GANODERMA ISOLATES FROM THE LOWER VOLTA RIVER BASIN OF GHANA: MOLECULAR IDENTIFICATION AND PHYLOGENETIC ANALYSIS, METABOLOMICS AND BIOLOGICAL ACTIVITY EVALUATION A Thesis Presented to the Board of Graduate Studies University of Ghana, Legon, Ghana. In Partial Fulfillment of the requirement for the Degree of Doctor of Philosophy (PhD) in Biochemistry By Gideon Adotey (M. Phil) Department of Biochemistry, Cell and Molecular Biology Faculty of Biological Sciences College of Basic and Applied Sciences University of Ghana Legon, Accra, Ghana University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh i DECLARATION I hereby declare that except for references to other people’s work, this exercise is a result of my own research, and this thesis, either in whole, or in part has not been presented for another degree. ……………………………………………………………… GIDEON ADOTEY (Student) ……………………………………………………………… PROF. ABRAHAM KWABENA ANANG (Supervisor) ……………………………………………………………… PROF. LAUD K.N. A. OKINE (Supervisor) ……………………………………………………………… PROF. W.S.K. GBEWONYO (Supervisor) University of Ghana http://ugspace.ug.edu.gh ii DEDICATION To my mother, Felicia Adzovi Bokor, my father, Adzorlolo Awuku Adotey And the entire Adotey Family for All your love and care University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENTS This study would not have been possible without the expert guidance and encouragement of my supervisors, Professor Abraham Kwabena Anang, Professor Laud K.N. A. Okine and Prof W.S.K. Gbewonyo, all of University of Ghana. I am grateful for their help in the design and execution of the entire research. I am especially thankful to Professor (Mrs.) Regina Appiah-Oppong, Head of Clinical Pathology Department (NMIMR) and the entire staff of Clinical Pathology Department (NMIMR) for their kindness and support. I am also grateful to Prof. Kwaku Kyeremeh of Department of Chemistry, University of Ghana for granting me permission to conduct gravity column chromatographic fractionation aspect of this research in his laboratory. My special thanks go to Prof. Vincent C. Lombardi of University of Nevada, Reno for donating human plasmacytoid dendritic cell (pDC) for this study. I am deeply indebted to Professor. Catherine M. Aime of Purdue University, Dr. Daniel Tura and Dr John C. Holliday of Aloha Medicinals, Nevada, United States, for the help in running the DNA sequencing. Again, from the Clinical Pathology Department of NMIMR, I wish to remember Mrs. Eunice Dotse, Mr. Ebenezer Ofori-Attah and Miss Abigail Anning, for their encouragement and assistance during the conduct of the work described in this thesis. I am also grateful to Professor Marcello Nicolleti and Dr. Claudio Frezza of University Sapienza, Rome, for the excellent facilities made available to me during my period of experiential learning. To all members of the Science Laboratory Technology Department, Accra Technical University, especially Mr. Paul Yirenkyi, Rev. Abraham Quarcoo and Mr. Ahmed Mohammed Gedel and special family friends, Modesta, Dieudonne, Virginia Aseye and Fiona Dziedzorm. University of Ghana http://ugspace.ug.edu.gh iv I finally wish to recognize the fine lecturers and Professors of the Department of Biochemistry, University of Ghana, without whose academic support and professional guidance, I would never have completed this Doctor of Philosophy degree programme. May God bountifully bless you all! University of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENTS DECLARATION……………………………………………………………………………………………i DEDICATION……………………………………………………………………………………………...ii ACKNOWLEDGEMENTS………………………………………………………………………………..iii TABLE OF CONTENTS…………………………………………………………………………………...v LIST OF FIGURES…………………………………………………………………………………………x LIST OF TABLES………………………………………………………………………………………..xiv ABBREVIATIONS……………………………………………………………………………………….xv ABSTRACT…………………………………………………………………………………………….xviii CHAPTER 1………………………………………………………………………………………………..1 INTRODUCTION………………………………………………………………………………………….1 1.1 Ganoderma:-The Ancient Biomedical Fungus……………………………………………………...2 1.2. Ganoderma Bioactive Metabolites………………………………………………………………….2 1.3. Health-Promoting Properties of Ganoderma……………………………………………………......4 1.3.1. Anticancer and Immunomodulatory Activities of Ganoderma……………………………………..5 1.4. Ganoderma and its Health Supplements……………………………………………………………5 1.5. Identification of Ganoderma Species………………………………………………………………6 1.6. Research Question………………………………………………………………………………….7 1.7. Aims and Objectives………………………………………………………………………………..8 17.1. Aim of Study………………………………………………………………………………………..8 1.7.1.1 Specific Objectives…………………………………………………………………………………8 1.8. Justification for the Study…………………………………………………………………………..8 CHAPTER 2………………………………………………………………………………………………10 LITERATURE