fig. 4.82B). Finally, method 3 resulted in formation of very thin and long needle-like crystals (Fig. 4.82C). Crystals obtained from method 2 were subjected to XRD analysis. For XRD analysis, the crystal with dimensions 0.24 × 0.05 × 0.05 mm3 was selected, and it revealed a crystal structure as shown in ORTEP style, which was later identified as echinodol (Fig. 4.83). Detailed crystal data can be found in supplementary data of appendix C. Fig. 4. 82. Crystallization of compound (4) using method 1 (A), method 2 (B), and method 3 (C). 193 Fig. 4. 83. ORTEP style image of compound (4). X-Ray crystal data: C32H50O4, Mr = 498.72, monoclinic, P21 (No.4), a= 7.9203(3) Å, b= 20.6269(8) Å, c= 17.4422(6) Å, b= 97.890 (2)°, a = g = 90°, V= 2822.58(18) Å3, T= 110(2) K, Z= 4, Z'= 2, µ(CuKa)= 0.585, 78352 reflections measured, 8399 unique (Rint = 0.0722) which were used in all calculations. The final wR2 was 0.1093 (all data) and R1 was 0.0429 (I≥2 s(I)). Structural elucidation studies confirmed that compound (4) is echinodol, a lanostane-type triterpene acetate which has been reported together with its chemically-modified derivatives from E. tinctorium (Bond et al., 1966). To date, there is no study which has reported the crystal structure of echinodol, and no study conducted to explore its bioactive potential. Therefore, further experiments were carried out to explore the growth-inhibitory potential of echinodol. 4.3.3.7. Growth-inhibitory effects of (4) on cancer cells Echinodol caused growth inhibition in HeLa cervical cancer cells (Fig. 4.84A) with an IC50 of 1.2 µM. It also showed anti-proliferative effects (Table 4.11) against U251 glioblastoma cells, SVG immortalized glial cells, RIE-1 Rat intestinal epithelial cells, U87 glioblastoma cells, HCT116 human colon cancer cells, and SW480 colorectal cancer cells with an IC50 of 4.6 µM, 4.09 µM, 2.28 µM, 5.41 µM, 5.37 µM, and 3.65 µM respectively. Results shown in Table 4.11 were derived from experiments performed by Dr. Chow Lee. 194 % Cell Viability (% Control) 100 50 0 0.1 1 10 100 Concentration (logµM) Fig. 4. 84. Effect of compound (4) at 48 h treatment on growth inhibition of HeLa cervical cancer cells. Result shown is a representative from three biological replicates. Table 4. 11. IC50 of compound (4) on multiple cell lines Cell Lines Average IC50 (µM)* SVG Immortalized glial cells 4.09 ± 0.387 U251 Glioblastoma cells 4.62 ± 0.664 U87 Glioblastoma cells 5.41 ± 1.80 RIE-1 Rat intestinal epithelial cells 2.28 ± 0.496 HCT116 Human colon cancer cells 5.37 ± 1.146 SW480 Colorectal cancer cells 3.65 ± 0.961 *Results obtained from three biological replicates (n=3). 4.3.3.8. Flow cytometry analysis of compound (4) Compound (4) induced apoptosis in U251 cells at 40 µM. After 24 h of treatment with 40 µM of (4), significant (p = 0.002) apoptosis was observed with 57 % apoptotic cells as compared to 4.6 µM and 10 µM treatments with 12.6 % and 13 % apoptotic cells respectively. After 48 h treatment with 40, 4.6 and 10 µM of compound (4), there were 61, 41, and 40 % of apoptotic cells respectively (Fig. 4.85). 195 Fig. 4. 85. Apoptosis induced by compound (4) at 4.6, 10 and 40 µM in U251 cells (A), % apoptotic cells at 24 h (B) and 48 h (C). Results in (B) and (C) are from three biological replicates (n=3). For statistical analysis, one-way ANOVA was used. * represents p = 0.002 and ** shows p < 0.0001. The cell cycle analysis after treatment with 40 µM of compound (4) for 24 h showed only a slight increase in S phase cells (Fig. 4.86A). In contrast, there was a significant increase (p = 00012) in cell population in S phase (Fig. 4.86B) after treatment with 40 µM dose of compound (4) for 48 h. The results indicated an S phase arrest induced by compound (4). 196 2June2021_U251Cellcycle_24h_DMSO control_24h.fcs Cell Cycle 45061 DMSO 4.0K G1= 47.5 % S= 45.2 % G2/M= 6.19 % 6.0K Compound (4) 4.0K G1= 45.4 % S= 46.8 % G2/M= 5.86 % Count 24h 6.0K Count A 2June2021_U251Cellcycle_24h_Echinodol 40uM_24h.fcs Cell Cycle 44216 2.0K 2.0K 4June2021_U251Cellcycle_48h_DMSO control_48h.fcs 0 Cell Cycle 0 50K 100K 150K 200K 250K 46680 0 3June2021_U251Cellcycle_48h_Echinodol 40uM_48h.fcs Cell 0Cycle 50K 100K 150K 200K 250K 40911 PI*-A :: PI*-A PI*-A :: PI*-A DMSO 6.0K RMSD : 4,80 %G1 : 47,5 %S : 45,2 %G2 : 6,19G1= 68.7 % G1 Mean :S= 65340 27.9 % G2 Mean : 128200 G2/M= 4.95 % G1 CV : 3,02 G2 CV : 2,48 % less G1 : 0,73 % greater G2 : 0,17 48h Count 4.0K 2.0K RMSD : Compound 8,27 (4) %G1 : 45,4 %S : 46,8 %G2 : 5,86 G1= 47.7 % G1 Mean : 68163 G2 MeanS= : 134164 43.8 % G1 CV : 2,66 8.86 % G2 CV : G2/M= 2,20 % less G1 : 1,83 % greater G2 : 0,13 5.0K 4.0K Count B 3.0K 2.0K 1.0K 0 0 0 50K 100K 150K 200K 250K 0 50K 100K PI*-A :: PI*-A 150K 200K 250K PI*-A :: PI*-A RMSD : 10,2 %G1 : 68,7 %S : 27,9 %G2 : 4,95 G1 Mean : 51370 G2 Mean : 99828 G1 CV : 5,43 G2 CV : 6,34 % less G1 : -1,74 % greater G2 : -0,066 RMSD : 6,60 Fig. 4. 86. Cell cycle analysis of compound (4) at%G140: 47,7 µM for 24 h (A) and 48 h (B) for n=3. Error %S : 43,8 %G2 : 8,86 G1 Mean : 57627 G2 Mean : 112376 G1 CV : 3,39 G2 CV : 3,65 % less G1 : 0,71 % greater G2 : -0,044 bars represent standard deviation. One-way ANOVA was used for statistical analysis. ** represent p = 0.0002 and * shows p = 0.0012. 4.3.3.9. HPLC and ESI-MS analysis of PK5 PK5 purified from hexane layer using semi-preparative HPLC column was reinjected to check purity. The analysis discovered the presence of two peaks in the purified fraction which were named as PK5a (later on renamed as compound (6)) and PK5b (later on renamed as compound (5)). Repurification of PK5 was carried out initially on Phenomenex Luna C18(2) HPLC column F C: C D ... D 2020 161H K5 C18 (1) 2021-03-16 12-46-22.D : 161H K5 C18 ===================================================================== A . : E : E A . I : EE H C : 1-A-01 I D : 3/16/2021 12:47:16 I : 1 I : 7.000 D I E ! A I : 8.000 A . : C: C D 1 D 2020 161H 18 J 2021. : 3/16/2021 12:46:15 E A : C: C D 1 D 2020 161H 18 J 2021. : 3/17/2021 1:22:16 E ( ) A I : ( ) ===================================================================== (Fig. 4.87) and later on method developed using Agilent Zorbax Poroshell C18 HPLC column (Fig. 4.88). Both columns were able to resolve the two peaks and Poroshell C18 column also reduced the analysis time. DAD1 C, S g=210,4 Ref= ff (Meh ee _Dec2020\161He PK5 e f _C18 (1) 2021-03-16 12-46-22.D) 34.142 mAU 39.012 350 300 250 1 11.849 100 50 13.129 150 2 200 6.552 D 0 5 10 15 20 25 30 35 ================================================================================== F I ================================================================================== F , ID1 ( ). ================================================================================== F B E # # ----- ----- ---------- ---------- ---------- ---------- ------------- -----------1 1 1 1600.00 33.000 34.600 2 1 2 2100.00 37.900 40.000 ================================================================================== ===================================================================== A ===================================================================== Fig. 4. 87. Repurification of PK5 on Phenomenex Luna C18(2). 197 B : : 1.0000 40 mi Last changed : 3/15/2021 2:41:40 PM by SYSTEM Analysis Method : C:\ChemData\1\Methods\DEF_LC.M Last changed : 4/23/2021 10:10:19 AM by SYSTEM (modified after loading) Additional Info : Peak(s) manually integrated DAD1 C, Sig=210,4 Ref=off (Mehreen_Dec2020\161HexPK5repurify_C18poroshell (1) 2021-03-15 14-42-01.D) mAU 1750 1250 13.023 11.629 1500 1000 2.385 750 500 250 0 2 4 6 8 10 DAD1 D, Sig=230,4 Ref=off (Mehreen_Dec2020\161HexPK5repurify_C18poroshell (1) 2021-03-15 14-42-01.D) min 12 11.629 mAU 1750 1500 1250 1000 500 13.024 2.386 750 250 0 2 4 6 8 10 min 12 ===================================================================== Area Percent ReportZorbax Poroshell C18. Fig. 4. 88. Repurification of PK5 on Agilent ===================================================================== Signal (5) and was reinjected into HPLC to check for purity as well PK5b was renamed :as compound Sorted By Multiplier Dilution Sample Amount: Do not use Multiplier : 1.0000 : 1.0000 : 1.00000 [ng/ul] (not used in calc.) & Dilution Factor with ISTDs as for ESI-MS analysis. HPLC DAD spectrum showed a 100 % pure peak of compound (5) at 254 and 230 nm (Fig. 4.89). ESI-MS (Fig. 4.90) showed a small parent molecular ion peak with Signal 1: DAD1 C, Sig=210,4 Ref=off Peak RetTime Type Width Area Height Area m/z# 497 [M+H] and[min] its fragment base peak with m/z 437 [M+H]. The fragment at 437 was [min] [mAU*s] [mAU] % ----|-------|----|-------|----------|----------|--------| 1 2.385 VB R 0.0673 1387.31384 272.95090 5.1841 2 11.629 BB 0.1772 1.21004e4 1044.18567 45.2170 3 13.023 BBA 0.2624 1.32730e4 743.08844 49.5989 possibly the deacetylated form of compound (5). Page LEE_HPLC 4/23/2021 10:10:22 AM SYSTEM Fig. 4. 89. HPLC DAD spectrum of compound (5). 198 1 of 2 A B Fig. 4. 90. Positive mode ESI-MS of compound (5) in scan mode 100-1000 Da (A) and SIM mode (B). 4.3.3.10. Structural elucidation of (5) by NMR spectroscopy Most of the signals from the 1H NMR, 13C NMR, 1H-13C HSQC and HMBC, 1H-1H COSY and NOESY of compound (5) shown in Figures 4.91-4.96 & Tables 4.12 & 4.13, resembled the NMR data of compound (4), indicating structural similarity of both compounds. This was later confirmed from the crystal structure. Compound (4) has a hydroxy at carbon 3 which is replaced by a carbonyl group in compound (5). Therefore, (5) has two carbonyl groups present which was also confirmed from the 13C NMR data (Fig. 4.90 & Table 4.13). 199                                                                        18 12 19 2 3 11 17 13  31 21  20 22 23 24 10 5 4 9 6 8 7 25  26 16  14 1   32 27 15  30   29 28                                                !!"!! !#!$%% "#! "#%% %% %  Fig. 4. 91. 1H NMR of compound (5). Table 4. 12. 1H NMR chemical shift data of compound (5) Number 1 J value (Hz) 1a 1b 2a/b 5 6a/b H Chemical shift δ ppm (#, multiplicity) 2.1 (1H, m) 1.69-1.62 (>6H, m) 1.69-1.62 (>6H, m) 1.69-1.62 (>6H, m) 2.1-2.25 (m) 7a 7b 11a/b 2.6 (1H, ddd) 2.3-2.29 (2H, m) 2.1-2.25 (m) 15.8, 11.5, 7 12a 12b 15a 15b 16 17 18 19 20 21 22 23 24 26 27 28 29 30 1.87 (2H, qd) 1.69-1.62 (>6H, m) 2.38-2.29 (2H, m) 1.18 (1H) 3.85 (1H, td) 1.69-1.62 (>6H, m) 0.75 (3H, s) 1.07 (3H, s) 1.87 (2H, qd) 0.9 (3H, d) 4.44 (1H, t) 4.10 (1H, t) 5.04 (1H, dt) 1.71 (3H, s) 1.70 (3H, s) 1.07 (3H, s) 1.06 (3H, s) 1.14 (3H, s) 10.1, 6.8 Comments Signal overlap with CD3CN solvent peak Signal overlap Signal overlap Signal overlap with H-11a/b and water peak Signal overlap with H-6a/b and water peak Signal overlap with H-20 Signal overlap 10.1, 4.8 Signal overlap 10.1, 6.8 6.5 9.5 9.1 8.7, 1.5 200 Signal overlap with 12a 32 1.9 (3H) Signal behind CD3CN solvent peak Table 4. 13. Chemical shift data of compound (5) from 13C NMR Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 13 C Chemical shift δ (ppm) 35.4 19.1 216.5 43.5 51.0 26.5 34.1 133.8 134.8 36.9 19.9 29.7 49.1 47.0 35.5 82.5 53.2 16.0 25.4 38.8 14.5 77.7 78.1 123.2 137.3 24.8 17.6 27.0 20.6 17.7 170.2 20.0 Functionality CH2 CH2 C=O °4 C CH CH2 CH2 °4 C °4 C °4 C CH2 CH2 °4 C °4 C CH2 CH CH CH3 CH3 CH CH3 CH CH =CH °4 C CH3 CH3 CH3 CH3 CH3 C=O CH3 201 12 11 19 2 3 29 17 13 20 22 23 9 10 5 4 25 26  16  27  30 7 6   24 15 8   14 1                                31 21 18  32             28                                                  32 18 12 19 2 3 29 11 13 17 31 14 1 10 5 4 9 6 8 7 15 20 22 23 24 25                              Fig. 4. 92. 13C NMR spectrum of compound (5). 21  26 16 27 30 28 Fig. 4. 93. 1H-1H COSY spectrum of compound (5). 202 32 31 21 18 12 11 19 2 3 17 13 20 22 23 14 1 10 5 4 29 9 30 7 6 26 25 27 15 8 24 16 28 Fig. 4. 94. 1H-1H NOESY spectrum of compound (5). 32 31 21 18 12 19 2 3 29 11 13 17 14 1 10 5 4 9 6 8 7 15 20 22 23 24 25 26 16 27 30 28 Fig. 4. 95. 1H-13C HSQC spectrum of compound (5). 203 32 31 21 18 12 19 2 3 29 11 13 17 14 1 10 5 4 9 6 8 7 15 20 22 23 24 25 26 16 27 30 28 Fig. 4. 96. 1H-13C HMBC spectrum of compound (5). 4.3.3.11. Crystallization and crystal structure of compound (5) Colorless crystals were obtained from both method 2 and 3 (Fig. 4.97B & C). The crystals obtained from method 1 were very small (Fig. 4.97A). The crystals from method 2 and 3 qualified for XRD analysis. For XRD analysis, a single flat crystal with dimensions 0.28 × 0.24 × 0.06 mm3 was used. The XRD analysis revealed the final crystal structure of compound (5) (Fig. 4.97D). Detailed crystal data can be found in supplementary data of appendix C. 204 B A C D Fig. 4. 97. Crystals of (5) obtained from method 1 (A), method 2 (B) and method 3 (C), and ORTEP style image of (5) from X ray crystallography (D). Crystal Data. C32H48O4, Mr = 496.70, orthorhombic, P212121 (No. 19), a = 8.0284(2) Å, b = 10.6283(3) Å, c = 32.8305(9) Å, a = b = g = 90°, V = 2801.37(13) Å3, T = 110(2) K, Z = 4, Z' = 1, µ(CuKa) = 0.589, 51100 reflections measured, 5125 unique (Rint = 0.0369) which were used in all calculations. The final wR2 was 0.0725 (all data) and R1 was 0.0281 (I≥2 s(I)). The FTIR, NMR, ESI-MS along with the crystal data confirmed compound (5) to be echinodone, which is a lanostane-type triterpenoid. One of the previous studies has reported that echinodone was isolated from organic extracts of E. tsugicola fruiting bodies (Kanematsu et al., 1972). E. tsugicola is one of the phylogenetically related species of E. tinctorium. Echinodone shares the same nucleus as echinodol with the exception of a hydroxyl group replaced by a carbonyl group in the structure. To date, there is no study conducted to explore the bioactive potential of echinodone. 4.3.3.12. Growth inhibitory activity of (5) Compound (5) caused growth inhibition in U251 cells with an IC50 of 5.45 µM + 1.76 (Fig. 4.98). The IC50 of compound (5) was comparable to compound (4) in inhibiting U251 cells. Due 205 to their structural similarities, both molecules likely inhibit the growth of U251 cells via a similar % Cell Viability (% Control) pathway. 