REVIEW………………………………………………………………………………....10 University of Ghana http://ugspace.ug.edu.gh vi 2.1 Ganoderma and Historical Account……………………………………………………………….11 2.2. Ganoderma Systematics and Phylogenetics……………………………………………………….12 2.2.1 Traditional Systematics……………………………………………………………………………13 2.2.2 Molecular Systematics…………………………………………………………………………….13 2.2.3 Ganoderma Phylogenetics………………………………………………………………………...15 2.3 Bioactive Molecules of Ganoderma……………………………………………………………….19 2.3.1 Ganoderma Polysaccharides………………………………………………………………………19 2.3.1.1 Ganoderma β-D-glucans…………………………………………………………………………..20 2.3.1.2 Ganoderma Heteropolysaccharides……………………………………………………………….21 2.4 Ganoderma Proteins……………………………………………………………………………….21 22.4.1 Ganoderma Glycoproteins………………………………………………………………………...21 2.4 2 Ganoderma Lectins………………………………………………………………………………..21 2.4 3. Ganoderma Protease Inhibitors……………………………………………………………………22 2.5. Ganoderma Triterpenes………………………………………………………………………........22 2.6. Ganoderma Steroids……………………………………………………………………………….23 2.7. Ganoderma Alkaloids……………………………………………………………………………..24 2.8. Ganoderma Biopharmacological Activities……………………………………………………….25 2.8.1. Anticancer Activities of Ganoderma………………………………………………………………26 2.8.2. Immunomodulatory Activities…………………………………………………………………….28 2.8.3. Anti-Oxidative Activities………………………………………………………………………….30 2.8.4. Anti-viral Activity…………………………………………………………………………………32 2.8.5. Anti-inflammatory………………………………………………………………………………...33 2.9. Principles and Theories of Metabolomics…………………………………………………………34 2.9.1. What metabolomics?........................................................................................................................34 2.9.2. Metabolomics Analytical Tools…………………………………………………………………...35 University of Ghana http://ugspace.ug.edu.gh vii 2.9.3. Metabolomics Approaches and Application………………………………………………………36 2.9.4. Statistical Analysis and Data Visualization……………………………………………………….37 CHAPTER 3………………………………………………………………………………………………39 MATERIALS AND METHODS………………………………………………………………………….39 3.1. Chemicals and Reagents…………………………………………………………………………..40 3.2. Molecular Identification and Phylogenetic Analysis……………………………………………..40 3.2.1. Origin, Collection and Sampling of Mushroom Fruit Bodies…………………………………….40 3.2.2. Fungal Tissue Isolation……………………………………………………………………………41 3.2.3. DNA Extraction...............................................................................................................................41 3.2.4. Polymerase Chain Reactions (PCR)……………………………………………………………....42 3.2.5. Cycle Sequencing…………………………………………………………………………………43 3.2.6. DNA Sequence Comparison………………………………………………………………………43 3.2.7. Molecular Phylogenetic Analyses…………………………………………………………………43 3.3. LC-MS-based Metabolomics Analysis of Mycelial Biomass …………………………………….44 3.3.1. Ganoderma Samples for Metabolome Study……………………………………………………..44 3.3.2. Mycelia Biomass Production……………………………………………………………………..44 3.3.3. Sample Preparation for LC-MS Analysis…………………………………………………………44 3.3.4. LC-MS Analysis…………………………………………………………………………………..45 3.3.5. LC-MS Data Processing…………………………………………………………………………...45 3.3.6. Identification of Metabolites………………………………………………………………………45 3.4. Evaluation of Biological Activity…………………………………………………………………46 3.4.1. Cell viability Inhibition……………………………………………………………………………46 3.4.2. Production of Mycelia Biomass…………………………………………………………………...46 3.4.3. Mycelial Biomass Extraction……………………………………………………………………...46 3.4.4. Column Chromatographic fractionation…………………………………………………………..47 University of Ghana http://ugspace.ug.edu.gh viii 3.4.5. In-vitro Cell Viability (MTT) Assay……………………………………………………………....48 3.4.5.1. Cell Cultures Used in the Study…………………………………………………………………..48 3.4.5.2. Cell Viability (MTT) Assay………………………………………………………………………49 CHAPTER 4………………………………………………………………………………………………50 RESULTS………………………………………………………………………………………………....50 4.1. Molecular Identification and Phylogenetic Analysis……………………………………………...51 4.1.1. Origin and Sampling of Ganoderma Isolates……………………………………………………...51 4.1.2. Sequence Generation……………………………………………………………………………....53 4.1.3. DNA Sequence Comparisons by BLASTn………………………………………………………..58 4.1.4. DNA Sequence and Data Sets for Phylogenetic Analysis………………………………………...58 4.1.5. Phylogenetic Analysis ……………………………………………………………………………..59 4.1.5.1. ITS2 Phylogenetic Analysis………………………………………………………………………60 4.1.5.1.2. ITS2 RNA Secondary Structure Analysis………………………………………………………63 4.1.5.2. ITS Phylogenetic Analysis………………………………………………………………………..64 4.1.5.3. LSU Phylogenetic Analysis………………………………………………………………………66 4.2. LC-MS Metabolomics Study………………………………………………………………………68 4.2.1. Metabolomics Comparison………………………………………………………………………..68 4.2.2. Identification of Compounds……………………………………………………………………....70 4.3. Biological Activity Evaluation Studies……………………………………………………………78 4.3.1. Cytotoxic Effect of Mycelial Biomass…………………………………………………………….78 4.3.2. The cytotoxic Effect of Solvent Fractions………………………………………………………...80 4.3.3. Cytotoxic effect of GL-C2 subfractions………………………………………………………….. 