100 50 0 10 100 Concentration (logµM) Fig. 4. 98. Growth inhibition caused by compound (5) after 48 h treatment in U251 cells. Result shown is a representative from three biological replicates. 4.3.3.13. HPLC analysis of PK5a Once it was repurified from compound (5), PK5a was reinjected into HPLC to determine its F % purity. Fig. 4.99 shows the spectrum where(1) two distinct peaks can be seen; (6) C: C D ... M D HPLC 2020 161H 5 2of (6) C18A 2021-03-30 10-45-02.D : 161H 5 2 C18A retained at 11.9 min and another compound retained at 2.3 min. This indicated that (6) was ===================================================================== A . : EM : EM A . I : LEE H LC L : 1-A-01 I D : 3/30/2021 10:45:59 AM I : 1 I : 7.000 D I E ! A I : 12.000 A . M : C: CHEMDA A 1 ME H D MEH EE DEC2020 161H C18A M L : 3/30/2021 10:44:21 AM EM A M : C: C D 1 M DEF LC.M L : 3/31/2021 11:10:37 AM EM ( ) degraded into an undesirable compound probably due to its sensitivity to light, air or having been 2021.M kept in liquid storage for too long. DAD1 C, S g=210,4 Ref= ff (Me ee _Dec2020\161He P 5a2 _P C18A (1) 2021-03-30 10-45-02.D) 4 6 ee _Dec2020\161He P 5a2 _P 8 C18A (1) 2021-03-30 10-45-02.D) 11.947 2.327 mAU 350 300 250 200 1 150 100 50 0 2 DAD1 D, S g=230,4 Ref= ff (Me 10 12 11.947 mAU mi 700 2.327 600 500 400 300 1 D 200 100 0 2 4 6 8 10 ================================================================================== F I ================================================================================== F , ID1 ( ). ================================================================================== F L B E M # # ----- ----- ---------- ---------- ---------- ---------- ------------- -----------1 1 1 699.83 11.000 11.700 ================================================================================== ===================================================================== A ===================================================================== 12 mi Fig. 4. 99. HPLC DAD spectrum of PK5a. In order to determine the stability issues of (6), the freshly repurified (6) was kept in dark (wrapped with aluminum foil), cold and lyophilized on the same day after its collection. The M D lyophilized (6) was:resuspended in acetonitrile and injected into HPLC for determination of % B : 1.0000 : 1.0000 206 A M : : & D 1.00000 F I / D ( .) purity (Fig. 4.100). PK5a was then renamed as compound (6). The HPLC DAD spectrum of compound (6) revealed a pure peak which indicated that it was a sensitive compound that might D F C: C D ...M D 2020 161H 5 2 C18A (1) 2021-03-31 11-05-49.D N : 161H 5degradation. 2 C18A have undergone The positive mode ESI-MS indicated a molecular ion peak with ===================================================================== A . : EM : EM A . I : LEE H LC L : 1-A-01 I D : 3/31/2021 11:06:44 AM I : 1 I : 7.000 D I E ! A I : 20.000 A . M : C: C D 1 M M D 2020 161H C18A M L : 3/31/2021 11:20:21 AM EM ( ) A M : C: C D 1 M DEF LC.M L : 3/31/2021 11:21:16 AM EM ( ) m/z 441 [M+H] (Fig. 4.101). Compound (6) has the same mass as deacetoxyechinodol which was isolated from E. tsugicola, so it is likely that it is the same compound present here in E. 2021.M tinctorium. DAD1 C, S =210,4 Re = (Me ee _Dec2020\161He P 5a2 da _P C18A (1) 2021-03-31 11-05-49.D) DAD1 D, S 2 =230,4 Re = (Me 4 6 ee _Dec2020\161He P 5a2 da _P 8 10 C18A (1) 2021-03-31 11-05-49.D) DAD1 F, S 2 =260,4 Re = (Me 4 6 ee _Dec2020\161He P 5a2 da _P 8 10 C18A (1) 2021-03-31 11-05-49.D) 11.649 mAU 800 600 1 400 200 0 mi 12 mi 12 mi 11.648 mAU 12 1500 1 1000 500 0 11.648 mAU 1 150 125 100 75 50 25 0 2 4 6 8 10 ================================================================================== F I ================================================================================== F , ID1 ( ). ================================================================================== F L B E M # # ----- ----- ---------- ---------- ---------- ---------- ------------- -----------1 1 1 800.00 10.700 11.500 ================================================================================== Fig. 4. 100. HPLC DAD spectrum of compound (6). A LEE H LC 3/31/2021 11:21:25 AM EM 1 B Fig. 4. 101. ESI-MS spectrum of compound (6) in scan mode (A) and SIM mode (B). 207 2 4.3.3.14. Melting point determination Melting point for compound (2) was determined as 208.3-209.5 ºC. Compound (4) on the other hand had melting point of 228.5-230 ºC. Both melting points had narrow range indicating the purity of compounds. Additionally, a high melting point suggested the structural stability and complexity of compounds (2) and (4). 4.3.3.15. HPLC and ESI-MS analysis of DEE layer The HPLC spectrum of DEE showed several peaks (Fig. 4.102) at 230 nm. The mass of the retained peaks was determined in the positive mode ESI. The ESI-MS analysis revealed common m/z ions as that of compounds found in hexane layer which included m/z 287 Da at 14.5 min, 439 Da at 17.5 min, and 437 Da at 21.4 min. Since these compounds were also identified from the hexane layer, the DEE layer was not pursued further. Fig. 4. 102. HPLC analysis of DEE. A summary of all the small molecules isolated from E. tinctorium is shown in Table 4.14. Table 4. 14. Small molecules isolated from E. tinctorium # Compound name Chemical class Molar mass (g/mol) 124 FTIR NMR 1 Orcinol Phenol derivative Yes 1 2 bis(2,4dihydroxy6methylphen yl) methane 3 Unknown Diphenyl methane derivative 260 Yes NA 506 Yes 13 Crystal structure 1 13 H, C, DEPT135, H- C 1 13 1 1 HSQC, H- C HMBC, H- H 1 1 COSY, H- H NOESY 1 13 1 13 H, C, DEPT135, H- C 1 13 1 1 HSQC, H- C HMBC, H- H 1 1 COSY, H- H NOESY No 1 No 13 1 13 H, C, DEPT135, H- C 1 13 1 1 HSQC, H- C HMBC, H- H 1 1 COSY, H- H NOESY 208 Yes 4 Echinodol Lanostane triterpenoi ds 498 5 Echinodone Lanostane triterpenoi ds 496 6 Deacetoxye chinodol Lanostane triterpenoi ds 440 4.3.3.16. 1 Yes 13 1 13 H, C, DEPT135, H- C 1 13 1 1 HSQC, H- C HMBC, H- H 1 1 COSY, H- H NOESY 1 13 1 13 H, C, DEPT135, H- C 1 13 1 1 HSQC, H- C HMBC, H- H 1 1 COSY, H- H NOESY Yes Yes No Target prediction of compounds 1-5 Target prediction can be done with biochemical techniques and computational tools. In the past, biochemical techniques were considered more reliable. Nowadays, computational prediction methods offer a fast, cheap and accurate target prediction. Some of the available web tools include SEA (Keiser et al., 2009), SwissTargetPrediction (Gfeller et al., 2014), HitPick (Liu et al., 2013), TarPred (Liu et al., 2015), and MolTarPred (Peon et al., 2019). Amongst these, MolTarPred is the most accurate user-friendly web tool due to a large number of targets, types of organisms, and reliability score to identify the predictions (Peon et al., 2019). Due to these advantages, the MolTarPred program was used to predict the molecular targets for compounds (1), (2), (4), and (5). Generally, a score of 3 or more indicates a reliable target prediction. For compound (1), 30 targets were identified by MolTarPred with a reliability score equal to and less than 2, except for one predicted target named prelamin-A/C which had a reliability score of 3. Prelamin-A/C is a protein identified in humans and is present in the nuclear lamina to promote nuclear stability (Mattioli et al., 2011). For compound (2), a total of 16 targets were identified by MolTarPred with a low reliability of prediction score (1-2). Surprisingly, all the identified targets had low reliability of prediction. This is likely due to the fact that compound (2) has not been studied before or the targets have not been identified in any organisms. For compound (4), 5 targets were identified, two of them with a reliability of prediction score 1 and one with a score 2. One predicted target, Prostaglandin E synthase enzyme, had a reliability score 3 and another protein target 7-dehydrocholesterol reductase had a reliability 209 score of 4. The 7-dehydrocholesterol reductase is an important terminal enzyme in biosynthesis of cholesterol. Cholesterol biosynthesis is important for neuronal development, which is sometime disrupted due to certain genetic defects. One such genetic defect is associated with Smith-Lemli-Opitz syndrome (SLOS), caused by gene mutation that encodes for 7dehydrocholesterol reductase thereby inhibiting the conversion of 7-dehydrocholesterol to cholesterol (Horling et al., 2012). Compound (4) could be a possible lead molecule for treatment of such neurological disorders. Another reliable protein target for compound (4) was prostaglandin E synthase enzyme, involved in cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) synthesis. Prostaglandins play important role in inflammation. PGE2 has been identified as a novel target against inflammation and cancer, including brain cancers (Murakami & Kudo, 2006). Medulloblastoma (MB) is a common malignant brain cancer that occurs in children; it is associated with overexpressed COX-2 and PGE2 synthase (Baryawno et al., 2008). Compound (4) has shown promising effect in glioblastoma cells and could be a potential target to inhibit the PGE2 synthase and thereby address MB. For compound (5), 11 molecular targets were predicted with a reliability score 1 for 10 targets, 2 for one target and 3 for another target named Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, also known as PPIase Pin1 and Rotamase Pin1, is a protein target identified in humans. Pin1 is an isomerase enzyme that catalyzes the cis/trans isomerization. Pin1 is believed to be an attractive target in cancer research due to its over expression in various cancers, especially brain cancers (Bao et al., 2004). The role of Pin1 in regulating cell differentiation has been identified through multiple molecular pathways (Lu, 2003). According to one study, knockdown of Pin1 reduced the tumorigenic features of glioblastoma cells through apoptosis and reduced cell migration (Atabay et al., 2015). Compound (5) has shown growth inhibition in glioblastoma cells and acting through Pin1 identified protein target could be one of the possible mechanisms for inducing apoptosis. A summary of the predicted molecular targets for compounds (1), (2), (4) and (5) is shown in Table 4.15. 210 Table 4. 15. Predicted molecular targets of compounds from E. tinctorium using MolTarPred Compound# Predicted molecular target Reliability of Function prediction 1 Prelamin-A/C 3 Nuclear stability 2 No reliable target <3 NA 4 7-dehydrocholesterol 4 Cholesterol biosynthesis Prostaglandin E synthase 3 COX derived PGE2 synthesis Rotamase Pin1 3 Cis/trans isomerization, reductase 5 Regulates cell differentiation 4.4. Conclusion Compounds (1-6) were successfully isolated from organic extracts of E. tinctorium. (1), (4), (5), and (6) are known compounds previously isolated from other species. Compound (4) was also previously isolated from E. tinctorium. I report here, for the first time, the isolation of compound (2) from a natural origin. There were no previous reports on bioactivity analysis of molecules (2), (4), and (5), therefore, the growth-inhibitory potential was investigated here. All the compounds were obtained with high purity at multiple wavelengths and their structure was successfully elucidated using FTIR, ESI-MS, XRD and NMR analyses. Using MolTarPred program, potential protein targets that may play a role in their growth-inhibitory activity, were identified for compounds (1), (2), (4), and (5). Further research is clearly required to determine whether these proteins are true molecular targets for these compounds. In addition, animal studies are needed to assess the potential anti-cancer activity of these compounds, especially for compounds (4) and (5). Appendix C. Supplementary data Supplementary material related to this chapter can be found in Appendix C. 4.5. 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Ye, Y., Josten, I., Arnold, N., Steffan, B., & Steglich, W. (1996). Isolation of a fluorone pigment from the Indian paint fungus Echinodontium tinctorium and Pyrofomes albomarginatus. Tetrahedron, 52(16), 5793-5798. 213 Appendix C. Supplementary data Compound (2) Crystal Data and Experimental Compound (2) Experimental. Single yellow irregular crystals of compound (2) recrystallised from ethanol by slow evaporation. A suitable crystal with dimensions 0.12 × 0.10 × 0.04 mm3 was selected and mounted on a mylar loop in oil on a Bruker APEX-II CCD diffractometer. The crystal was kept at a steady T = 90(2) K during data collection. The structure was solved with the XT (Sheldrick, 2015) solution program using Intrinsic Phasing methods and by using Olex2 (Dolomanov et al., 2009) as the graphical interface. The model was refined with XL (Sheldrick, 2015) using full matrix least squares minimisation on F2. Crystal Data. C16H20O5, Mr = 292.32, triclinic, P-1 (No. 2), a = 8.8847(5) Å, b = 9.2916(5) Å, c = 9.4831(4) Å, a = 97.485(4)°, b = 105.756(4)°, g = 105.320(4)°, V = 709.25(7) Å3, T = 90(2) K, Z = 2, Z' = 1, µ(CuKa) = 0.838, 9454 reflections measured, 1829 unique (Rint = 0.0628) which were used in all calculations. The final wR2 was 0.0968 (all data) and R1 was 0.0378 (I > 2(I)). 