86 CHAPTER 5………………………………………………………………………………………………95 DISCUSSION ……………………………………………………………………………………………..95 CHAPTER 6……………………………………………………………………………………………..111 University of Ghana http://ugspace.ug.edu.gh ix SIGNIFICANCE, LIMITATION, SUGGESTION AND CONCLUSION………………………….......111 6.1 Significance……………………………………………………………………………………....112 6.2 Limitation……………………………………………………………………………………….. 112 6.3 Suggestion………………………………………………………………………………………..113 6.4 Conclusion……………………………………………………………………………………….113 REFERENCES…………………………………………………………………………………………..114 University of Ghana http://ugspace.ug.edu.gh x LIST OF FIGURES Figure 1. Typical Ganoderma polysaccharide structure…………………………………………………..20 Figure 2. Typical lanostane structure………………………………………………………………………22 Figure 3. Examples of Ganoderma steroids: (1) ergosterol peroxide and (2) ergosterol…………………..24 Figure 4. Structures of lucidimine A, B, C and D…………………………………………………………..25 Figure 5. Locational map of sample collection site……………………………………………..................41 Figure 6. A flow chart of silica column chromatographic fractionation of Ganoderma LVRB-9 ethanol extract……………………………………………………………………………………………………...48 Figure 7. Collected Ganoderma specimen. A: Ganoderma isolate LVRB-1 growing on dead Azadirachta indica collected from Agortigagorme; B: Ganoderma isolate LVRB-2 growing on dead Acacia spp. collected from Degorme; C: Ganoderma isolate LVRB-9 growing at the base of Mangifera indica collected from Kizito Campus; D: Ganoderma isolate LVRB-14 growing on dead Baphia nitida collected from a farm in Lukunu; E: Ganoderma isolate LVRB-16 growing on at the base of Mangifera indica collected from Lukunu and F: Ganoderma isolate LVRB-17 collected from Azaglo Torkor………………………..53 Figure 8. Bayesian tree showing position of collected Ganoderma compared to ITS2 sequences at GenBank. Branch node values represent Bayesian posterior probability (BPP). Tramete hirsuta from India and Phillipnes used as outgroups…………………………………………………………………………….....62 Figure 9. RNA secondary structures of ITS2 of Ganoderma isolates……………………………………63 Figure 10. Bayesian phylogenetic tree showing position of collected Ganoderma compared to ITS sequence data at GenBank. Branch node values represent Bayesian poterior probability (BPP). Trametes hirsute from India and Philippines used as out groups……………………………………………………………..65 University of Ghana http://ugspace.ug.edu.gh xi Figure 11. Bayesian phylogenetic tree showing position of collected Ganoderma samples compared to LSU sequence data of Ganoderma in GenBank. Branch node values represent Bayesian posterior probability (BPP) values. Trametes hirsuta from Germany and Russia used as outgroups…………………………….67 Figure 12. Total ion chromagram (TIC) of mycelia biomass. A: Ganoderma sample LVRB-1 (G. enigmaticum); B: Ganoderma sample LVRB-17 (G. resinaceum); C: Ganoderma sample LVRB-9 (Ganoderma weberianum-sichuanese)……………………………………………………………………68 Figure 13. An overlaid TIC presentation of mycelial biomass analyzed by LC-MS. Color code is the same as in Figure 12……………………………………………………………………………………………..69 Figure 14. Metabolomic differences between mycelia biomas analyzed. A: PLS-DA score plot and B: Heatmap representation of sample. G1 and G1.1 correspond to two different Ganoderma mycelia biomass sample 1; G9 and G9.1 represent two Ganoderma mycelia sample 9; G17 and G17.1 are two different Ganoderma mycelia biomass sample 17………………………………......................................................70 Figure 15. Structures of triterpenoids compounds in mycelial biomass……………………………………71 Figure 16. Electron ion chromatogram (EIC) for ganoderic acid C6……………………………………….72 Figure 17. Electron ion chromatogram (EIC) for ganoderenic acid K……………………………………...73 Figure 18. Electron ion chromatogram (EIC) for ganoderic acid AM1…………………………………….74 Figure 19. Electron ion chromatogram (EIC) for ganoderenic acid D……………………………………...75 Figure 20. Electron ion chromatogram (EIC) for ganoderenic acid A……………………………………...76 Figure 21. Electron ion chromatogram (EIC) for ganoderic acid G………………………………………..77 Figure 22. Mycelia biomass crude ethanol extract (GL-CO1) cytotoxic effect on PC-3, Jurkat, pDC and normal Chang liver cell lines evaluated by MTT assay…………………………………………………….78 Figure 23. Curcumin cytotoxic effect on Jurkat, PC-3, pace and normal Chang liver cell lines evaluated by MTT assay………………………………………………………………………………………………....79 Figure 24. Mycelia biomass of Ganoderma LRVB-9 solvent fraction GL-C1 cytotoxic effect on Jurkat, PC- 3, pDC and Chang liver cell lines