214 R1=3.78% Formula C16H20O5 -3 Dcalc./ g cm 1.369 -1 0.838 µ/mm Formula Weight 292.32 Colour yellow Shape irregular 3 Size/mm 0.12×0.10×0.04 T/K 90(2) Crystal System triclinic Space Group P-1 8.8847(5) a/Å 9.2916(5) b/Å 9.4831(4) c/Å 97.485(4) a/° ° 105.756(4) b/ ° 105.320(4) g/ 3 709.25(7) V/Å Z 2 Z' 1 Wavelength/Å 1.54178 Radiation type CuKa 4.965 Qmin/° ° 56.046 Qmax/ Measured Refl's. 9454 Ind't Refl's 1829 Refl's with I > 1440 2(I) Rint 0.0628 Parameters 213 Restraints 0 Largest Peak 0.238 Deepest Hole -0.192 GooF 1.032 wR2 (all data) 0.0968 wR2 0.0880 R1 (all data) 0.0552 R1 0.0378 Structure Quality Indicators Reflections: Refinement: A yellow irregular-shaped crystal with dimensions 0.12 × 0.10 × 0.04 mm3 was mounted on a mylar loop in oil. Data were collected using a Bruker APEX-II CCD diffractometer equipped with an Oxford Cryosystems low-temperature device operating at T = 90(2) K. Data were measured using f and w scans of 1.0 ° per frame for between 20 and 60 s using CuKa radiation (microfocus sealed X-ray tube, 45 kV, 0.60 mA). The total number of runs and images was based on the strategy calculation from the program APEX3. The maximum resolution that was achieved was Q = 56.046° (0.93 Å). The unit cell was refined using SAINT (Bruker, V8.40A, after 2013) on 3706 reflections, 39% of the observed reflections. Data reduction, scaling and absorption corrections were performed using SAINT (Bruker, V8.40A, after 2013). The final completeness is 99.00 % out to 56.046° in Q. A multi-scan absorption correction was performed using SADABS-2016/2 (Bruker, 2016/2) was used for absorption correction. wR2(int) was 0.0727 before and 0.0613 after correction. The ratio of minimum to maximum transmission is 0.8672. The l/2 correction factor is not present. The absorption coefficient µ of this material is 0.838 mm-1 at this wavelength (l = 1.54178Å) and the minimum and maximum transmissions are 0.837 and 0.967. The structure was solved and the space group P-1 (# 2) determined by the XT (Sheldrick, 2015) structure solution program using Intrinsic Phasing methods and refined by full matrix least squares minimisation on F2 using version 2018/3 of XL (Sheldrick, 2015). All non-hydrogen atoms were refined anisotropically. Most hydrogen atom positions were calculated geometrically and refined using the riding model, but all O—H hydrogen atoms were located in difference maps and refined freely. Table 1: Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2×103) for compound (2). Ueq is defined as 1/3 of the trace of the orthogonalised Uij. Atom O1 O2 O3 O4 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 x 580(2) 3683(2) -915(2) -798(2) 1860(3) 2170(3) 3445(3) 4413(3) 4097(3) 2800(3) 2432(3) 1517(3) 2130(3) 1290(3) -154(3) -831(3) -1(3) y 3378.3(19) 42.2(18) 2376(2) 2338.6(19) 2816(3) 1728(3) 1178(3) 1727(3) 2825(3) 3393(3) 4630(3) 4093(3) 4729(3) 4141(3) 2937(3) 2338(3) 2942(3) z 2520.0(19) 1807.1(18) 9523.2(18) 4547.1(17) 3052(2) 2135(3) 2730(3) 4229(3) 5146(3) 4574(2) 5512(3) 6589(2) 8144(2) 9095(3) 8525(2) 6996(3) 6055(2) 215 Ueq 23.5(4) 24.6(4) 22.3(4) 25.5(4) 19.1(6) 21.1(6) 21.2(6) 21.2(6) 19.8(6) 19.6(6) 20.4(6) 18.9(5) 20.0(6) 21.0(6) 18.7(6) 21.8(6) 20.4(6) Atom C14 C15 O5 C16 x 5184(3) 3656(3) -3333(2) -2582(3) y 3420(3) 6104(3) -67(2) -667(3) z 6760(3) 8811(3) 2718(2) 1755(3) Ueq 24.3(6) 25.7(6) 36.6(5) 33.8(7) Table 2: Anisotropic Displacement Parameters (×104) for compound (2). The anisotropic displacement factor exponent takes the form: -2p2[h2a*2 × U11+ ... +2hka* × b* × U12] Atom O1 O2 O3 O4 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 O5 C16 U11 26.1(10) 20.9(10) 20.4(10) 23.3(9) 16.7(13) 20.6(13) 19.5(13) 18.8(13) 17.1(13) 22.0(13) 21.7(13) 19.8(13) 19.2(13) 20.8(13) 19.6(13) 17.2(13) 21.8(14) 21.2(13) 25.5(14) 23.9(10) 30.2(15) U22 31.6(10) 28.0(10) 27.1(10) 34.0(10) 23.4(13) 25.3(14) 21.6(13) 24.5(14) 21.4(13) 19.5(13) 21.6(13) 22.4(13) 24.4(13) 27.1(14) 24.0(13) 27.4(14) 27.7(14) 31.0(15) 28.4(14) 42.9(11) 40.8(16) U33 14.3(9) 23.4(10) 15.0(9) 12.6(9) 19.1(13) 15.5(13) 22.7(14) 21.4(14) 20.4(13) 16.3(13) 16.8(13) 15.2(13) 17.7(13) 13.5(12) 16.4(13) 18.1(13) 10.7(13) 19.3(13) 18.4(13) 36.1(11) 31.8(16) U23 6.7(8) 1.5(8) 4.8(8) 2.5(8) 10.5(11) 6.4(11) 5.0(11) 9.5(11) 8.3(11) 6.2(10) 5.8(10) 6.2(10) 5.9(10) 2.5(10) 8.8(11) 4.1(11) 3.1(10) 7.8(11) 3.8(11) -6.1(9) 4.2(12) U13 4.2(7) 5.9(8) 5.2(8) 2.5(8) 6.5(11) 5.5(10) 10.3(11) 6.1(11) 7.8(11) 8.6(11) 5.3(10) 4.8(10) 5.4(10) 3.3(10) 7.5(10) 3.1(11) 1.7(11) 4.8(11) 4.5(11) 7.6(9) 16.5(13) U12 12.7(8) 8.4(8) 0.3(9) 2.8(8) 6.0(11) 3.4(11) 3.1(11) 7.1(11) 1.2(11) 1.1(11) 4.9(11) 7.4(11) 8.6(11) 7.9(11) 9.4(11) 5.3(11) 10.2(11) 6.3(11) 3.8(11) 8.4(9) 8.3(13) Table 3: Bond Lengths in Å for compound (2) Atom O1 O2 O3 O4 C1 C1 C2 C3 C4 C5 C5 Atom C1 C3 C11 C13 C2 C6 C3 C4 C5 C6 C14 Length/Å 1.376(3) 1.379(3) 1.384(3) 1.376(3) 1.379(3) 1.405(3) 1.377(3) 1.391(3) 1.389(3) 1.399(3) 1.508(3) Atom C6 C7 C8 C8 C9 C9 C10 C11 C12 O5 Atom C7 C8 C9 C13 C10 C15 C11 C12 C13 C16 Atom C3 O2 C2 Atom C2 C3 C3 Length/Å 1.520(3) 1.524(3) 1.410(3) 1.394(3) 1.395(3) 1.510(3) 1.377(3) 1.383(3) 1.387(3) 1.409(3) Table 4: Bond Angles in ° for compound (2) Atom O1 O1 C2 Atom C1 C1 C1 Atom C2 C6 C6 Angle/° 121.2(2) 116.2(2) 122.6(2) 216 Atom C1 C4 O2 Angle/° 119.0(2) 121.8(2) 117.8(2) Atom C2 C5 C4 C4 C6 C1 C5 C5 C6 C9 C13 C13 Atom C3 C4 C5 C5 C5 C6 C6 C6 C7 C8 C8 C8 Atom C4 C3 C6 C14 C14 C7 C1 C7 C8 C7 C7 C9 Angle/° 120.3(2) 120.5(2) 120.4(2) 119.4(2) 120.2(2) 120.0(2) 117.3(2) 122.6(2) 115.51(19) 122.6(2) 120.5(2) 116.9(2) Atom C8 C10 C10 C11 C10 C10 C12 C11 O4 O4 C12 Table 5: Torsion Angles in ° for compound (2) Atom Atom Atom Atom Angle/° O1 C1 C2 C3 -178.7(2) O1 C1 C6 C5 179.30(19) O1 C1 C6 C7 -3.1(3) O2 C3 C4 C5 -177.3(2) O3 C11 C12 C13 -179.5(2) C1 C2 C3 O2 177.21(19) C1 C2 C3 C4 -0.6(3) C1 C6 C7 C8 103.9(2) C2 C1 C6 C5 0.4(3) C2 C1 C6 C7 178.0(2) C2 C3 C4 C5 0.5(3) C3 C4 C5 C6 0.1(3) C3 C4 C5 C14 -179.0(2) C4 C5 C6 C1 -0.5(3) C4 C5 C6 C7 -178.1(2) C5 C6 C7 C8 -78.7(3) C6 C1 C2 C3 0.2(3) C6 C7 C8 C9 124.0(2) C6 C7 C8 C13 -56.8(3) C7 C8 C9 C10 -176.5(2) C7 C8 C9 C15 6.6(3) C7 C8 C13 O4 -5.6(3) C7 C8 C13 C12 175.4(2) C8 C9 C10 C11 -0.3(3) C9 C8 C13 O4 173.6(2) C9 C8 C13 C12 -5.3(3) C9 C10 C11 O3 178.5(2) C9 C10 C11 C12 -2.9(3) C10 C11 C12 C13 1.9(3) C11 C12 C13 O4 -176.7(2) C11 C12 C13 C8 2.3(3) C13 C8 C9 C10 4.3(3) C13 C8 C9 C15 -172.6(2) C14 C5 C6 C1 178.5(2) C14 C5 C6 C7 1.0(3) C15 C9 C10 C11 176.7(2) 217 Atom C9 C9 C9 C10 C11 C11 C11 C12 C13 C13 C13 Atom C15 C8 C15 C9 O3 C12 O3 C13 C8 C12 C8 Angle/° 121.1(2) 120.4(2) 118.4(2) 120.4(2) 118.0(2) 120.5(2) 121.5(2) 118.7(2) 122.2(2) 115.1(2) 122.8(2) Table 6: Hydrogen fractional atomic coordinates (×104) and equivalent isotropic displacement parameters (Å2×103) for compound (2). Atom x y z Ueq H1 100(40) 2970(40) 1530(40) 62(10) H2 4670(40) -30(30) 2160(30) 50(9) H3 -1750(40) 1650(40) 9080(30) 37(9) H4 -300(40) 2860(40) 3910(40) 64(10) H2A 1513.74 1362.92 1109.49 25 H4A 5295.44 1347.23 4628.04 25 H7A 1770.2 5112.97 4824.51 25 H7B 3486.61 5426.15 6102.02 25 H10 1716.37 4573.74 10142 25 H12 -1843.97 1528.5 6598.73 26 H14A 5966.09 2845.6 6992.88 37 H14B 4501.06 3294.83 7419.38 37 H14C 5789.11 4505.75 6915.21 37 H15A 4605.4 5838.8 8656.84 39 H15B 3850.4 6422.34 9888.59 39 H15C 3503.72 6943.8 8319.66 39 H5 -2440(50) 720(40) 3540(40) 67(10) H16A -1997.75 -1323.16 2235.79 51 H16B -1797.92 174.24 1548.26 51 H16C -3426.81 -1268.24 810.85 51 Ueq is defined as 1/3 of the trace of the orthogonalised Uij. Table 7: Hydrogen Bond information for compound (2) D H A d(D-H)/Å d(H-A)/Å d(D-A)/Å 1 O1 H1 O3 0.90(4) 1.81(4) 2.705(2) 2 O2 H2 O5 0.87(3) 1.72(4) 2.589(3) O3 H3 O23 0.82(3) 1.89(3) 2.710(2) O4 H4 O1 0.95(4) 1.77(4) 2.695(2) O5 H5 O4 0.99(4) 1.74(4) 2.699(2) 1+x,+y,-1+z; 21+x,+y,+z; 3-x,-y,1-z 218 D-H-A/deg 172(3) 173(3) 176(3) 166(3) 163(3) Compound (4) Crystal Data and Experimental Compound (4) Experimental. Single colourless needle crystals of compound (4) recrystallised from acetonitrile by slow evaporation. A suitable crystal with dimensions 0.24 × 0.05 × 0.05 mm3 was selected and mounted on a mylar loop in oil on a Bruker APEX II area detector diffractometer. The crystal was kept at a steady T = 110(2) K during data collection. The structure was solved with the XT 2018/2 (Sheldrick, 2015) solution program using Intrinsic Phasing methods and by using Olex2 (Dolomanov et al., 2009) as the graphical interface. The model was refined with XL (Sheldrick, 2015) using full matrix least squares minimisation on F2. Crystal Data. C32H50O4, Mr = 498.72, monoclinic, P21 (No. 4), a = 7.9203(3) Å, b = 20.6269(8) Å, c = 17.4422(6) Å, b = 97.890(2)°, a = g = 90°, V = 2822.58(18) Å3, T = 110(2) K, Z = 4, Z' = 2, µ(CuKa) = 0.585, 78352 reflections measured, 8399 unique (Rint = 0.0722) which were used in all calculations. The final wR2 was 0.1093 (all data) and R1 was 0.0429 (I≥2 s(I)). 219 R1= 4.29 % Formula C32H50O4 Dcalc./ g cm-3 1.174 -1 0.585 µ/mm Formula Weight 498.72 Colour colourless Shape needle 3 Size/mm 0.24×0.05×0.05 T/K 110(2) Crystal System monoclinic Flack Parameter -0.08(9) Hooft Parameter -0.03(7) Space Group P21 7.9203(3) a/Å 20.6269(8) b/Å 17.4422(6) c/Å ° 90 a/ ° 97.890(2) b/ ° 90 g/ 2822.58(18) V/Å3 4 Z 2 Z' Wavelength/Å 1.54178 Radiation type CuKa 2.557 Qmin/° ° 60.158 Qmax/ Measured Refl's. 78352 Indep't Refl's 8399 Refl's I≥2 s(I) 7934 Rint 0.0722 Parameters 675 Restraints 1 Largest Peak 0.188 Deepest Hole -0.202 GooF 1.131 wR2 (all data) 0.1093 wR2 0.1073 R1 (all data) 0.0464 R1 0.0429 Structure Quality Indicators Reflections: Refinement: A colourless needle-shaped crystal with dimensions 0.24 × 0.05 × 0.05 mm3 was mounted on a mylar loop in oil. Data were collected using a Bruker APEX II area detector diffractometer equipped with an Oxford Cryosystems low-temperature device operating at T = 110(2) K. Data were measured using f and w scans of 1.0 ° per frame for between 10 and 30 s using CuKa radiation (microfocus sealed X-ray tube, 45 kV, 0.60 mA). The total number of runs and images was based on the strategy calculation from the program APEX3. The maximum resolution that was achieved was Q = 60.158° (0.89 Å). The unit cell was refined using SAINT (Bruker, V8.40B, after 2013) on 9826 reflections, 13% of the observed reflections. Data reduction, scaling and absorption corrections were performed using SAINT (Bruker, V8.40B, after 2013). The final completeness is 99.90 % out to 60.158° in Q. A multi-scan absorption correction was performed using SADABS-2016/2 (Bruker, 2016/2) was used for absorption correction. wR2(int) was 0.1042 before and 0.0716 after correction. The ratio of minimum to maximum transmission is 0.8207. The l/2 correction factor is not present. The absorption coefficient µ of this material is 0.585 mm-1 at this wavelength (l = 1.54178Å) and the minimum and maximum transmissions are 0.797 and 0.971. The structure was solved and the space group P21 (# 4) determined by the XT 2018/2 (Sheldrick, 2015) structure solution program using Intrinsic Phasing methods and refined by full matrix least squares minimisation on F2 using version 2018/3 of XL (Sheldrick, 2015). The material crystallizes with two molecules in the asymmetric unit, with the two molecules forming a hydrogen bonded dimer. All non-hydrogen atoms were refined anisotropically. Most hydrogen atom positions were calculated geometrically and refined using the riding model, however the hydroxyl hydrogen in each molecule was located in a difference map and refined freely. The value of Z' is 2. This means that there are two independent molecules in the asymmetric unit. The Flack parameter was refined to -0.08(9) (Parsons, 2013). Determination of absolute structure using Bayesian statistics on Bijvoet differences using the Olex2 results in -0.03(7). The absolute configurations of the different stereocenters are: C3: S, C5: R, C10: S, C13: R, C14: S, C16: R, C17: R, C20: S, C22: R, C23: S C35: S, C37: R, C42: S, C45: R, C46: S, C48: R, C49: R, C52: S, C54: R, C55: S Note: The Flack parameter is used to determine chirality of the crystal studied, the value should be near 0, a value of 1 means that the stereochemistry is wrong and the model should be inverted. A value of 0.5 means that the crystal consists of a racemic mixture of the two enantiomers. 220 Figure 1: ORTEP-style image of one molecule in the asymmetric unit. Some hydrogen atoms removed for clarity. Figure 2: ORTEP-style image of the second molecule in the asymmetric unit. Some hydrogen atoms removed for clarity. Table 8: Fractional atomic coordinates (×104) and equivalent isotropic displacement parameters (Å2×103) for compound (4). Atom O1 O2 O3 O4 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 x 2760(4) 7599(3) 11982(3) 11784(4) 6511(5) 5568(5) 3657(6) 3125(5) 4185(5) 3759(5) 4418(5) 6126(5) 6884(5) 6154(5) 8590(5) 9400(5) 8842(5) 6828(5) 6482(5) 8027(5) 9175(5) 9594(5) 7057(6) y 5699.0(16) 6635.9(14) 6237.2(14) 5206.7(15) 5646(2) 5646(2) 5700(2) 6302(2) 6323(2) 6896(2) 6780(2) 6437.2(19) 6178.8(19) 6251(2) 5824(2) 5768(2) 6311(2) 6325(2) 6824(2) 6773(2) 6241(2) 6950(2) 6841(2) z -687.3(17) 5646.1(15) 6510.8(15) 6914.5(18) 906(2) 86(2) 86(2) 497(2) 1324(2) 1811(2) 2660(2) 2805(2) 2234(2) 1376(2) 2399(2) 3253(2) 3743(2) 3640(2) 4260(2) 4895(2) 4631(2) 3482(2) 1059(3) 221 Ueq 30.5(7) 22.0(6) 21.4(6) 31.1(7) 22.8(9) 26.2(10) 24.3(9) 21.2(9) 19.7(8) 24.9(9) 26.8(10) 19.2(9) 18.2(8) 19.4(8) 22.4(9) 22.2(9) 18.1(8) 18.2(8) 24.1(9) 20.7(9) 18.9(8) 25.1(9) 27.3(10) Atom C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 O5 O6 O7 O8 C33 C34 C35 C36 C37 C38 C39 C40 C41 C42 C43 C44 C45 C46 C47 C48 C49 C50 C51 C52 C53 C54 C55 C56 C57 C58 C59 C60 C61 C62 C63 C64 x 10903(5) 12169(5) 10451(5) 9135(5) 8620(5) 9308(5) 10780(5) 8699(6) 1237(5) 3299(6) 6044(5) 12490(5) 14056(5) 8847(4) 640(3) 2448(3) 3255(4) 8648(5) 9377(5) 8160(5) 6393(5) 5707(5) 3965(5) 3155(5) 4398(5) 6095(5) 6926(5) 7324(5) 6561(5) 4760(5) 3655(5) 1836(5) 1872(5) 3667(5) 4861(5) 7233(6) 3918(5) 5685(5) 2543(5) 699(5) -414(5) -1404(5) -1684(6) -2356(6) 5225(5) 6477(6) 3578(5) 2808(5) 2602(6) y 6267(2) 5762(2) 6166(2) 6654(2) 6485(2) 6701(2) 7165(2) 6461(2) 6224(3) 6914(2) 5675(2) 5726(2) 5884(2) 4372.8(17) 3941.3(14) 3526.9(14) 4465.0(15) 4034(2) 4032(2) 4359(2) 4030(2) 3973.6(19) 3641(2) 3768(2) 3803.0(19) 3786.0(19) 3680.1(19) 3823(2) 3863(2) 3583.5(19) 3933.1(19) 3684(2) 3568(2) 3734.6(19) 2852(2) 2946(2) 3459(2) 3589(2) 3807(2) 3770(2) 4240(2) 4132(2) 3488(2) 4681(2) 4493(2) 3391(2) 4675(2) 3905(2) 3556(2) z 5136(2) 4940(3) 5953(2) 6201(2) 6969(2) 7664(2) 7799(2) 8394(3) 593(3) 14(3) 3873(2) 6959(2) 7512(2) 6744.3(16) 907.0(15) -867.9(15) -1367.2(17) 4611(2) 5468(2) 5939(2) 5866(2) 4988(2) 4824(2) 3992(2) 3405(2) 3608(2) 