evaluated by MTT assay………………………………………………..81 University of Ghana http://ugspace.ug.edu.gh xii Figure 25. Mycelia biomass of Ganoderma LRVB-9 solvent fraction GL-C2 inhibitory effect on Jurkat, PC-3, pDC and Chang liver cell lines evaluated by MTT assay……………………………………………82 Figure 26. Mycelia biomass of Ganoderma LRVB-9 solvent fraction GL-C3 inhibitory effect on viability of Jurkat, PC-3, pDC and Chang liver cell lines evaluated by MTT assay…………………………………83 Figure 27. Mycelia biomass of Ganoderma LRVB-9 solvent fractions GL-C4 inhibitory effect on Jurkat, PC-3, pDC and Chang liver cell lines evaluated by MTT assay……………………………………………84 Figure 28. Mycelia biomass of Ganoderma LRVB-9 solvent fractions GL-C5 inhibitory effect on Jurkat, PC-3, pDC and Chang liver cell lines evaluated by MTT assay……………………………………………84 Figure 29. Cultured mycelia biomass of Ganoderma LRVB-9 solvent fractions GL-C6 inhibitory effect on Jurkat, PC-3, pDC and Chang liver cell lines evaluated by MTT assay……………………………………85 Figure 30. Mycelial biomass of Ganoderma LRVB-9 solvent fractions GL-C7 inhibitory effect on Jurkat, PC-3, pDC and Chang liver cell line evaluated by MTT assay……………………………………………..85 Figure 31. Mycelia biomass of Ganoderma LVRB-9 sub-fractions GL-C2-C1 cytotoxic effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by MTT assay……………………………………………………………………………………………………….86 Figure 32. Mycelial biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C2 cytotoxic effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by MTT assay……………………………….87 Figure 33. Mycelial biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C3 cytotoxic effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by MTT assay……………………………………88 Figure 33. Mycelial biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C3 cytotoxic effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by MTT assay……………………………………89 Figure 35. Mycelia biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C5 cytotoxic effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by mitochondrial activity using the MTT assay………………………………………………….................................................................................89 University of Ghana http://ugspace.ug.edu.gh xiii Figure 36. Mycelial biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C6 inhibitory effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by MTT assa………………...........................90 Figure 37. Mycelial biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C7 inhibitory effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by MTT assay……………………………….91 Figure 38. Mycelial biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C8 inhibitory effect on Jurkat and PC-3, pDC and normal Chang liver cell lines evaluated by MTT assay……………………………….92 Figure 39 Mycelial biomass of Ganoderma LVRB-9 sub-fraction GL-C2-C8 inhibitory effect on Jurkat and PC-3, pDC and normal Chang liver cell line evaluated by MTT assay…………………………………….93 University of Ghana http://ugspace.ug.edu.gh xiv LIST OF TABLES Table 1. Collection details and morphological features of Ganoderma isolates…………………….........52 Table 2. ITS2 sequence matching results of Ganoderma isolatesLVRB-1, LVRB-2, LVRB-14, LVRB-16 and LVRB-17………………………………………………………………………………………….......54 Table 3. ITS sequence matching results of Ganoderma isolate LVRB-2, LVRB-9 and LVRB-17………55 Table 4. nLSU sequence matching results of Ganoderma isolates LVRB-1, LVRB-2, LVRB-9, LVRB-14, LVRB-16 and LVRB-1756…………………………………………………………. …………………....56 Table 5. ITS2, ITS and nLSU Ganoderma sequences from the Lower Volta River Basin of Ghana and GenBank accession numbers of other isolates used in this study…………………………………………59 Table 6. Details of triterpenoids identified…………………………………………………………….....72 Table 7. IC50 value and selectivity index (SI) of fractions of ethanol extract of cultured Ganoderma LVRB-9 mycelial biomass on Jurkat, PC-3, pDC and Chang liver cell line……………………………...80 Table 8. IC50 values and selectivity index (SI) of sub-fractions of ethanol extract of cultured Ganoderma LVRB-9 mycelial biomass on Jurkat, PC-3, pDC and Chang liver cell line………………...93 University of Ghana http://ugspace.ug.edu.gh xv ABBREVIATIONS ACEI angiotensin-converting enzyme inhibitors ADP Adenosine diphosphate AMA amanitin AMEA antibiotic malt extract agar AP-1 activator protein-1 APBP acidic protein bound polysaccharide ARB angiotensin receptor blockers ARS Apostles Revelation Society Bax Bcl-2-associated X Bcl-2 B-cell lymphoma 2 Bcl-xL B-cell lymphoma-extra large CM-GL carboxymethylated G. lucidum Covid-19 Coronavirus disease 2019 COX-2 cyclooxygenase-2 CREB cyclic element-binding protein D1 domain 1 D2 domain 2 D3 domain 3 DCs dendritic cells DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPPH 1,1-diphenyl-2-picrylhydrazyl DWV deformed wing virus EI-MS Electron Ionization-Mass Spectrometry ELISA enzyme-linked immunosorbent assay EPS exopolysaccharide ESI Electrospray Ionization EV71 Enterovirus 71 FIP -LZ-8 fungal immunomodulatory proteins-ling-zhi-8 FIP-gap1 fungal immunomodulatory proteins G. applanatum 1 FIP-gap2 fungal immunomodulatory proteins G. applanatum 2 FIP-gsi, fungal immunomodulatory proteins-G. sinense FIP-gts fungal immunomodulatory proteins-G. tsugae FIPs fungal immunomodulatory proteins FIPs fungal immunomodulatory proteins G0 gap 0 GA-Me ganoderic acid Me G-CSF granulocyte colony-stimulating factor GLIS G. lucidum immunomodulating substance GLPS G. lucidum polysaccharides GLPss58 G. lucidum polysaccharides sulfated 58 GLTs G. lucidum triterpenoids GM-CSF granulocyte-macrophage colony-stimulating factor GMI G. microsporum GPS Ganoderma polysaccharides GSPS G. sinense polysaccharide University of Ghana http://ugspace.ug.edu.gh xvi GTs Ganoderma triterpenoids Hep G2 human hepatocellular carcinoma HL-60 human promyelocytic leukemia cells HMG-CoA β-Hydroxy β-methylglutaryl-CoA HPBLs human peripheral blood lymphocytes HPLC high performance liquid chromatography HSV-1 herpes simplex virus type 1 HSV-2 herpes simplex virus type 2 IC50 half maximal inhibitory concentration IFN-1 interferon-1 IFN-α interferon-alpha IFN-γ interferon-gamma IL-17A interleukin-17A IL-17F interleukin-17F IL-1β interleukin-1 β IL-2 interleukin-2 IL-22 interleukin-22 IL-23 interleukin-23 IL-6 interleukin-6 ITS internal transcribed spacer ITS internal transcribed spacer 1 ITS1 internal transcribed spacer 3 ITS2 internal transcribed spacer 2 ITS4 internal transcribed spacer 4 kDa kilodalton LC-MS Liquid chromatography–mass spectrometry LPS lipopolysaccharide LSU large subunit LSV Lake Sinai virus LVRB-1 Lower Volta River Basin -1 LVRB-14 Lower Volta River Basin -14 LVRB-16 Lower Volta River Basin -16 LVRB-17 Lower Volta River Basin -17 LVRB-2 Lower Volta River Basin -2 LVRB-9 Lower Volta River Basin -9 LZ-8 ling-zhi-8 LZP-F3 ling-zhi polysaccharide fraction 3 m/z mass-to-charge ratio MAPK mitogen-activated protein kinase MCF-7 human breast adenocarcinoma M-CSF macrophage colony-stimulating factor MDA-MB-231 human breast cancer cells MDR multidrug resistance MEA malt extract agar MHC histocompatibility complex MMP-9 matrix metalloproteinase-9 MTT 3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide NCBI National Center for Biotechnology Information NF-κB) nuclear factor-κB University of Ghana http://ugspace.ug.edu.gh xvii NK natural killer nLSU nuclear large subunit NMR Nuclear magnetic resonance NO nitric oxide NOS nitric oxide synthase nSSU nuclear small subunit PAI-1 plasminogen activator inhibitor-1 PBS phosphate buffered saline PCR Polymerase Chain Reactions pDC plasmacytoid dendritic cell PGE2 prostaglandin E 2 PI3K phosphoinositide 3-kinase PLS-DA partial least squares-discriminate analysis PMA phorbol-12-myristate-13-acetate rDNA ribosomal deoxyribonucleic acid rLZ-8 recombinant ling-zhi-8 RPMI-1640 Roswell Park Memorial Institute-1640 S-180 Sarcoma 180 SeGLP-2B-1 Se-enriched G. lucidum polysaccharide-2B-1 S-GL sulfated G. lucidium SI selectivity index Th1 T helper 1 TIC total ion chromatograms TNF-α tumor necrosis factor alpha tPA tissue plasminogen activators UC urothelial carcinoma uPA urokinase-plasminogen activator) vRNA viral RNA University of Ghana http://ugspace.ug.edu.gh xviii ABSTRACT Ganoderma, a cosmopolitan genus of polypore mushroom, is known to have a number of interesting medicinal properties. The Lower Volta River Basin is reportedly rich in several species of polypore mushrooms resembling Ganoderma. Despite the medicinal importance, the Ganoderma mushroom isolates obtained from this river basin have not been well studied. In this present research study, sequence analysis of the internal transcribed spacer 2 (ITS 2), complete internal transcribed spacer (ITS) and the nuclear large subunit (nLSU) was used to identify collected Ganoderma from this riverine Lower Volta Basin. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to study the chemical constituents of the mycelial biomass of these Ganoderma mushrooms and the effect of their extracts and fractions on the human carcinoma cell line PC-3 and two human lymphoma cell lines; Jurkat, derived from a T cell leukemia and plasmacytoid dendritic cell (pDC) derived from acute leukemia evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The result of the sequence analysis revealed that the Ganoderma sample designated Ganoderma sample 2 belongs G. mbrekobenum species whereas three of the Ganoderma muhrooms belong to the species G. enigmaticum. The sequence analysis further demonstrated that Ganoderma sample coded Ganoderma sample 17 belongs to the species G. resinaceum whereas the sample designated Ganoderma sample 9 belongs to G. weberianum-sichuanese species complex. Thus, the native Ganoderma mushrooms collected in the present study belong to four mushroom species, namely G. mbrekobenum, G. enigmaticum, G. resinaceum and G. weberianum-sichuanese species complex. The current data on molecular identity and phylogeny of Ganoderma mushrooms from Ghana would be helpful in future studies relating to molecular evolution and medical implications of Ganoderma isolates from different regions of Ghana and other part of the world. The total ion chromatogram (TIC) data demonstrated an interesting metabolic profile difference, suggesting UPLC-Q-TOF-MS could be used to differentiate between Lower Volta River Basin Ganoderma isolates based on their mass spectra. The PLS-DA score plot of the mycelial biomass was separated into three distinct clusters, consistent with the phylogenetic analysis in the current study University of Ghana http://ugspace.ug.edu.gh xix which showed that the Ganoderma mushrooms used in the current metabolomic study belong to three different species. UPLC-Q-TOF-MS analysis revealed the presence of six lanostane-triterpenoids in the mycelia biomas of three Ganoderma mushrooms. Ganoderenic acid A, Ganoderenic acid D, Ganoderic C6 and Ganoderic acid G were identified in the mycelia biomass by comparing their mass spectra with pure reference compounds. The remaining two (Ganoderenic acid K and Ganoderic acid AM1), due to absence of reference pure compounds, were annotated by comparing their mass spectra with Ganoderma lanostane triterpenoids previously reported in literature. The result of the biological activity evaluation showed the fraction GL-C2 significantly (≤ 0.05%) inhibited the proliferation and survival of the three cancer cell lines, PC-3, pDC and Jurkat with increasing concentrations and with IC50 values of 27.73±5.25, 21.31±2.40 and 17.09±0.86 μg/mL, respectively compared to Chang liver cells (CVCL_0238) with an IC50 value of 75.41±1.95 μg/mL. The study further demonstrated that the subfraction GL-C2-C1 from GL-C2 demonstrated a potent cytotoxic effect against PC-3 with IC50 value of 3.24± 0.10 μg/mL compared to curcumin with IC50 = 5.13± 0.86 μg/mL. This finding suggests that the subfraction GL-C2-C1 could be an excellent candiadate for developing new treatment option for prostate cancer prevention or treatment. The results also revealed that the subfractions GL-C2-C4 and GL-C2-C5 potently inhibited the growth and survivl of pDC with IC50 values of 19.95±0.50 and 13.57±2.14 μg/mL, respectively, suggesting GL-C2-C4 and GL-C2-C5 may be useful in modulating the production of type I interferon (IFN-1) by suppressing the viability of pDCs and may thereby be useful in developing biopharmaceuticals for treating disorders associated with pDCs. Thus, the current findings demonstrated that specific mycelial fractions are selectively cytotoxic to the three human cancer cell lines suggesting their potential efficacy in the treatment of malignancies. Future study with other cancer cell lines, primary pDCs, T cells, B cell and macrophages as well as animal models is worthy of investigation. The isolation of the bioactive compounds in GL-C2- C1 may lead to a novel bioactive compound that can be used in developing new treatment for prostate cancer whereas novel bioactive compounds from GL-C2-C4 and GL-C2-C5 may lead to a novel compound for developing new treatment for disorders associated with pDC. University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER 1 INTRODUCTION University of Ghana http://ugspace.ug.edu.gh 2 1.1 Ganoderma: - The Ancient Biomedical Fungus Medicinal mushrooms have been used for long because of their novel pharmaceutical attributes and have gained increasing attention in health research (Cheung et al., 2010; Zhang et al., 2016). Several medicinal mushroom species have been developed into dietary supplements for health maintenance or therapeutic “agents” for prevention or treatment of chronic disorders and neurodegenerative diseases (Zhang et al., 2016). In China, Japan and Korea, Ganoderma mushroom is one of such medicinal mushrooms that plays a major role in their traditionl medical system because of its health-promoting properties (Xu et al., 2011; Wachtel-Galor et al., 2011). In Namibia for example, Ganoderma mushrooms are burnt and the smoke is inhaled for relieving flu. This ancient biomedical fungus Ganoderma is regarded in some cultural practices as a symbol of ‘longevity and immortality’ (Wasser, 2005; Halpern, 2007; Lin, 2009). Ganoderma was, therefore, represented in different ancient Chinese art work most probably because of its medicinal and cultural