4447(2) 3002(2) 2148(2) 2014(2) 2575(2) 2272(2) 1404(2) 1238(2) 2153(2) 4541(3) 451(2) 222(3) -103(2) 114(2) -370(2) -1032(2) -1421(3) -1469(3) 6248(2) 6311(2) 2478(2) -1449(2) -2209(2) Ueq is defined as 1/3 of the trace of the orthogonalised Uij. 222 Ueq 20.1(8) 27.9(10) 20.1(9) 21.6(9) 22.6(9) 22.7(9) 24.3(9) 29.5(10) 33.2(11) 30.3(10) 22.3(9) 21.0(9) 26.1(10) 28.2(7) 22.5(6) 22.4(6) 30.9(7) 22.3(9) 24.4(9) 20.1(9) 19.3(9) 17.4(8) 24.1(10) 23.0(9) 18.4(8) 17.6(8) 19.0(9) 20.4(9) 21.2(9) 18.0(8) 18.0(9) 22.2(9) 19.9(9) 17.4(8) 23.2(9) 28.3(10) 20.8(9) 27.3(10) 18.8(8) 20.7(9) 22.3(9) 23.5(9) 30.9(10) 31.4(11) 28.1(10) 27.0(10) 21.6(9) 23.7(10) 30.1(10) Table 9: Anisotropic displacement parameters (×104) for compound (4). The anisotropic displacement factor exponent takes the form: -2p2[h2a*2 × U11+ ... +2hka* × b* × U12] Atom O1 O2 O3 O4 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 O5 O6 O7 O8 C33 C34 C35 C36 C37 C38 C39 C40 C41 C42 U11 37.0(18) 13.3(14) 12.9(13) 25.4(16) 23(2) 30(2) 31(2) 23(2) 19(2) 23(2) 23(2) 17(2) 18(2) 19(2) 19(2) 15(2) 14.3(19) 14.3(19) 21(2) 22(2) 16(2) 24(2) 28(2) 17(2) 17(2) 12.4(19) 16(2) 16(2) 19(2) 23(2) 28(2) 24(2) 40(3) 16(2) 18(2) 21(2) 24.7(16) 17.7(14) 29.5(16) 38.2(18) 14(2) 15(2) 19(2) 16(2) 17(2) 16(2) 18(2) 19(2) 20(2) 16(2) U22 33.0(19) 31.3(17) 24.0(15) 25.6(18) 23(2) 27(2) 23(2) 24(2) 20(2) 24(2) 32(3) 14(2) 18(2) 21(2) 25(2) 23(2) 20(2) 18(2) 27(2) 21(2) 19(2) 25(2) 25(2) 18(2) 35(3) 22(2) 25(2) 27(2) 23(2) 23(2) 36(3) 50(3) 23(2) 27(2) 28(2) 30(3) 40.4(19) 33.8(17) 23.4(15) 27.7(19) 30(2) 30(2) 22(2) 22(2) 16(2) 33(3) 34(2) 17(2) 14(2) 19(2) U33 U23 19.5(16) -4.5(14) 20.7(15) -1.8(12) 25.7(15) 3.1(13) 39.2(18) 8.0(14) 23(2) -2.0(18) 22(2) -4.8(18) 19(2) -1.1(18) 16(2) -0.2(17) 19(2) 0.7(17) 26(2) -3.3(18) 25(2) -11.8(19) 26(2) -0.7(17) 19(2) -3.0(17) 19(2) 1.5(17) 23(2) -1.6(18) 29(2) -0.3(18) 19(2) 1.9(17) 22(2) -2.8(17) 23(2) -3.8(18) 18(2) -1.2(17) 22(2) -0.7(17) 25(2) 1.0(18) 28(2) 3.3(19) 25(2) 1.4(17) 31(2) -2(2) 24(2) 1.3(17) 23(2) -0.7(17) 24(2) 1.7(18) 26(2) 3.1(18) 26(2) -2.6(18) 24(2) 3.3(19) 25(2) -5(2) 26(2) 2.1(19) 24(2) -0.6(18) 18(2) 2.4(18) 26(2) 2.2(19) 18.2(15) 0.4(13) 15.4(14) -2.2(12) 14.3(14) -1.6(12) 28.2(17) -0.5(13) 22(2) 0.5(18) 28(2) 0.2(19) 19(2) 3.5(17) 20(2) 1.6(17) 20(2) 1.8(16) 23(2) 3.2(18) 17(2) -0.8(18) 19(2) -0.7(16) 19(2) -0.5(16) 22(2) -0.7(16) 223 U13 -3.6(13) -0.6(11) -3.6(11) -6.3(13) 5.5(17) 5.9(18) 2.3(18) 1.3(16) 1.6(16) -0.5(18) -0.8(18) 0.6(17) 2.6(16) 3.8(16) 4.7(17) 5.6(17) -1.5(15) 1.3(15) -2.0(17) 0.5(16) 1.7(15) -1.7(18) 1.0(19) 1.3(16) 1.3(18) -4.5(16) -2.0(17) 0.6(17) 1.8(17) -2.0(18) 2.3(19) -1.7(18) -2(2) 1.6(17) 3.7(16) -0.6(18) -2.3(12) 0.3(11) 2.7(11) 9.6(14) 1.9(17) 2.3(17) 1.8(16) 3.2(16) 3.5(16) 4.3(17) 5.9(16) 4.3(16) 4.7(16) 2.3(16) U12 2.3(15) 2.3(12) -2.9(12) -4.4(14) 4.9(18) 2(2) -2.3(19) 4.6(18) 1.7(18) 6.8(18) 12.2(19) 2.2(16) -2.3(16) -1.4(17) 4.5(18) 2.3(17) 0.2(17) 2.0(17) 6.7(18) 3.3(17) 0.1(17) -3.8(18) -4.4(19) -1.1(17) 4.3(19) -0.8(16) -1.5(17) -3.5(17) 4.6(17) 0.8(18) 0.4(19) 3(2) 5(2) -1.1(18) 2.6(19) 2.3(18) -9.0(15) -0.6(12) -2.6(12) -3.7(14) -0.6(17) -2.7(17) -2.1(17) -1.2(17) 1.7(16) -3.1(18) -3.6(18) -1.6(17) 0.2(16) 1.0(16) Atom C43 C44 C45 C46 C47 C48 C49 C50 C51 C52 C53 C54 C55 C56 C57 C58 C59 C60 C61 C62 C63 C64 U11 18(2) 15(2) 18(2) 12.4(19) 17(2) 17(2) 19(2) 26(2) 34(3) 25(2) 26(2) 23(2) 22(2) 21(2) 20(2) 28(2) 25(2) 24(2) 31(2) 21(2) 20(2) 32(2) U22 20(2) 25(2) 18(2) 23(2) 31(2) 24(2) 17(2) 22(2) 23(2) 20(2) 33(3) 21(2) 24(2) 25(2) 32(2) 36(3) 43(3) 40(3) 25(2) 24(2) 30(3) 36(3) U33 24(2) 24(2) 19(2) 19(2) 18(2) 19(2) 17(2) 21(2) 27(2) 17(2) 25(2) 12(2) 16(2) 21(2) 20(2) 27(2) 26(2) 19(2) 24(2) 20(2) 22(2) 22(2) U23 -3.0(17) 0.3(18) -2.7(16) -1.1(17) -2.0(17) 0.2(17) -1.5(16) -0.6(18) 1.0(19) -2.4(17) -4.0(19) -4.4(16) -2.3(17) -2.3(17) 1.9(18) -3(2) 4(2) -5.1(19) 5.9(18) -2.2(17) 3.1(19) -2(2) U13 4.4(16) 6.8(16) 6.2(16) 3.8(16) 2.2(16) 1.1(16) 5.2(16) 2.6(17) 1(2) 4.1(17) 9.4(18) 3.7(16) 2.2(16) 2.5(18) 7.1(18) -1.6(19) 2.6(19) 1.0(18) -0.6(18) 2.7(16) 5.9(17) 3.0(18) U12 1.0(17) -1.9(17) -3.3(16) -1.1(16) -4.4(18) -3.6(17) -3.3(16) 0.9(18) 8.6(19) 2.4(17) 3.5(19) -3.5(17) -3.9(18) -2.3(17) -3.0(18) -8(2) 3(2) 8(2) -8.7(19) 2.2(18) 5.9(19) 3(2) Table 10: Bond Lengths in Å for compound (4). Atom O1 O2 O2 O3 O3 O4 C1 C1 C2 C3 C4 C4 C4 C5 C5 C6 C7 C8 C8 C9 C9 C10 C11 C12 C13 C13 Atom C3 C16 C23 C22 C31 C31 C2 C10 C3 C4 C5 C28 C29 C6 C10 C7 C8 C9 C14 C10 C11 C19 C12 C13 C14 C17 Length/Å 1.435(5) 1.426(5) 1.447(5) 1.453(5) 1.342(5) 1.206(5) 1.519(6) 1.540(6) 1.518(6) 1.522(6) 1.567(5) 1.536(6) 1.534(6) 1.520(6) 1.557(5) 1.520(6) 1.517(6) 1.342(6) 1.504(6) 1.536(5) 1.529(6) 1.551(6) 1.542(6) 1.511(6) 1.580(5) 1.541(5) Atom C13 C14 C14 C15 C16 C17 C20 C20 C22 C23 C24 C25 C25 C31 O5 O6 O6 O7 O7 O8 C33 C33 C34 C35 C36 C36 224 Atom C18 C15 C30 C16 C17 C20 C21 C22 C23 C24 C25 C26 C27 C32 C35 C48 C55 C54 C63 C63 C34 C42 C35 C36 C37 C60 Length/Å 1.541(6) 1.544(6) 1.555(6) 1.536(6) 1.536(6) 1.523(5) 1.516(6) 1.532(6) 1.551(6) 1.495(6) 1.334(6) 1.502(6) 1.506(6) 1.499(6) 1.435(5) 1.437(5) 1.434(5) 1.447(5) 1.340(5) 1.211(5) 1.526(6) 1.539(6) 1.510(6) 1.544(6) 1.558(5) 1.542(6) Atom C36 C37 C37 C38 C39 C40 C40 C41 C41 C42 C43 C44 C45 C45 Atom C61 C38 C42 C39 C40 C41 C46 C42 C43 C51 C44 C45 C46 C49 Length/Å 1.527(6) 1.532(5) 1.561(5) 1.527(6) 1.516(5) 1.343(6) 1.511(6) 1.536(5) 1.535(5) 1.539(6) 1.530(6) 1.527(5) 1.574(5) 1.534(6) Atom C45 C46 C46 C47 C48 C49 C52 C52 C54 C55 C56 C57 C57 C63 Atom C50 C47 C62 C48 C49 C52 C53 C54 C55 C56 C57 C58 C59 C64 Atom C12 C17 C17 C18 C8 C8 C8 C15 C15 C30 C16 O2 O2 C17 C16 C20 C20 C17 C21 C21 O3 O3 C20 O2 O2 C24 C25 C24 C24 C26 O3 O4 O4 C55 Atom C13 C13 C13 C13 C14 C14 C14 C14 C14 C14 C15 C16 C16 C16 C17 C17 C17 C20 C20 C20 C22 C22 C22 C23 C23 C23 C24 C25 C25 C25 C31 C31 C31 O6 Length/Å 1.529(6) 1.553(5) 1.539(6) 1.537(6) 1.528(5) 1.524(5) 1.531(6) 1.531(6) 1.560(5) 1.493(6) 1.323(6) 1.495(6) 1.507(6) 1.497(6) Table 11: Bond angles in ° for compound (4). Atom C16 C31 C2 C3 O1 O1 C2 C3 C3 C3 C28 C29 C29 C6 C6 C10 C7 C8 C9 C9 C14 C8 C8 C11 C1 C1 C9 C9 C9 C19 C9 C13 C12 C12 Atom O2 O3 C1 C2 C3 C3 C3 C4 C4 C4 C4 C4 C4 C5 C5 C5 C6 C7 C8 C8 C8 C9 C9 C9 C10 C10 C10 C10 C10 C10 C11 C12 C13 C13 Atom C23 C22 C10 C1 C2 C4 C4 C5 C28 C29 C5 C5 C28 C4 C10 C4 C5 C6 C7 C14 C7 C10 C11 C10 C5 C19 C1 C5 C19 C5 C12 C11 C14 C17 Angle/° 109.0(3) 117.8(3) 113.5(3) 111.2(3) 111.4(3) 108.1(3) 113.6(3) 108.0(3) 107.6(4) 111.1(3) 108.0(3) 113.9(3) 108.0(4) 113.8(3) 109.9(3) 117.2(3) 110.5(3) 114.5(3) 122.8(4) 120.9(3) 115.9(3) 122.5(3) 121.6(4) 115.8(3) 107.6(3) 108.6(3) 111.6(3) 107.8(3) 107.0(3) 114.3(3) 117.2(3) 112.1(3) 108.6(3) 118.5(3) 225 Atom C18 C14 C18 C14 C13 C15 C30 C13 C30 C13 C14 C15 C17 C15 C13 C13 C16 C22 C17 C22 C20 C23 C23 C22 C24 C22 C23 C26 C27 C27 C32 O3 C32 C48 Angle/° 108.1(3) 98.5(3) 110.9(3) 112.0(3) 110.3(3) 119.9(3) 106.1(3) 101.9(3) 105.9(3) 112.8(3) 105.0(3) 114.1(3) 111.0(3) 106.2(3) 102.0(3) 126.4(3) 108.9(3) 103.1(3) 114.6(3) 111.3(3) 109.2(3) 106.2(3) 115.0(3) 109.4(3) 106.6(3) 112.0(3) 127.5(4) 124.5(4) 121.3(4) 114.1(4) 111.1(4) 124.1(4) 124.8(4) 109.9(3) Atom C63 C34 C35 O5 O5 C34 C35 C60 C60 C61 C61 C61 C36 C38 C38 C39 C40 C41 C41 C46 C40 C40 C43 C33 C33 C41 C41 C41 C51 C44 C45 C44 C44 Atom O7 C33 C34 C35 C35 C35 C36 C36 C36 C36 C36 C36 C37 C37 C37 C38 C39 C40 C40 C40 C41 C41 C41 C42 C42 C42 C42 C42 C42 C43 C44 C45 C45 Atom C54 C42 C33 C34 C36 C36 C37 C35 C37 C35 C37 C60 C42 C36 C42 C37 C38 C39 C46 C39 C42 C43 C42 C37 C51 C33 C37 C51 C37 C41 C43 C46 C49 Angle/° 118.4(3) 112.7(3) 110.4(3) 110.9(3) 107.9(3) 113.4(3) 107.7(3) 106.4(3) 108.8(3) 111.2(3) 114.8(3) 107.6(3) 117.5(3) 113.6(3) 109.3(3) 110.5(3) 115.0(3) 122.7(4) 120.1(3) 116.9(3) 122.5(3) 121.6(4) 115.8(3) 108.0(3) 108.9(3) 111.2(3) 107.5(3) 106.4(3) 114.8(3) 118.0(3) 111.5(3) 108.6(3) 117.5(3) Atom C44 C49 C50 C50 C40 C40 C40 C47 C62 C62 C48 O6 O6 C49 C48 C52 C52 C49 C49 C53 O7 O7 C52 O6 O6 C56 C57 C56 C56 C58 O7 O8 O8 Table 12: Torsion angles in ° for compound (4). Atom O1 O1 O1 O2 O2 O2 O3 O3 C1 C1 C2 C2 C2 C2 C2 Atom C3 C3 C3 C16 C16 C23 C22 C22 C2 C2 C1 C1 C1 C3 C3 Atom C4 C4 C4 C17 C17 C24 C23 C23 C3 C3 C10 C10 C10 C4 C4 Atom C5 C28 C29 C13 C20 C25 O2 C24 O1 C4 C5 C9 C19 C5 C28 Angle/° -176.8(3) 66.9(4) -51.1(4) 156.5(3) -67.9(4) -147.7(4) 177.0(3) -65.0(4) -179.8(3) 57.7(5) 52.3(4) 170.4(3) -71.9(4) -52.5(4) -168.9(4) 226 Atom C45 C45 C45 C45 C46 C46 C46 C46 C46 C46 C47 C48 C48 C48 C49 C49 C49 C52 C52 C52 C54 C54 C54 C55 C55 C55 C56 C57 C57 C57 C63 C63 C63 Atom C50 C46 C46 C49 C45 C47 C62 C45 C45 C47 C46 C47 C49 C47 C45 C45 C48 C53 C54 C54 C52 C55 C55 C54 C56 C54 C55 C58 C59 C59 C64 O7 C64 Angle/° 108.7(3) 99.2(3) 112.0(3) 110.6(3) 109.8(3) 119.2(3) 106.6(3) 101.9(3) 113.9(3) 105.7(3) 104.1(3) 114.0(3) 109.7(3) 107.2(3) 102.4(3) 126.2(3) 109.0(3) 114.0(3) 102.9(3) 110.5(3) 109.7(3) 105.8(3) 115.7(3) 112.2(3) 106.9(3) 109.3(3) 128.1(4) 125.4(4) 120.6(4) 113.9(4) 112.4(4) 123.7(4) 123.9(4) Atom C2 C3 C3 C4 C4 C4 C4 C5 C6 C6 C6 C6 C6 C7 C7 C7 C7 C7 C8 C8 C8 C8 C8 C9 C9 C9 C9 C10 C10 C10 C11 C11 C11 C11 C11 C11 C12 C12 C12 C12 C12 C13 C13 C13 C14 C14 C14 C14 C14 C14 C15 C15 C16 Atom C3 C4 C4 C5 C5 C5 C5 C6 C5 C5 C5 C7 C7 C8 C8 C8 C8 C8 C9 C9 C9 C9 C14 C8 C8 C8 C11 C1 C5 C9 C9 C9 C9 C12 C12 C12 C13 C13 C13 C13 C13 C14 C17 C17 C8 C8 C13 C13 C15 C15 C16 C16 O2 Atom C4 C5 C5 C6 C10 C10 C10 C7 C10 C10 C10 C8 C8 C9 C9 C14 C14 C14 C10 C10 C10 C11 C15 C14 C14 C14 C12 C2 C6 C11 C10 C10 C10 C13 C13 C13 C14 C14 C14 C17 C17 C15 C20 C20 C9 C9 C17 C17 C16 C16 C17 C17 C23 Atom C29 C6 C10 C7 C1 C9 C19 C8 C1 C9 C19 C9 C14 C10 C11 C13 C15 C30 C1 C5 C19 C12 C16 C13 C15 C30 C13 C3 C7 C12 C1 C5 C19 C14 C17 C18 C8 C15 C30 C16 C20 C16 C21 C22 C10 C11 C16 C20 O2 C17 C13 C20 C22 Angle/° 73.1(4) -178.9(3) 51.0(5) 163.2(4) -50.8(4) -171.3(3) 69.9(5) 37.6(5) 177.3(3) 56.8(4) -62.0(4) -8.6(6) 179.0(4) 4.6(6) -178.8(4) -151.2(4) -33.3(5) 86.4(4) -146.3(4) -28.3(5) 95.0(4) 0.6(6) -150.8(4) 36.3(5) 154.1(4) -86.2(4) -26.2(5) -57.5(5) -63.2(4) 177.5(3) 36.9(5) 154.9(3) -81.8(4) 54.4(4) 165.5(3) -67.3(4) -59.8(4) 171.8(3) 58.6(4) -165.2(3) 70.1(5) -28.7(4) -58.5(5) -179.6(4) 176.6(4) -6.8(6) -48.6(4) -173.3(4) -124.0(4) -1.4(4) 31.9(4) 167.6(3) -56.8(4) 227 Atom C16 C16 C16 C17 C17 C17 C17 C17 C18 C18 C18 C18 C18 C20 C20 C21 C21 C22 C22 C22 C23 C23 C23 C23 C28 C28 C29 C29 C30 C31 C31 O5 O5 O5 O6 O6 O6 O7 O7 C33 C33 C34 C34 C34 C34 C34 C34 C35 C35 C36 C36 C36 C36 Atom O2 C17 C17 C13 C13 C13 C20 C20 C13 C13 C13 C13 C13 C22 C22 C20 C20 O3 O3 C23 O2 O2 C24 C24 C4 C4 C4 C4 C14 O3 O3 C35 C35 C35 C48 C48 C55 C54 C54 C34 C34 C33 C33 C33 C35 C35 C35 C36 C36 C37 C37 C37 C37 Atom C23 C20 C20 C14 C14 C14 C22 C22 C14 C14 C14 C17 C17 C23 C23 C22 C22 C31 C31 C24 C16 C16 C25 C25 C5 C5 C5 C5 C15 C22 C22 C36 C36 C36 C49 C49 C56 C55 C55 C35 C35 C42 C42 C42 C36 C36 C36 C37 C37 C38 C42 C42 C42 Atom C24 C21 C22 C8 C15 C30 O3 C23 C8 C15 C30 C16 C20 O2 C24 O3 C23 O4 C32 C25 C15 C17 C26 C27 C6 C10 C6 C10 C16 C20 C23 C37 C60 C61 C45 C52 C57 O6 C56 O5 C36 C37 C41 C51 C37 C60 C61 C38 C42 C39 C33 C41 C51 Angle/° -178.2(3) 179.7(3) 58.6(4) 176.2(3) 47.7(4) -65.4(4) -174.9(3) -55.6(4) 59.5(4) -69.0(4) 177.9(3) 69.0(4) -55.7(5) 56.0(4) 174.0(3) 61.7(4) -178.9(3) 2.1(6) -178.6(3) 92.6(5) -175.3(3) 64.8(4) -0.3(7) -177.1(4) -62.7(5) 167.1(4) 57.2(5) -72.9(5) 89.4(4) -119.1(4) 116.3(4) -176.2(3) 67.3(4) -49.6(4) 153.5(3) -70.9(4) -144.1(4) 171.2(3) -70.4(4) -178.7(3) 59.6(5) 53.0(4) 170.8(3) -72.2(4) -52.9(4) -169.4(3) 73.7(4) 178.8(3) 49.5(4) 164.2(3) -50.0(4) -170.2(3) 71.7(4) 228 Atom C37 C38 C38 C38 C38 C38 C39 C39 C39 C39 C39 C40 C40 C40 C40 C40 C41 C41 C41 C41 C42 C42 C42 C43 C43 C43 C43 C43 C43 C44 C44 C44 C44 C44 C45 C45 C45 C46 C46 C46 C46 C46 C46 C47 C47 C48 C48 C48 C48 C49 C49 C49 C49 Atom C38 C37 C37 C37 C39 C39 C40 C40 C40 C40 C40 C41 C41 C41 C41 C46 C40 C40 C40 C43 C33 C37 C41 C41 C41 C41 C44 C44 C44 C45 C45 C45 C45 C45 C46 C49 C49 C40 C40 C45 C45 C47 C47 C48 C48 O6 O6 C49 C49 C45 C45 C45 C52 Atom C39 C42 C42 C42 C40 C40 C41 C41 C46 C46 C46 C42 C42 C42 C43 C47 C46 C46 C46 C44 C34 C38 C43 C42 C42 C42 C45 C45 C45 C46 C46 C46 C49 C49 C47 C52 C52 C41 C41 C49 C49 C48 C48 C49 C49 C55 C55 C52 C52 C46 C46 C46 C54 Atom C40 C33 C41 C51 C41 C46 C42 C43 C45 C47 C62 C33 C37 C51 C44 C48 C45 C47 C62 C45 C35 C39 C44 C33 C37 C51 C46 C49 C50 C40 C47 C62 C48 C52 C48 C53 C54 C42 C43 C48 C52 O6 C49 C45 C52 C54 C56 C53 C54 C40 C47 C62 O7 Angle/° 35.0(5) 178.7(3) 58.5(4) -59.6(4) -6.1(6) -179.5(4) 4.6(6) -179.9(4) -148.1(3) -31.2(5) 88.1(4) -148.7(4) -30.6(5) 92.8(4) -1.6(6) -151.0(4) 38.3(5) 155.2(4) -85.5(4) -23.6(5) -59.6(5) -62.5(4) 174.2(3) 35.5(5) 153.6(3) -83.0(4) 54.0(4) 165.5(3) -68.0(4) -61.8(4) 170.9(3) 57.6(4) -163.5(3) 71.5(5) -30.0(4) -57.1(5) -176.8(3) 177.8(4) -6.7(6) -46.8(4) -171.9(4) -120.5(3) 1.1(4) 29.2(4) 164.9(3) -51.9(4) -171.8(3) -179.3(3) 60.9(4) 175.0(3) 47.6(4) -65.6(4) -171.7(3) 229 Atom C49 C50 C50 C50 C50 C50 C52 C52 C53 C53 C54 C54 C54 C55 C55 C55 C55 C60 C60 C61 C61 C62 C63 C63 Atom C52 C45 C45 C45 C45 C45 C54 C54 C52 C52 O7 O7 C55 O6 O6 C56 C56 C36 C36 C36 C36 C46 O7 O7 Atom C54 C46 C46 C46 C49 C49 C55 C55 C54 C54 C63 C63 C56 C48 C48 C57 C57 C37 C37 C37 C37 C47 C54 C54 Atom C55 C40 C47 C62 C48 C52 O6 C56 O7 C55 O8 C64 C57 C47 C49 C58 C59 C38 C42 C38 C42 C48 C52 C55 Angle/° -52.1(4) 58.2(4) -69.1(4) 177.6(3) 71.0(4) -54.1(5) 49.6(5) 168.0(3) 66.2(4) -174.2(3) 2.2(6) -178.1(3) 94.2(5) -176.4(3) 63.4(4) 3.4(7) -176.0(4) -66.2(4) 164.5(3) 54.4(5) -74.9(4) 89.2(4) -117.5(4) 117.1(4) Table 13: Hydrogen Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2×103) for compound (4). Ueq is defined as 1/3 of the trace of the orthogonalised Uij. Atom H1 H1A H1B H2A H2B H3 H5 H6A H6B H7A H7B H11A H11B H12A H12B H15A H15B H16 H17 H18A H18B H18C x 2850(70) 7750.99 6178.58 5967.68 5826.94 3277.74 3807.98 4282.44 2508.56 4519.69 3566.88 9413.91 8431.98 9076.71 10656.93 6383.09 5415.76 8660.81 8647.59 10834.47 9108.39 9316.57 y 5300(30) 5618.94 5255.28 6015.17 5240.96 5312.41 5933.36 7294.68 6959.47 7203.73 6519.43 6047.02 5380.19 5347.1 5776.89 7266.42 6716.88 7193.76 5820.05 6946.03 7315.57 6993.77 z -910(30) 882.56 1178.56 -204.66 -178.43 360.36 1595.71 1631.98 1746.84 2929.53 2890.15 2111.05 2185.31 3463.09 3280.91 4040.16 4470.36 4917.8 4753.09 3623.99 3735.66 2919.04 230 Ueq 41(15) 27 27 31 31 29 24 30 30 32 32 27 27 27 27 29 29 25 23 38 38 38 Atom H19A H19B H19C H20 H21A H21B H21C H22 H23 H24 H26A H26B H26C H27A H27B H27C H28A H28B H28C H29A H29B H29C H30A H30B H30C H32A H32B H32C H5A H33A H33B H34A H34B H35 H37 H38A H38B H39A H39B H43A H43B H44A H44B H47A H47B H48 H49 H50A H50B H50C H51A H51B H51C x 8280.44 6579.34 6883.2 11406.89 11689.41 13229.71 12406.07 9999.05 9626.5 7695.49 11783.8 10484.97 11032.1 7686.36 8413.03 9602.42 574.38 819.87 1112.64 4469.53 3031.56 2507.5 4808.41 6282.41 6546.49 14647 14815.19 13729.8 9580(100) 9472.54 8498.9 9576.51 10484.5 7995.53 5503.13 4103.54 3208.54 2319.44 2519.65 8060.94 8072.32 6531.25 7294.98 1586.5 965.29 1657.75 3691.31 5421.75 3707.97 5518.58 7831.57 6136.58 7927.24 y 6822.54 7242.89 6830 6708.31 5327.97 5802.16 5829.77 5716.7 7100.97 6190.63 6939.12 7525.85 7331.85 6189.14 6831.66 6205.25 6128.29 6626.54 5867.24 6947.37 7296.67 6888.96 5682.02 5619.99 5314.82 6251.6 5506.43 5996.12 4700(40) 3824.13 4488.46 3579.35 4261.93 4816.72 4430.32 3168.61 3807.86 3420.04 4182.46 4208.1 3437.16 4321.26 3620.2 3277.3 4012.07 3097.14 4216.73 2645.32 2675.62 2765.06 2787.96 2722.24 2860.37 z 1247.38 1237.59 491.87 5098.92 4985.17 5298.84 4408.74 5992.88 6221.71 6962.34 8063.11 8120.34 7300.85 8260.1 8702.43 8693.72 88.86 798.74 951.81 -105.09 308.12 -468.71 3713.32 4435.19 3616.51 7313.31 7567.78 8017.94 6760(40) 4311.83 4431.06 5647.06 5541.47 5754.97 4805.79 4908.91 5186.18 3834.14 3977.98 3121.67 3067.47 1979.17 1832.43 2536.12 2354.44 1291.85 1180.92 1749.12 2138.62 2660.62 4122.28 4521.15 5040.73 231 Ueq 41 41 41 24 42 42 42 24 26 27 36 36 36 44 44 44 50 50 50 45 45 45 33 33 33 39 39 39 90(30) 27 27 29 29 24 21 29 29 28 28 24 24 25 25 27 27 24 21 35 35 35 42 42 42 Atom H52 H53A H53B H53C H54 H55 H56 H58A H58B H58C H59A H59B H59C H60A H60B H60C H61A H61B H61C H62A H62B H62C H64A H64B H64C x 3688.57 5942.19 5703.03 6542.26 2869.54 241.72 -411.13 -1204.12 -2908.85 -1121.66 -3584.96 -2021.2 -2082.36 5697.49 4084.45 5150.76 7276.89 5342.35 6864.75 3024.44 2923.18 4736.82 2371.2 3651 1648.74 y 2982.21 4052.72 3453.37 3342.83 4273.56 3321.43 4671.78 3493.47 3398.01 3149.42 4599.75 4710.2 5088.36 4560.34 4302.48 4909.81 3097 3192.25 3472.91 4865.35 4783.25 4847.16 3096.88 3598.28 3745.99 z 440.57 272.31 -315.2 563.1 -132.43 16.82 -179.06 -1910.19 -1525.39 -1083.53 -1510.85 -1987.87 -1191.01 6791.17 6218.99 5975.72 6104.73 6252.46 6859.96 2893.98 1976.45 2502.59 -2125.73 -2444.54 -2554.13 Ueq 25 41 41 41 23 25 27 46 46 46 47 47 47 42 42 42 41 41 41 32 32 32 45 45 45 Table 14: Hydrogen bond information for compound (4). D O1 O5 H H1 H5A A O8 O4 d(D-H)/Å 0.91(6) 0.88(8) d(H-A)/Å 1.95(6) 2.02(8) d(D-A)/Å 2.858(4) 2.876(4) 232 D-H-A/deg 175(5) 162(7) Compound (5) Crystal Data and Experimental Compound (5) Experimental. Single colourless plate crystals of compound (5) recrystallised from acetonitrile by slow evaporation. A suitable crystal with dimensions 0.28 × 0.24 × 0.06 mm3 was selected and mounted on a mylar loop in oil on a Bruker APEX II area detector diffractometer. The crystal was kept at a steady T = 110(2) K during data collection. The structure was solved with the XT 2018/2 (Sheldrick, 2015) solution program using Intrinsic Phasing methods and by using Olex2 (Dolomanov et al., 2009) as the graphical interface. The model was refined with XL (Sheldrick, 2015) using full matrix least squares minimisation on F2. Crystal Data. C32H48O4, Mr = 496.70, orthorhombic, P212121 (No. 19), a = 8.0284(2) Å, b = 10.6283(3) Å, c = 32.8305(9) Å, a = b = g = 90°, V = 2801.37(13) Å3, T = 110(2) K, Z = 4, Z' = 1, µ(CuKa) = 0.589, 51100 reflections measured, 5125 unique (Rint = 0.0369) which were used in all calculations. The final wR2 was 0.0725 (all data) and R1 was 0.0281 (I≥2 s(I)). 233 R1= 2.81 % Formula C32H48O4 Dcalc./ g cm-3 1.178 -1 0.589 µ/mm Formula Weight 496.70 Colour colourless Shape plate 3 Size/mm 0.28×0.24×0.06 T/K 110(2) Crystal System orthorhombic Flack Parameter 0.03(5) Hooft Parameter 0.04(4) Space Group P212121 a/Å 8.0284(2) b/Å 10.6283(3) c/Å 32.8305(9) ° 90 a/ ° 90 b/ ° 90 g/ V/Å3 2801.37(13) Z 4 Z' 1 Wavelength/Å 1.54178 Radiation type CuKa 2.692 Qmin/° ° 68.209 Qmax/ Measured Refl's. 51100 Indep't Refl's 5125 Refl's I≥2 s(I) 4993 Rint 0.0369 Parameters 335 Restraints 0 Largest Peak 0.165 Deepest Hole -0.137 GooF 1.040 wR2 (all data) 0.0725 wR2 0.0718 R1 (all data) 0.0290 R1 0.0281 Structure Quality Indicators Reflections: Refinement: A colourless plate-shaped crystal with dimensions 0.28 × 0.24 × 0.06 mm3 was mounted on a mylar loop in oil. Data were collected using a Bruker APEX II area detector diffractometer equipped with an Oxford Cryosystems low-temperature device operating at T = 110(2) K. Data were measured using f and w scans of 1.0 ° per frame for 10 s using CuKa radiation (microfocus sealed X-ray tube, 45 kV, 0.60 mA). The total number of runs and images was based on the strategy calculation from the program APEX3. The maximum resolution that was achieved was Q = 68.209° (0.83 Å). The unit cell was refined using SAINT (Bruker, V8.40B, after 2013) on 9123 reflections, 18% of the observed reflections. Data reduction, scaling and absorption corrections were performed using SAINT (Bruker, V8.40B, after 2013). The final completeness is 100.00 % out to 68.209° in Q. A multi-scan absorption correction was performed using SADABS-2016/2 (Bruker, 2016/2) was used for absorption correction. wR2(int) was 0.0906 before and 0.0516 after correction. The ratio of minimum to maximum transmission is 0.8607. The l/2 correction factor is not present. The absorption coefficient µ of this material is 0.589 mm-1 at this wavelength (l = 1.54178Å) and the minimum and maximum transmissions are 0.831 and 0.965. The structure was solved and the space group P212121 (# 19) determined by the XT 2018/2 (Sheldrick, 2015) structure solution program using Intrinsic Phasing methods and refined by full matrix least squares minimisation on F2 using version 2018/3 of XL (Sheldrick, 2015). All nonhydrogen atoms were refined anisotropically. Hydrogen atom positions were calculated geometrically and refined using the riding model. The absolute configuration was determined on the basis of the refined Flack parameter value, 0.03(5). C5, C10, C13, C14, C16, C17, C21, C22, and C24 were assigned configurations of R, S, R, S, R, R, S, R, and S, respectively. Determination of absolute structure using Bayesian statistics on Bijvoet differences using the Olex2 results in 0.04(4). Note: The Flack parameter is used to determine chirality of the crystal studied, the value should be near 0, a value of 1 means that the stereochemistry is wrong and the model should be inverted. A value of 0.5 means that the crystal consists of a racemic mixture of the two enantiomers. Figure 3: ORTEP-style image of compound (5), with most hydrogen atoms removed, for clarity. 234 Table 15: Fractional atomic coordinates (×104) and equivalent isotropic displacement parameters (Å2×103) for compound (5). Ueq is defined as 1/3 of the trace of the orthogonalised Uij. Atom O1 O3 O4 O5 C20 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C21 C23 C22 C24 C25 C26 C27 C28 C29 C30 C31 C32 x 7721(2) 3937.5(14) -316.4(14) -144.5(17) 6253(2) 5530(2) 5887(3) 7041(2) 7307(2) 6673(2) 6472(2) 6346(2) 5332(2) 4809(2) 5119(2) 3792(2) 3033(2) 3109(2) 4948(2) 4996(2) 3533(2) 2829(2) 1879(2) 3511(2) 1105(2) 274(2) 1332(2) 2406(2) 2846(2) 2073(2) 2550(2) 664(2) 6479(3) 9194(2) -892(2) -2606(2) y 5637.3(14) 4951.2(11) 5921.3(11) 7553.6(13) 6624.2(17) 6963.0(17) 6736.6(18) 5667.6(17) 4584.2(16) 4887.9(15) 3716.0(15) 4086.8(15) 5255.8(14) 6024.9(15) 5742.6(15) 7210.4(16) 7357.1(16) 6121.0(14) 5625.1(14) 4594.5(15) 4908.3(15) 6169.1(15) 5185.5(16) 5160.4(18) 6321.7(15) 7578.8(17) 6158.0(15) 5024.5(16) 5167.4(15) 4652.7(16) 5044.2(18) 3726.9(19) 3414.1(17) 4353(2) 6679.3(17) 6275(2) z 4649.5(4) 7990.8(3) 8248.7(4) 8686.4(4) 7067.2(5) 5529.2(5) 5077.8(5) 4979.9(5) 5283.6(5) 5722.4(5) 5986.7(5) 6433.9(5) 6508.4(5) 6211.4(5) 5758.5(5) 6297.9(5) 6730.3(5) 6964.4(5) 6946.0(5) 7283.4(5) 7566.6(4) 7428.4(5) 6768.6(5) 5586.2(5) 7619.3(5) 7529.3(6) 8080.0(5) 8216.0(5) 8656.9(5) 8974.4(5) 9399.2(5) 8948.4(6) 5093.1(5) 5306.3(6) 8546.8(5) 8674.3(6) Ueq 39.0(4) 18.1(2) 21.7(3) 33.6(3) 22.1(4) 24.9(4) 28.9(4) 23.8(4) 21.5(4) 17.7(3) 19.6(3) 20.0(3) 16.6(3) 17.7(3) 18.8(3) 22.2(4) 20.8(4) 16.2(3) 16.4(3) 20.5(3) 17.0(3) 16.5(3) 21.2(3) 25.4(4) 18.2(3) 24.9(4) 18.0(3) 17.5(3) 18.7(3) 21.0(4) 26.3(4) 32.0(4) 28.9(4) 32.5(4) 23.5(4) 36.5(5) Table 16: Anisotropic displacement parameters (×104) for compound (5). The anisotropic displacement factor exponent takes the form: -2p2[h2a*2 × U11+ ... +2hka* × b* × U12] Atom O1 O3 O4 O5 C20 C1 U11 49.3(9) 16.5(6) 13.6(5) 28.6(7) 15.0(8) 31.8(10) U22 40.6(8) 22.4(5) 29.0(6) 36.3(7) 26.8(8) 21.8(8) U33 26.9(7) 15.3(5) 22.6(6) 35.8(7) 24.6(8) 21.1(9) U23 8.6(6) -1.4(5) -5.2(5) -15.0(6) -4.8(7) 4.9(7) 235 U13 14.5(6) 0.5(4) 2.5(5) 4.7(6) -1.5(7) 0.3(7) U12 3.4(7) 4.0(5) -0.9(5) 2.8(6) 0.8(7) 2.2(7) Atom C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C21 C23 C22 C24 C25 C26 C27 C28 C29 C30 C31 C32 U11 35.6(10) 25.0(9) 22.1(9) 16.9(8) 21.6(8) 21.1(8) 14.5(7) 15.3(7) 18.9(8) 25.0(9) 21.2(9) 14.8(8) 16.5(8) 23.6(9) 19.3(8) 14.4(8) 19.0(8) 19.7(9) 13.6(8) 20.2(9) 12.4(8) 14.8(8) 15.6(8) 19.0(8) 33.7(10) 29.5(10) 43.1(11) 25.5(10) 18.4(8) 22.9(10) U22 28.6(9) 26.7(9) 24.2(9) 19.6(8) 17.6(8) 20.4(8) 16.5(7) 17.4(7) 19.0(8) 19.3(8) 18.4(8) 15.3(7) 14.6(7) 20.2(8) 17.3(7) 15.4(7) 26.0(9) 32.7(9) 20.2(8) 26.7(9) 20.8(8) 19.2(8) 19.6(8) 23.9(8) 25.6(9) 40.9(11) 24.4(9) 44.2(11) 31.3(9) 51.7(13) U33 22.4(9) 19.8(8) 18.0(8) 16.8(7) 19.8(8) 18.6(8) 18.9(8) 20.4(8) 18.6(7) 22.3(8) 22.9(8) 18.5(8) 18.0(7) 17.6(8) 14.5(7) 19.7(8) 18.5(7) 23.8(8) 20.8(8) 27.7(9) 20.8(8) 18.4(7) 21.1(8) 19.9(8) 19.7(8) 25.7(9) 19.1(8) 27.7(9) 20.9(8) 34.9(10) U23 9.4(7) 2.2(7) 1.4(7) 0.3(6) 0.6(6) 2.7(6) 1.5(6) 1.4(6) 2.7(6) 5.0(6) 2.5(6) 0.6(6) 1.5(6) 2.2(6) -0.3(6) -1.2(6) -2.3(7) 0.2(7) -3.2(6) -0.8(7) -3.8(6) -2.3(6) -5.8(6) -2.2(7) -3.0(7) 5.8(8) -2.4(7) 0.9(8) -1.6(7) -2.8(9) U13 0.2(8) 1.0(7) 1.5(7) -0.8(6) 1.7(7) 1.5(6) -0.4(6) -1.0(6) -1.7(6) -0.7(7) -1.3(7) -1.5(6) -1.1(6) 1.2(7) -0.8(6) -2.4(6) -1.8(6) -4.9(7) -2.2(6) -0.2(7) 2.3(6) 1.8(6) -1.3(6) 0.5(7) 1.7(7) 0.1(8) 2.3(8) 6.6(8) 0.8(7) 9.2(8) U12 0.4(8) -9.2(7) -1.3(7) -1.9(6) 2.4(7) 3.7(7) -1.4(6) -1.0(6) 0.6(7) 4.1(7) 6.2(7) 0.6(6) 2.5(6) 7.7(7) 1.0(6) 0.1(6) -3.6(7) 0.3(7) -0.2(6) 6.4(7) -0.5(6) -1.4(6) 1.9(6) 3.4(7) 4.0(8) -8.5(9) -1.8(8) 6.6(9) 7.2(8) 4.1(9) Atom C8 C8 C9 C9 C10 C11 C12 C13 C13 C13 C14 C15 C16 C17 C21 C21 C22 C24 Atom C9 C14 C10 C11 C19 C12 C13 C14 C17 C18 C15 C16 C17 C21 C23 C22 C24 C25 Table 17: Bond lengths in Å for compound (5). Atom O1 O3 O3 O4 O4 O5 C20 C1 C1 C2 C3 C4 C4 C4 C5 C5 C6 C7 Atom C3 C16 C24 C22 C31 C31 C14 C2 C10 C3 C4 C5 C29 C30 C6 C10 C7 C8 Length/Å 1.215(2) 1.4310(18) 1.4369(19) 1.457(2) 1.349(2) 1.197(2) 1.544(2) 1.529(2) 1.536(2) 1.501(3) 1.538(2) 1.562(2) 1.543(2) 1.536(3) 1.527(2) 1.548(2) 1.524(2) 1.506(2) 236 Length/Å 1.340(2) 1.521(2) 1.537(2) 1.528(2) 1.539(2) 1.553(2) 1.523(2) 1.569(2) 1.541(2) 1.542(2) 1.558(2) 1.534(2) 1.523(2) 1.528(2) 1.522(2) 1.533(2) 1.547(2) 1.498(2) Atom C25 C26 Atom C26 C27 Length/Å 1.331(2) 1.505(2) Atom C26 C31 Atom C28 C32 Atom C12 C17 C17 C18 C20 C20 C8 C8 C8 C15 C16 O3 O3 C17 C16 C16 C21 C17 C23 C23 O4 O4 C21 O3 O3 C25 C26 C25 C25 C28 O4 O5 O5 Atom Atom C13 C18 C13 C14 C13 C18 C13 C14 C14 C13 C14 C15 C14 C20 C14 C13 C14 C15 C14 C13 C15 C14 C16 C15 C16 C17 C16 C15 C17 C13 C17 C21 C17 C13 C21 C22 C21 C17 C21 C22 C22 C21 C22 C24 C22 C24 C24 C22 C24 C25 C24 C22 C25 C24 C26 C27 C26 C28 C26 C27 C31 C32 C31 O4 C31 C32 Length/Å 1.502(3) 1.501(3) Table 18: Bond Angles in ° for compound (5). Atom C16 C31 C2 C3 O1 O1 C2 C3 C3 C29 C30 C30 C30 C6 C6 C10 C7 C8 C7 C9 C9 C8 C8 C11 C1 C1 C1 C9 C9 C19 C9 C13 C12 C12 Atom Atom O3 C24 O4 C22 C1 C10 C2 C1 C3 C2 C3 C4 C3 C4 C4 C5 C4 C29 C4 C5 C4 C3 C4 C5 C4 C29 C5 C4 C5 C10 C5 C4 C6 C5 C7 C6 C8 C14 C8 C7 C8 C14 C9 C10 C9 C11 C9 C10 C10 C5 C10 C9 C10 C19 C10 C5 C10 C19 C10 C5 C11 C12 C12 C11 C13 C14 C13 C17 Angle/° 107.94(11) 118.94(13) 112.49(14) 116.25(15) 119.24(16) 119.77(17) 120.92(15) 113.44(14) 106.31(14) 113.58(14) 106.74(15) 108.06(14) 108.41(16) 112.96(13) 110.51(13) 117.06(13) 110.11(13) 113.94(13) 118.42(13) 123.70(14) 117.80(14) 122.30(15) 122.36(15) 115.28(13) 106.53(13) 110.12(13) 109.89(14) 108.59(12) 107.37(13) 114.32(14) 117.55(13) 111.04(13) 107.94(13) 117.68(13) Table 19: Torsion angles in ° for compound (5). Atom O1 O1 O1 O3 O3 O3 O4 O4 Atom C3 C3 C3 C16 C16 C24 C22 C22 Atom C4 C4 C4 C17 C17 C25 C24 C24 Atom C5 C29 C30 C13 C21 C26 O3 C25 Angle/° 167.76(16) -66.7(2) 48.9(2) 159.20(12) -70.16(15) -141.94(17) 165.24(12) -75.85(15) 237 Angle/° 108.67(13) 100.74(12) 109.91(13) 111.71(13) 113.43(13) 106.47(13) 106.46(13) 108.30(13) 119.02(13) 103.41(13) 105.01(13) 115.01(13) 110.24(12) 107.13(13) 102.20(12) 107.89(13) 122.75(13) 106.54(13) 114.25(14) 110.07(14) 106.66(13) 105.18(13) 116.06(13) 111.74(13) 107.51(13) 109.35(13) 127.26(15) 119.55(16) 125.18(16) 115.20(15) 110.19(16) 124.70(17) 125.10(17) Atom C20 C1 C1 C2 C2 C2 C2 C2 C2 C3 C3 C4 C4 C4 C4 C5 C6 C6 C6 C6 C6 C7 C7 C7 C7 C7 C8 C8 C8 C8 C8 C9 C9 C9 C9 C10 C10 C10 C11 C11 C11 C11 C11 C11 C12 C12 C12 C12 C12 C13 C13 C13 C14 Atom C14 C2 C2 C1 C1 C1 C3 C3 C3 C4 C4 C5 C5 C5 C5 C6 C5 C5 C5 C7 C7 C8 C8 C8 C8 C8 C9 C9 C9 C9 C14 C8 C8 C8 C11 C1 C5 C9 C9 C9 C9 C12 C12 C12 C13 C13 C13 C13 C13 C14 C17 C17 C8 Atom C15 C3 C3 C10 C10 C10 C4 C4 C4 C5 C5 C6 C10 C10 C10 C7 C10 C10 C10 C8 C8 C9 C9 C14 C14 C14 C10 C10 C10 C11 C15 C14 C14 C14 C12 C2 C6 C11 C10 