importance (Wasser, 2005; Halpern, 2007). The inclusion in the Chinese pharmacopoeia dating several years back (Halpern, 2007; Chen et al., 2012) and recently in Herbal pharmacopoeia and therapeutic compendium of America (Upton, 2000) strongly highlighted the medicinal and economic importance of Ganoderma as a biomedical fungus. It has been reported that ancient Malaysian traditional healers cut Ganoderma mushrooms into pieces and worn round the neck of children in the form of strings for treating epilepsy (Tan, 2015). However, in modern traditional medicine Ganoderma mushrooms, have been cited for treating chronic fatigue syndrome, diabetes, hepatitis, lower cholesterol level, prevent formation of blood clot and tumor growth (Halpern, 2007). 1.2. Ganoderma Bioactive Metabolites Modern biochemical studies revealed the mycelia, fruit bodies or spores of Ganoderma mushrooms are rich in diverse biologically active compounds, including polysaccharides, triterpenoids, proteins, steroids, sterols, University of Ghana http://ugspace.ug.edu.gh 3 nucleotides, fatty acids and vitamins. These biologically active compounds are reported to have a number of interesting biopharmaceutical properties (Radwan et al., 2011; Xu et al., 2011; Ahmad et al., 2018). Ganoderma is considered by many as a cell factory for producing pharmacologically active compounds. Ganoderma polysaccharides (GPS), for example, have been shown to activate a number of important immune cells in the body (Xu et al., 2011). The activated cells include T lymphocytes, macrophages and natural killer (NK) cells. Numerous authors reported that the activation of these cells by GPS leads to production of IL-6, IL-12 and IFN-γ in these cells. GPS are interestingly known to inhibit mast cells but activate lymphocytes and complement system (Min et al., 2001, Seo et. al., 2009). Ganoderma triterpenoids (GTs), on the other hand, possess wide spectrum of biological activities. The most important biological activities of GTs include antitumour, antiviral, antihypertensive, antiangiogenic, immunomodulating, antihepatitis, antioxidant, anticomplement, and antimicrobial (Akihisa et al., 2007; Boh et al., 2007 and Xu et al., 2011). Dudhgaonkar et al. (2009) reported that GTs unlike GPS have been shown to remarkably suppressed the secretion of TNF-α, IL-6, NO and PGE(2)) from lipopolysaccharide (LPS)-stimulated murine RAW264.7 cells In another similar study, GLTs have been shown to suppress in RAW264.7 cells the expression of inducible nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide (NO) and cyclooxygenase 2 (COX-2), the enzyme catalyzing the synthesis of the proinflammatory mediators, prostaglandins (Bhardwaj et al., 2014). These authors observed that the antiinflammatory and antiproliferative actions of GLT on cells such as macrophages was mediated through the inhibition of NF-kappaB and activator protein-1 (AP-1) signaling pathways. Thus, while GPS activate secretion of inflammatory cytokines, GTs suppress the secretion of cytokines. The opposing role of GPS and GTs, with regard to secretion of inflammatory cytokines has, therefore, made Ganoderma mushroom an interesting biomedical fungus to study. Numerous ergostane bioactive compounds with multifaceted biopharmacological activities have been isolated from Ganoderma mushrooms and they include antitumour (Chen et al., 2009; Cui et al., 2010), antiaging (Weng et al., 2010), antiinflammatory (Akihisa et al., 2007), anticomplement (Seo et al., 2009), and cholesterol lowering University of Ghana http://ugspace.ug.edu.gh 4 (Kim, 2010). Β-sitosterol and its related glycosides is another important class of bioactive compounds found in Ganoderma mushrooms. Β-sitosterol and its glycosides are known to have biological activity against breast, prostate, colon, lung, stomach and ovarian cancer cells (Bin et al., 2015). Other interesting biological activities of β-sitosterol include mosquito larvicidal, trypanocidal, and neutralizing effect on viper and and cobra venom among others (Saeidnia et al., 2014). Silva et al. (2003), in another interesting study, isolated two novel cerebrosides from some species of Ganoderma mushrooms and the isolated cerebrosides have been shown to have inhibitory effect against DNA polymerase, which may lead to cell death. In addition, several biologically active long chain fatty acids, including nonadecanoic, heptadecanoic and hexadecanoic acids with antitumour proliferation effect have been isolated from some species of Ganoderma mushrooms (Fukuzawa et al., 2008; Gao et al., 2012). Nucleosides and nucleobases are another interesting class of bioactive compounds isolated from some Ganoderma mushrooms (Gao et al., 2007). Generally, nucleosides and nucleobases from different