C10 C10 C13 C13 C13 C14 C14 C14 C17 C17 C15 C21 C21 C9 Atom C16 O1 C4 C5 C9 C19 C5 C29 C30 C6 C10 C7 C1 C9 C19 C8 C1 C9 C19 C9 C14 C10 C11 C20 C13 C15 C1 C5 C19 C12 C16 C20 C13 C15 C13 C3 C7 C12 C1 C5 C19 C14 C17 C18 C20 C8 C15 C16 C21 C16 C23 C22 C10 Angle/° 98.72(15) -160.60(18) 22.3(3) 61.22(18) 178.79(15) -63.13(19) -15.2(2) 110.37(19) -134.06(18) 163.39(14) 33.3(2) 163.70(14) -56.02(18) -174.60(13) 65.56(19) 39.36(19) 172.78(13) 54.20(17) -65.65(17) -10.4(2) 172.76(14) 3.5(3) -179.50(15) 97.32(16) -140.39(14) -22.8(2) -141.46(17) -25.2(2) 98.92(18) -13.4(2) -141.16(14) -79.68(18) 42.61(19) 160.19(15) -13.4(2) -46.5(2) -62.98(17) 163.79(14) 41.36(19) 157.65(14) -78.26(17) 51.80(17) 164.82(14) -69.52(18) 50.23(17) -67.71(15) 165.13(13) -162.53(14) 76.6(2) -21.07(16) -65.25(19) 172.99(13) -179.65(14) 238 Atom C14 C14 C14 C14 C14 C15 C15 C16 C16 C16 C16 C17 C17 C17 C17 C17 C18 C18 C18 C18 C18 C21 C21 C23 C23 C22 C22 C22 C24 C24 C24 C24 C29 C29 C30 C30 C31 C31 Atom C8 C13 C13 C15 C15 C16 C16 O3 O3 C17 C17 C13 C13 C13 C21 C21 C13 C13 C13 C13 C13 C22 C22 C21 C21 O4 O4 C24 O3 O3 C25 C25 C4 C4 C4 C4 O4 O4 Atom C9 C17 C17 C16 C16 C17 C17 C24 C24 C21 C21 C14 C14 C14 C22 C22 C14 C14 C14 C17 C17 C24 C24 C22 C22 C31 C31 C25 C16 C16 C26 C26 C5 C5 C5 C5 C22 C22 Atom C11 C16 C21 O3 C17 C13 C21 C22 C25 C23 C22 C20 C8 C15 O4 C24 C20 C8 C15 C16 C21 O3 C25 O4 C24 O5 C32 C26 C15 C17 C27 C28 C6 C10 C6 C10 C21 C24 Angle/° -2.7(2) -45.56(14) -166.40(14) -130.26(14) -7.35(17) 33.39(16) 164.02(13) -55.80(16) -175.79(13) 176.62(13) 54.86(16) -73.71(15) 168.35(12) 41.18(14) -163.17(12) -46.39(18) 169.64(13) 51.70(17) -75.47(16) 72.41(15) -48.43(19) 47.64(18) 166.55(14) 72.45(16) -170.76(14) 0.3(3) 179.72(14) 96.6(2) -169.67(13) 69.11(16) -171.68(16) 5.3(3) 41.8(2) -88.22(18) -78.47(18) 151.46(16) -123.96(15) 112.24(15) Table 20: Hydrogen fractional atomic coordinates (×104) and equivalent isotropic displacement parameters (Å2×103) for compound (5). Ueq is defined as 1/3 of the trace of the orthogonalised Uij. Atom H20A H20B H20C H1A H1B H2A H2B H5 x 7372.64 6064.44 6149.19 4582.14 6515.41 4813.76 6365.32 7594.83 y 6266.48 6884.24 7356.23 7552.98 7362.26 6582.29 7517.17 5376.68 z 7041.18 7349.85 6887.43 5555.19 5656.52 4937.91 4962.05 5852.34 239 Ueq 33 33 33 30 30 35 35 21 Atom H6A H6B H7A H7B H11A H11B H12A H12B H15A H15B H16 H17 H18A H18B H18C H19A H19B H19C H21 H23A H23B H23C H22 H24 H25 H27A H27B H27C H28A H28B H28C H29A H29B H29C H30A H30B H30C H32A H32B H32C x 5455.27 7440.28 5842.76 7483.54 2869.48 4515.48 3652.8 1858.85 4858.77 6063.77 2652.38 3561.69 736.93 2112.99 2006.25 2580.1 3280.84 3648.82 369.26 1018.85 -769.95 37.59 1796.18 1759.65 3780.91 3553.75 2771.52 1637.02 -341.39 958.95 454.14 5270.53 6732.34 6909.12 9612.77 9420.57 9751.89 -3149.93 -3263.58 -2524.26 y 3254.61 3152.17 3381.04 4221.19 7245.2 7947.83 8012.15 7632.83 3746.34 4623.25 4254.22 6845.68 5420.65 4334.48 5206.6 5741.81 4364.35 5004.11 5631.85 8265.24 7639.95 7640.28 6949.77 4229.22 5683.08 5568.29 4294.13 5525.18 4098.61 2961.34 3516.42 3537.76 2672.38 3288.74 4128.27 3664.86 5119.66 5836.02 7016.75 5708.39 z 5904.47 5946.83 6587.44 6542.09 6098.26 6246.22 6881.85 6706.85 7163.37 7433.81 7526.62 7541.22 6841.16 6868.26 6471.75 5629.04 5725.95 5293.96 7516.71 7610.08 7682.78 7237.12 8199.18 8176.88 8716.58 9388.45 9564.16 9521.56 9067.64 9098.36 8662.37 5082.26 5259.28 4816.65 5035.57 5496.97 5400.87 8447.91 8749.13 8908.88 240 Ueq 24 24 24 24 27 27 25 25 25 25 20 20 32 32 32 38 38 38 22 37 37 37 22 21 22 39 39 39 48 48 48 43 43 43 49 49 49 55 55 55 Citations of crystal data O.V. Dolomanov and L.J. Bourhis and R.J. Gildea and J.A.K. Howard and H. Puschmann, Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., (2009), 42, 339-341. Parsons, S., Flack, H.D., and Wagner, T., Acta Cryst., (2013), B69 249-259 Sheldrick, G.M., XT, Acta Cryst., (2015), A71, 3-8. Sheldrick, G.M., XL, Acta Cryst., (2015), C71, 3-8. Software for the Integration of CCD Detector System Bruker Analytical X-ray Systems, Bruker AXS., Madison, WI (after 2013). 241 Chapter 5: Discussion 5.1. Overview of Echinodontium tinctorium Echinodontium tinctorium (E. tinctorium) is a wood-decaying fungus from the western US and Canada that colonizes aged and decayed trees. It is found growing as a hoof-shaped conk on trees, mostly hemlocks (Ye et al., 1996). Like many other North American mushrooms, E. tinctorium has not been explored much for its bioactivities and bioactive compounds. At the outset of this research, only two small molecules, echinotinctone and echinodol (Ye et al., 1996), and one polysaccharide (AIPetinc, Javed et al., 2019) had been isolated from E. tinctorium. AIPetinc was isolated from the alkali extracts of E. tinctorium (Javed et al., 2019). Echinotinctone and echinodol had never been reported to have any bioactivity, whereas AIPetinc is a large polysaccharide isolated from alkali extracts, and has anti-inflammatory activity (Javed et al., 2019). Due to the limited prior studies, E. tinctorium was therefore considered a good resource for the potential discovery of novel bioactive compounds. In addition, based on the literature review described in chapter 1, there is high potential in discovering novel compounds in mushrooms native to North America. 5.2. Overview of project The current research carried out in this thesis was aimed at providing scientific evidence of the presence of immuno-stimulatory and growth-inhibitory compounds in E. tinctorium. The project was mostly based on using bioactivity-guided fractionation approach. Initially, a group of compounds were isolated from the powdered mushroom via extraction. Small molecules were then mostly obtained from the organic extracts 80% ethanol and 50% methanol, while polysaccharides were mainly extracted using water or 5% sodium hydroxide. Further separation of compounds was achieved using several purification techniques. The separation of compounds 242 was based on size, polarity and charge. High-resolution chromatographic techniques were then employed to achieve purity. At each step of purification, bioactivity was confirmed and bioactive fractions were subjected to the next purification steps. Once pure compounds were obtained, bioactivity was again confirmed, structure was elucidated and molecular targets for small molecules were predicted, when possible. The hypothesis proposed at the start of the research project was tested with the help of several research objectives, sub-divided into three (Chapters 2, 3 and 4) experimental chapters. 5.3. Key findings of the biomolecules isolated from E. tinctorium In chapter 2, a novel immuno-stimulatory large molecular weight polysaccharide (EtISPFa) with an estimated molecular weight of 1354 kDa was isolated from the aqueous extracts of E. tinctorium mushroom. EtISPFa was successfully purified using Sephadex LH-20 SEC, Sephadex DEAE AEC, Sephacryl S-500 HR SEC and HPLC BioSEC-5. Chemical characterization of EtISPFa was carried out by GC-MS for monosaccharide content and linkage analysis, FTIR for presence of functional groups and NMR for absolute configuration. EtISPFa comprises of a high abundance of glucose (66.2%) and glucuronic acid (10.1%) and a relatively lower percentage of mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), and rhamnose (3.1 %), and traces of fucose (1.8 %) and arabinose (0.2 %). EtISPFa had abundant 3-linked glucose (19.8 %), 4-linked glucuronic acid (10.8 %), 6-linked glucose (10.7 %) and 3,6-linked glucose which suggested the main backbone to be of 3-linked glucose residues; NMR confirmed the linkages and final ßconfiguration of EtISPFa. EtISPFa was capable of inducing the production of several cytokines. This chapter was published in early 2021 in Carbohydrate Polymers (Zeb et al., 2021). In chapter 3, a growth-inhibitory polysaccharide EtGIPL1a with a weight average molecular weight of 275 kDa was isolated from the methanol extracts of E. tinctorium. EtGIPL1a was 243 successfully purified by multiple size exclusion and ion exchange chromatographic techniques. Structural elucidation was accomplished by GC-MS, NMR and FTIR. GC-MS revealed the monosaccharide content as well as linkages present in EtGIPL1a. The most prominent monosaccharides present in EtGIPL1a included glucose (54.3 %), galactose (19.6 %), mannose (11.1 %), fucose (10.3 %), along with small amounts of glucuronic acid (4 %) and rhamnose (0.6 %). The most abundant glycosidic linkages in EtGIPL1a were 3-linked glucose (28.9 %), 6linked glucose (18.3 %) and galactose (3.9 %), 3,6-linked glucose (13 %) and 2, 6-linked galactose (2.6 %), 4-linked glucuronic acid (9.2 %) and glucose (1.7 %), 3-linked fucose (2.5 %) and 6-linked mannose (2.4 %). Most of the terminal sugars comprised of glucose (15.3 %), mannose (1.3 %) and fucose (0.9 %). The final β-configuration of EtGIPL1a was confirmed from NMR. Chemical characterization data altogether suggested that EtGIPL1a constitutes a sugar backbone of β-1-3-linked glucose. Biological characterization of EtGIPL1a included growthinhibition against multiple cancer cell lines with IC50 ranging from 50.6-1446 nM. EtGIPL1a showed promising effects against U251 glioblastoma cells with an IC50 of 193 nM and was further investigated for mechanism. Flow cytometry analysis revealed that EtGIPL1a induced apoptosis in U251 glioblastoma cells and showed significant DNA fragmentation in cell cycle analysis which is considered a hallmark of apoptosis. This was further confirmed with the increased expression of apoptosis protein marker cleaved caspase-3 in U251 cells after treatment with EtGIPL1a. This paper will be submitted shortly to Scientific Reports. In chapter 4, six small molecules (1-6) were isolated and characterized from organic extracts of E. tinctorium. Isolation and purification were achieved through multiple purification strategies including phase separation, Sephadex LH-20 SEC, thin layer chromatography, silica column chromatography and HPLC. Structural elucidation was carried out by ESI-MS, FTIR, NMR and 244 X-Ray crystallography. Compounds (1), (4), (5) and (6) were known compounds identified as orcinol, echinodol, echinodone, and deacetoxyechinodol respectively. Compound (2) turned out to be a new compound which has never been isolated from natural sources. Other than (4), previously isolated from E. tinctorium, three other compounds (1, 5, 6) have been reported from other mushroom species. Despite the identification of (4) and the three compounds from other species, their bioactivity have never been previously reported and were therefore investigated. Compounds (2, 4, 5) exhibited growth-inhibitory activity against HeLa human cervical cancer cells and U251 human glioblastoma cells. Compound (4) also showed promising effects against other cancer cell lines. Additionally, compounds (2) and (4) induced apoptosis in U251 cells. XRay crystal structures of compounds (2), (4) and (5) were reported for the first time. In order to facilitate the final discussion of my thesis findings, I am providing in the following paragraphs the context on advances in the metabolism of sugar chemistry, biosynthetic gene clusters for enzymes involved in biosynthesis of polysaccharides and small molecules, chemical synthesis of isolated molecules, bioactive compounds produced by phylogenetically related and unrelated fungi, structure comparison of isolated molecules to existing drugs, possible molecular pathways of apoptosis in glioblastoma and approaches to molecular target prediction of compounds. 5.4. Biosynthesis of molecules using biosynthetic gene cluster Bioactive compounds including polysaccharides and lanostane-type triterpenes have been identified from mushrooms, however the molecular understanding of biosynthesis for these molecules at the molecular level remains limited. Advances in the genome and transcriptome sequencing as well as functional annotation tools has enabled researchers to predict the gene clusters for biosynthesis of bioactive molecules from mushrooms. Gene clusters for bioactive 245 compounds have recently been investigated for secondary metabolites from Aspergillus fumigatus (Lind et al., 2017), Ganoderma lucidum (Ye et al., 2018), Flammulina filiformis and Hericium erinaceus (Zhang et al., 2019) for triterpenoid biosynthesis. To further understand the molecular basis of bioactive potential of isolated compounds, the genome of the targeted mushroom would need to be sequenced, after which transcriptome sequencing is carried out. RNA sequence reads are mapped to the respective genome using TopHat software. Functional annotation of predicted genes can be performed in NCBI NonRedundant Protein Sequence Database (NR), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), SwissProt, and Clusters of Orthologous Groups (COG) (Chen et al., 2020). Gene clusters for biosynthesis can be reliably predicted by antiSMASH 3.0 and SMURF software and the genes for the enzymes related to biosynthesis of bioactive compounds were identified by homology searches using the genomes of respective mushrooms using BlastP (Chen et al., 2020). Medema et al (2011) gave a more accurate software strategy using software for identification and analysis of gene clusters for secondary metabolites. Based on the given strategy, genes were predicted by Glimmer 3 by employing genome sequence data (FASTA, GBK or EMBL files) and gene clusters were identified. HMMer3 was used to generate profile Hidden Markov Models (pHMMs) based on amino acid sequences of all protein encoding genes. A pHMMs library was generated for signature genes involved in biosynthesis of bioactive compounds (Medema et al., 2011). This suggests that genome sequencing of bioactive mushrooms will provide detailed insights into the genetic basis of therapeutic potential contained in those mushrooms. Another interesting combinatorial biosynthesis approach has been employed for generation of new bioactive compounds against cancers, where selected genes are inactivated giving rise to 246 mutant strains, which then undergo muta-synthesis to generate new product. Alternatively, genes of a biosynthetic pathway are expressed in a producer of a structurally related compound to generate a new compound. Olano and others (2009) reviewed biosynthetic gene clusters of antitumor compounds isolated from actinomycetes. Some of the common compounds included Actinomycin D, Bleomycin A, Doxorubicin, Daunorubicin, and many others. 5.4.1. Biosynthesis of isolated polysaccharides Polysaccharides are structurally complex molecules and understanding the structureactivity relationship is challenging. Structurally, polysaccharides are condensation polymers of varying monosaccharides. The biosynthesis of polysaccharides involves formation of nucleotide sugar precursors, assembly of monosaccharide residues and polymerization of repeating units. The biosynthesis starts with the phosphorylation of glucose to glucose-6-phosphate (Glc-6-P) which then isomerizes to Fructose-6-phosphate (Fru-6-P). Fru-6-P phosphorylates to fructose1,6-Bisphosphate and continues with the metabolic cycle. Fru-6-P isomerizes to mannose-6phosphate and Glc-6-P is converted to Glc-1-P. After multiple steps of conversions with isomerases, epimerases, mutases and dehydratases, as shown in Fig.1, the monosaccharides are added and, after repeating units of monosaccharides, the polysaccharide molecule is formed. The enzymes critical for polysaccharide biosynthesis are phosphoglucomutases (PGM), UDPGlucopyrophosphorylase (UGP) and phosphoglucoisomerase (PGI). PGM catalyzes the conversion of Glc-6-P to glucose-1-phosphate (Glc-1-P), UGP catalyzes conversion of Glc-1-P to UDP-Glucose and PGI isomerizes Glc-6-P to Fru-6-P. These enzymes were identified by homology sequence search using BLAST and are mentioned in previously published studies (Zhang et al., 2019). This biosynthesis has been deduced by researchers (Wang et al., 2017; Ye et al., 2018; Zhang et al., 2019) for polysaccharides from other mushroom species and can be 247 applied to the existing polysaccharides isolated form E. tinctorium. The scheme for biosynthesis mentioned by multiple researchers (Wang et al., 2017; Ye et al., 2018; Zhang et al., 2019) has been combined and summarized in Fig. 5.1. It has been reported that changes in the enzymes critical for biosynthesis of polysaccharides including PGM, UGP and PGI, was correlated with the mole percentage of galactose and mannose in Ganoderma lucidum (Peng et al., 2015). It is likely that the upregulation or down regulation of genes involved in biosynthesis of polysaccharides generates diverse polysaccharides with variable length and monosaccharide composition. Unlike small molecules, to the best of my knowledge, the biosynthesis of bioactive polysaccharides has never been described in literature. The inability to abundantly produce bioactive polysaccharides with little variability and high purity has in large part impeded the advancement of polysaccharide science and adoption of complex carbohydrate in Western medicine (Persin et al., 2011). 248 Glucose Glucokinase Mannose-6-P Phosphomannose isomerases Phosphogluco mutase Glucose-1-P Phosphoglucoisomerases Fructose-6-P Phosphomannose mutase UGP(UDP-Glc phosphorylase) UDP-Glucose Phosphofructokinase Mannose-1-P UDP-Galactose epimerase Fructose-1,6-BisP GDP-Mannose dehydratase GDP-Mannose UDPGalactose Pyruvate dehydratase GDP-4-keto-6 deoxy mannose Glucose-6-P GDP-Mannose Lactate UDP-Glucose UDP-Galactose UDP-Glucose dehydrogenase UDP-GlcA UDP-GlcA UDP-Glucuronate decarboxylase GDP-4k-6DMannose epimerase UDP-Xylose GDP-Fucose Repeating unit UDP-Xylose 4epimerase UDP-Arabinose POLYSACCHARIDE Fig. 5. 1. Schematic representation of biosynthesis of polysaccharides (Summarized from Wang et al., 2017; Ye et al., 2018; Zhang et al., 2019) 5.4.2. Biosynthesis of small molecules isolated from E. tinctorium 5.4.2.1. Biosynthesis of Orcinol (1) Compound (1) identified as orcinol is one of the small molecules isolated from E. tinctorium. Biosynthesis of orcinol has been proposed in literature where it can be biosynthesized by sequential condensations of malonyl-CoA and acetyl-CoA. The scheme was given by Taura and others (Taura et al., 2016). Orcinol synthase was identified as novel plant type III polyketide synthase (PKS) and considered essential in biosynthesis of orcinol. After multiple sequential condensations, a tetraketide-CoA (methyl tetra-ß-ketide CoA) is formed which then spontaneously or non-enzymatically undergoes cyclization in a step wise manner by thioester hydrolysis, aldol condensation, decarboxylation and aromatization (Fig. 5.2). 249 Acetyl-CoA Aromatization Orcinol 3x MalonylCoA Thioester hydrolysis Aldol condensation Methyltetra-ẞ-ketide-CoA OH -CO2 Fig. 5. 2. Biosynthesis of orcinol (1) (Taura et al., 2016) Another study identified the gene clusters for synthesis of orcinol from a pathogenic fungus Fusarium graminearum (Jørgensen et al., 2014). The PKS14 gene cluster was identified to be responsible for producing orsellinic acid and orcinol. It was also hypothesized that orcinol is produced from orsellinic acid by decarboxylation (Fig. 5.3). According to Jørgensen et al. (2014), there were seven genes identified near PKS14, amongst which one gene encoded for a carboxylase, enzyme likely to catalyze conversion of orsellinic acid to orcinol. Since, polyketides including orcinol can be biosynthesized by iterative decarboxylative Claisen condensation in the presence of polyketide synthases (PKS), an alternate synthesis strategy was proposed where polyketide backbone could be synthesized by non-decarboxylative Claisen condensation in the presence of polyketoacyl-CoA thiolases (PKTs) (Tan et al., 2020). Carboxylase -CO2 Orcinol Orsellinic acid Fig. 5. 3. Biosynthesis of orcinol from decarboxylation of orsellenic acid (Jørgensen et al., 2014) 250 5.4.2.2. Biosynthesis of diphenylmethane derivative (2) Biosynthesis of compound (2) has never been investigated due to the fact that it is isolated for the first time from a natural source. Therefore, gene clusters for its biosynthesis need to be identified from the genome of E. tinctorium. It is likely that PKS14 gene cluster is involved. It is also possible that both orsellinic acid and orcinol are biosynthesized and combined to give a keto dimer which is then reduced via some reductases enzymes to give compound (2). 5.4.2.3. Biosynthesis of lanostane-type triterpenes Biosynthesis of triterpenoids takes place by cyclization of squalene. Studies have shown that triterpene backbone can be biosynthesized through two major pathways in plants: the mevalonic acid (MVA) pathway and the methyl erythritol 4-phosphate (MEP) pathway. The MEP pathway does not exist in fungi (Shi et al., 2010). Genetic analysis of G. lucidum has revealed genes involved in MVA pathway (Liu et al., 2012), suggesting that triterpenoids from G. lucidum are biosynthesized via MVA pathway. From the genetic investigation and KEGG gene functional analysis, triterpenoid biosynthesis pathway was summarized (Fig. 5.4) for ganoderic acids isolated from G. lucidum. Likewise, the gene clusters for the enzymes involved in triterpenes biosynthesis were also identified in Flammulina filiformis (Chen et al., 2020) and Wolfiporia cocos (Shu et al., 2013). Compound (4), (5) and (6) are lanostane-type triterpenes isolated from E. tinctorium with structural resemblance to ganoderic acids. It is likely that the isolated compounds are biosynthesized via the MVA pathway to obtain the backbone of triterpenoid structure. 251 Acetyl-CoA Acetyl-CoA acetyltransferase AcetoacetylCoA 3-Hydroxy-3-methylglutaryl - 3-Hydroxy-3methylglutaryl-CoA CoA synthase Mevalonate 3-Hydroxy-3-methylglutaryl -CoA reductase Mevalonate kinase Mevalonate-5-P Squalene Farnesyldiphosphate synthase Squalene synthase Farnesyl-PP Pyrophosphomevalonate decarboxylase Isopentenyl-PP Phosphomevalonate kinase Mevalonate-5-PP Squalene monooxygenase 2,3-Epoxysqualene 2,3-oxidosqualene lanosterol cyclase Cytochrome P450 Lanosterol Triterpenes (variable) Fig. 5. 4. Mevalonic acid pathway for biosynthesis of triterpenes (adapted from Liu et al., 2012) 5.5. Chemical synthesis of isolated molecules 5.5.1. Chemical synthesis of isolated small molecules For compound (1) identified as orcinol, Collie proposed a scheme for chemical synthesis of orcinol (Staunton, J., & Weissman, 2001), involving hydrolysis of dihydro acetic acid to an intermediate that undergoes cyclization and rearrangement to give orcinol as shown in Fig. 5.5. H2O -H2O rearrangement BaH(OH)2 Dihydroacetic acid 5-methylcyclohexa-4-ene1,3-dione Octane-2,4,6-trione Orcinol Fig. 5. 5. Collie’s synthesis scheme for orcinol (1) For compound (2), several diphenylmethane derivatives were chemically synthesized (by reaction of 5-methylresorcinol with para-formaldehyde in the presence of formic acid. This resulted in formation of ortho-ortho form type A, para-para from type B, ortho-para type C and xanthene type D products (Fig. 5.6). Compound (2) was categorized as ortho-ortho type A 252 product. The resulting products were separated by silica column chromatography and HPLC (Matubara et al., 1998). HCOOH Formic acid 5-Methylresorcinol ortho-ortho form Type A Dimer Type A Compound (2) Fig. 5. 6. Chemical synthesis of diphenylmethane derivative compound (2) 5.5.2. Chemical synthesis of polysaccharides Researchers have attempted to synthesize simple carbohydrates although they have faced challenges: lower yields, longer reaction times even for single coupling and finding a single strategy for synthesis. In addition to this, there are also challenges associated with regioselective protection and stereoselective glycosylation for synthesizing complex carbohydrates (Guo, 2008; Kulkarni et al., 2018). This shows the complexity of chemical synthesis for small and simple carbohydrates, making it almost impossible to predict the chemical synthesis outcome of a complex polysaccharide. Several attempts have been made in the past to synthesize polysaccharides by self-condensation and have resulted in low yield and degree of polymerization due to some side reactions that caused chain termination (Schuerch, 1973). One of the reviews highlights the use of one-pot protection glycosylation strategies to obtain complex carbohydrates (Kulkarni et al., 2018). Chemoenzymatic synthesis is another approach that has come forward in recent years for synthesis of complex sugars which occurs in the presence of enzymes (Li et al., 2019). In addition to the aforementioned challenges, there is high molecular weight, variable monosaccharide composition, complex hyper-branched structures, protecting multiple reactive hydroxyl groups, and difficulty in defining the absolute structure of polysaccharides that limits researchers to chemically synthesize complex carbohydrates. 253 5.6. Compounds produced by phylogenetically related versus unrelated fungi Researchers have paid attention to the study of bioactive compounds from fungi and often neglected the importance of fungal hosts which might be a significant source of these bioactive compounds. Fungi have a symbiotic relationship with their hosts (or substrates), whereby the host provides the fungus a suitable habitat with plentiful resources and in return, the fungi produce bioactive compounds that may help the host against biotic and abiotic stresses and also supplements growth. It is most likely that the compounds produced by phylogenetically unrelated fungi are similar to that of its host (Kanematsu & Natori, 1972; Xia et al., 2014). This is based on the examples found in the literature of the different fungi growing on the same host plant, as described in detail in the next few paragraphs. 5.6.1. Phylogenetically unrelated fungi During evolutionary history, fungi have undergone molecular changes in order to adapt to the microenvironments of their habitats. Many studies on endophytic fungi isolated from the same plant host have revealed the presence of the same type of compounds; these have the ability to fight cancer and include paclitaxel, vincristine, vinblastine, camptothecin, and podophyllotoxin, as summarized in Table 5.1. Zhao et al. (2010) have reported 19 taxonomically different classes of fungi having the ability to produce the same bioactive compound “Paclitaxel”, a well-known anticancer drug; most of these fungi are hosted by the same plant genus Taxus (yew) (Zhao et al., 2010; Kumara et al., 2014). Another study conducted on numerous phylogenetically different fungi isolated from Nothapodytes nimmoniana (Icacinaceae) host plant was found to produce the same anticancer compound “camptothecin” (Gurrudatt et al., 2010; Shweta et al., 2010). Other examples include Echinolactone A and D produced by two wood-decaying fungi, Echinodontium japonicum and Granulobasidium vellereum (Nord, 2014; Suzuki et al., 2005). 254 One possible reason why endophytic fungi of different lineages produce similar compounds on the same host might be due to the relationship between these fungi and their host, (whether mutualistic or symbiotic) and could be related to some evolutionary molecular mechanisms. There are two possible mechanisms. The first mechanism is the acquisition of gene clusters or DNA segments from the host plant by the resident fungus. It is important to note that when a fungus establishes a relationship with a host, it usually extends its network and become part of the host. As a result of this, the fungal resident acquires the gene clusters present on plasmids of its host, which are responsible for producing those bioactive compounds. Over time, those plasmids containing gene clusters carry the information for biosynthesis of bioactive compounds in the fungi (Kumara et al., 2014). Staniek et al. (2009) identified the presence of two genes; txs and baps, which were involved in the biosynthesis of Taxol (Staniek et al., 2009). The second mechanism could be the independent evolution of genes that are responsible for the production of bioactive compounds. Based on a study conducted on three endophytic fungi, it was revealed that certain genes have independently evolved in the fungi that have a low homology with the txs and baps genes in Taxol biosynthesis (Xiong et al., 2013). E. tinctorium, a wood-rotting fungus native to western US and Canada causes decay of living trees (Abies grandis) and other conifers such as Western hemlock (Tsuga heterophylla) (Ye et al., 1996). Ye et al. (1996) have isolated an orange pigment named echinotinctone from E. tinctorium, which is also isolated from another fungus Pyrofomes albomarginatus (Ye et al., 1996). The common fact to note is that both fungi are isolated from the wood-rotting polypores found on the same host tree, Abies grandis. In the light of the above-presented evidence, it is possible that the compounds produced from both of these fungi are a contribution from the genetic machinery of the host. In this case, the compounds produced could be “host-derived”. 255 Table 5. 1. Host-derived compounds from phylogenetically unrelated fungi Compound Paclitaxel Fungi Host Cladosporium cladosporioides, Taxus (Yew) Taxomyces andreanae, Taxomyces sp., Alternaria sp., Aspergillus niger var. taxi, Botrytis sp., Botryodiplodia theobromae, Fusarium mairei, Fusarium solani, Metarhizium anisopliae, Papulaspora sp., Pestalotiopsis microspora Podophyllotoxin Alternaria sp., Penicillium sp., Sinopodophyllum Phialocephala fortinii, Trametes hirsuta Camptothecin Neurospora sp., Nothapodytes Entrophospora infrequens Gibberellic acid Fusarium proliferatum Orchid roots Vincristine Vinblastine Alternaria sp., Fusarium oxysporum Catharanthus roseus Huperzine A Blastomyces sp., Botrytis sp. Echinotinctone E. tinctorium, Pyrofomes albomarginatus Phlegmariurus cryptomerianus Abies grandis Reference Staniek et al., 2009; Zhao et al., 2010 Yang et al., 2003; Zhao et al., 2010 Rehman et al., 2008; Puri et al., 2005 Bomke and Tudzynski, 2009 Guo et al., 1998; Zhang et al., 2000 Ju et al., 2009 Ye et al., 1996 5.6.2. Phylogenetically related fungi Phylogenetically related fungi share a close resemblance to each other based on morphological and molecular characters. Because they are genetically close to each other, they might have the ability to produce similar compounds. However, reports in the literature are not numerous. For instance, G. lucidum and other species in the same genus produce the same type of polysaccharides, FIPs and small molecules. The triterpenes and polysaccharides are deemed to be the primary bioactive compounds of Ganoderma (Xia et al., 2014). Another example is Hypomyces subiculosus (Reeves et al., 2008) and Hypomyces trichothecoides (Nair et al., 1980), where both species produce the same compound, Hypothemycin. 256 E. tsugicola is found in Japan and is placed in the same phylogenetic group as E. tinctorium. From my investigation on small molecules described in chapter 4, E. tinctorium contained three lanostane-type triterpenes: echinodol, echinodone and deacetoxyechinodol. It is interesting to note that the latter two compounds have also been isolated from E. tsugicola providing evidence that the same genus produce some similar sets of compounds. In contrast to this, echinodol identified from E. tinctorium is not produced by E. tsugicola (Kanematsu & Natori, 1972). These examples show that phylogenetically-related fungi can also produce the same compounds and, in that case, these are “fungal-derived compounds”. 5.7. Future studies on isolated compounds Given the large number of bioactive compounds found in E. tinctorium, it would be profitable to investigate related species. Based on the phylogenetic classification mentioned in Chapter 1, species within the same genus as E. tinctorium can be potential source of similar compounds. These include E. tsugicola on Tsuga and Abies in Japan that shares many similarities with E. tinctorium found on western hemlock (Tsuga) and grand fir (Abies) in Western America. This study has already confirmed the presence of echinodone and deacetoxyechinodol from both species. Other members of the same genus including E. ballouii on Chamaecyparis in Eastern North America and E. ryvardenii on Juniperus in Europe are found on different hosts. These species may produce interesting compounds on alternate coniferous hosts. In contrast to this, Echinodontiellum japonicum is the only member of the Echinodontiaceae on a hardwood host (Quercus) so it is likely to produce different compounds. Species representatives of Amylostereum and Larssoniporia, placed in the Echinodontiaceae by Liu et al. (2017), also deserve further investigation. Finally, although two species included in the Echinodontiaceae by Gross (1964) have since be placed in other genera in the separate family 257 Bondarzewiaceae, Lauriliella taxodii and Laurilia sulcata, their occurrence of conifer hosts may make them worthy of further study. The anti-proliferative compounds including EtGIPL1a and small molecules (2), (4), and (5) isolated from E. tinctorium have already shown promising effects against cancer cell lines. Compound (2) is a diphenylmethane derivative. An anticancer small molecule A-007 (4,4’Dihydroxybenzophenone-2,4-dinitrophenylhydrazone) for advanced breast cancer melanomas and non-Hodgkin’s lymphoma showed promising results in phase I clinical trials (Morgan et al., 2003). A-007 structure was compared with Tamoxifen, a well-known drug for treatment of breast cancer. Both molecules have similarities in the skeletal backbones of their chemical structure. Both A-007 and tamoxifen contain diphenylmethane chemical moiety and therefore compound (2) can be related with these molecules for its anticancer potential. Structural comparison of both molecules with compound (2) is shown in Fig. 5.7. A-007 Compound (2) Tamoxifen Fig. 5. 7. Structure comparison of A-007, Tamoxifen and compound (2) Some of the well-known antihistamines are also diphenylmethane derivatives. These include hydroxyzine, diphenhydramine, orphenadrine, cetirizine and ebastine. These drugs share the same basic structural unit (Fig. 5.8). It is possible that compound (2) might also possess other 258 biological activities in addition to growth-inhibitory activity. Therefore, it will be interesting to test compound (2) for action on histamine receptors. Hydroxyzine Diphenhydramine Orphenadrine Ebastine Cetirizine Fig. 5. 8. Structures of anti-histamine drugs with diphenylmethane backbone From flow cytometry analysis, compounds (2) and (4) have shown ability to induce apoptosis in U251 glioblastoma cells. Further investigations are clearly required to determine the specific proteins and the molecular pathways involved in their induction apoptosis in U251 cells. The next section focuses on the known possible pathways as well as targets of apoptosis in glioblastoma. The proteins upregulated in those pathways can be investigated in order to determine the molecular pathway that is targeted by EtGIPL1a and small molecules (2), (4) and (5). 259 5.8. Glioblastoma and molecular mechanisms of apoptosis Glioblastoma is a very aggressive primary malignant tumor of the brain and it accounts for 16 % of the primary central nervous system tumors. Apoptosis, programmed cell death, is essential in maintaining homeostasis between cell survival and cell death. A number of molecular pathways have been reviewed for apoptosis in glioblastoma (Fig. 5.9). Apoptosis can occur through the extrinsic pathway (TRAIL/FasL death receptor cascade) or the intrinsic pathway (mitochondrial pathway). The major molecular targets of bioactive molecules against glioblastoma have been identified by multiple studies (Eisele & Weller, 2013; dos Santos Fernandes et al., 2019; Pearson & Regad, 2017; Trjo-Solis et al., 2018; Valdes-Rives et al., 2017) and includes death receptors DR4/DR5 and FasR, TGF-ß, p53, tyrosine kinase receptors (TKR), P13k/Akt/mTOR, and NF-kB. TRAIL-DR4/5 pathway is targeted by Taxol and TKR pathway is targeted by Axitinib, Imatinib, Niclosamide, NVP-BKM120A, CCT128930. NF-kB is a target for Bortezomib, DHNEQ and BAY-11-7082. Finally, mTOR is a target for Everolimus, Sirolimus, Temsirolimus, and Lonafarnib. 260 L Fa s TR AI L SR FA 5 4/ DR p53 FADD Bid PUMA/NOXA M ito tBid Bax Caspase 8 Bax Bak ch on dr ia CytC Caspase 3 Apaf-1 CytC Procaspase-9 Caspase 9 PIP3 PIP2 Akt mTOR TGF-β Smads PDK1 Apoptosis PI3k Nucleus T KR Transcription control (IAPs) NFkB active Ikk complex α,β,ϒ Transcription control (Bax, Bak, PUMA, BcL-2) NFkB inactive p53 TN FR 1 TN Fα Fig. 5. 9. Molecular targets of bioactive molecules for apoptosis in glioblastoma 5.9. Molecular target prediction and their importance Drug-target interaction has been predicted by biochemical and computational tools, where computational target deconvolution is considered faster, cost effective and considerably reliable in the current era. Predicting targets for the lead molecules is one of the essential steps in drug discovery process. The importance of predicting drug targets is to identify the receptors that a drug molecule will effectively bind to produce a response. It can also determine whether the drug has the ability to associate with multiple targets for producing multiple effects. The common computational tools currently available for target prediction include TarPred, MolTarPred, SEA, HitPick and SwissTargetPrediction with varying target coverage in their database. TarPred offers 261 target coverage of 533 whereas MolTarPred 4553, SEA 1400, HitPick 1375, and SwissTargetPrediction have 2686 targets (Peon et al., 2019). MolTarPred is a more user-friendly computational tool for molecular target prediction with large target coverage and also offers reliability of prediction score, ensuring how accurate a predicted target is. Therefore, for the small molecules (1), (2), (4) and (5) isolated from E. tinctorium, MolTarPred program was used to predict their molecular targets. A summary of isolated compounds with their predicted targets and reliability of prediction score is shown in chapter 4 Table 16. Interestingly, compound (2) did not have a reliable predicted target. This could be due to the fact that it has not been isolated before and therefore the database did not have structurally-related molecules which could interact the same way with the targets as (2). In contrast, it could also be that (2) in its current form does not have an effective target and it is possible that it will bind more effectively to predicted targets after chemical modification. This approach is also used to generate more effective and less toxic drug alternatives. Molecular targets predicted for lanostane-type triterpenes (4) and (5) were different although the structure of (4) and (5) differ by only one functional group, suggesting that structurallyrelated compounds can have diverse interactions with the target receptors. Compound (4) also has structural similarity to cholesterol and, not surprisingly the predicted target was 7dehydrocholesterol reductase, a terminal enzyme for biosynthesis of cholesterol. The structural resemblance to cholesterol explains the high prediction score of compound (4) towards this enzyme. The other predicted targets for (4) and (5) have been linked to brain cancers which to some extent could explain the growth-inhibitory effects of both compounds in U251 glioblastoma cells. The results of molecular target prediction using computational tools can then 262 be used to confirm whether or not these results can be reproduced and confirmed by biochemical testing. 5.10. Experimental approaches to identify molecular targets of isolated compounds Small molecule drug development involves two main strategies: target-based drug discovery and phenotype-based drug discovery (Kubota et al., 2019; Shangugguan, 2021; Wilkinson, 2020). Target-based drug discovery is also recognized as “bottom-up strategy” and is a traditional approach where target is identified first followed by small molecule screening. Phenotype-based drug discovery also known as “target deconvolution strategy” has gained more popularity recently in the drug discovery process as it starts with small molecules screening through assays and, once a lead molecule is selected, the molecular targets are then identified (Kubota et al., 2019; Shangugguan, 2021; Wilkinson, 2020). Experimental profiling of predicted targets of compounds can be done by using the assays which can confirm the binding of isolated molecules to its predicted target molecule. For target deconvolution, target prediction is either indirectly assessed via gene expression profiling using microarrays, RNA sequencing and connectivity maps (Kubota et al., 2019; Wilkinson, 2020) or by directly finding the targets that bind to the small molecules which is achieved by chemical proteomics, cloning-based methods (phage display) and protein microarrays (Kubota et al., 2019). For target prediction, small molecules are derivatized with chemical or radioactive labels, probes with or without photo-crosslinking, drug target databases (therapeutic target database, ChEMBL database, BindingDB database, PharmGKB database, canSAR database, DrugBank), biological assays (DNA microarray, RT-PCR, RNA sequencing, and gene knockout), and machine-based methods (support vector machine and random forest algorithm) are employed (Shangugguan, 2021). 263 These approaches have been successful in identifying targets for small molecules like Dasatinib a protein tyrosine kinase inhibitor, Orlistat as an anti-obesity drug, Paraglycine for Parkinson’s disease, Showdomycin, a nucleoside antibiotic (Shu et al., 2013) and therefore, these target deconvolution approaches can be used in the future to experimentally determine the molecular targets of small molecules (1-6) isolated from E. tinctorium as described in this thesis. 5.11. References Bömke, C., & Tudzynski, B. (2009). 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