sources, including those from Ganoderma mushrooms, have been reported to modulate a number of human physiological processes (Jacobson et al., 2002, Sánchez-Pozo et al., 2002; Guo et al., 2013), which include antiplatelet aggregation, antiarrhythmic and antiseizure effects (Schmidt et al., 2000, Anfossi et al., 2002 and Wang et al., 2008). Similarly, proteins with remarkable immunomodulatory effects, for example, LZ-8, a 12 kDa protein, have been purified from the mycelium culture of G. lucidumWu et al. (2008), indicating G. lucidummay be good candidate for the prevention or treatment of cancer and autoimmune diseases. Besides the above biologically active compounds, trace minerals, including selenium and germanium, known to have anticancer effect, have been reported from some Ganoderma mushroom species. 1.3. Health-Promoting Properties of Ganoderma Sanodiya et al. (2009) and Ahmad et al. (2018) reported a wide range of striking therapeutic properties associated with Ganoderma mushrooms. These striking therapeutic properties include anticancer and mmunomodulatory University of Ghana http://ugspace.ug.edu.gh 5 among others (Ahmad et al., 2018) As a result of these striking medicinal properties mushrooms, belonging to the genus Ganoderma, are used in traditional medicine in many parts of the world (Radwan et al., 2011) 1.3.1. Anticancer and Immunomodulatory Activities of Ganoderma Lin and others (2004) showed that polysaccharides from Ganoderma mushrooms can induce macrophages or T lymphocytes to secrete TNF-α and IFN-γ to suppress tumour cells growth by apoptosis. Although most of the immunomodulatory attributes of Ganoderma mushroom is credited to Ganoderma polysaccharides, recent studies have shown that methanol or ethanol soluble extracts, containing triterpenes from Ganoderma, can enhance expression of cellular immune activity and antigen processing and presentation (Radwan et al., 2011). For example, administration of ganoderic acid Me (GA-Me) has been shown to increase significantly NK cell activity and production of IL-2 and IFN-γ through upregulation of nuclear factor-κB (NF-κB) (Radwan et al., 2011). In another study, GA-Me has been shown to reverse the multidrug resistance (MDR) in colon cancer cells by inducing apoptosis through upregulation of p-p53, Bax, caspase-3, caspase-9 and downregulation of Bcl-2 (Jiang et al., 2011). Similarly, ganoderic acid X, another lanostanoid triterpene, was shown to suppress topoisomerases and induce apoptosis in numerous tomour cell lines (Li et al., 2005). In a similar intriguing study, dried powder of G. lucidum, consisting of 13.5% polysaccharides and 6% triterpenes, was shown to suppres angiogenesis (Stanley et al., 2005). The finding suggested G. lucidum could inhibit prostate cancer and, therefore, may have use for reatment of angiogenesis-dependent prostate cancer (Stanley et al., 2005). These interesting anticancer and immune modulating activities may explain why Ganoderma polysaccharides and triterpenes are used as chemoimmunotherapeutic agents against some cancers. 1.4. Ganoderma and its Health Supplements Health supplements derived from Ganoderma mushrooms come in diverse and various forms such as coffee, tea, capsules or tablets (Lai et al., 2004, Singh et al., 2013) and are formulated from intact fruiting bodies that processed University of Ghana http://ugspace.ug.edu.gh 6 into capsules or tablets (Wachtel-Galor et al., 2011). These health supplements have gained wide acceptance in developed countries such as China, Japan, USA (Paterson (2006) and several developing countries including Ghana although theirv effectiveness hass not be established in properly controlled clinical study. However, Wang et al. (2012) reported health supplements from Ganoderma are used for enhancing cellular immune response or treating diseases associated with immune disorders. Nevertheless, it takes a long time to produce Ganoderma mushroom fruiting bodies and the cultivation technique does not always guarantee production of standardized products. As a result, liquid or solid fermentation techniques have been introduced to produce Ganoderma health supplements to meet the increasing demand and ensure quality products throughout the year (Wachtel-Galor et al., 2011). In liquid or solid fermentation approach, Ganoderma health supplements are formulated from either (i) dried and powdered mycelia or (ii) pulverised mycelial and mushroom primordia combinations (Wachtel-Galor et al., 2011). Gano UltraTM, manufactured by Aloha Medicinals Inc, a U.S. biotechnology company, is one of such Ganoderma mycelial-based health supplements. This supplement, according to the manufacturer, is a combination product of the mycelium, primordia, fruiting body, spores, and extracellular compounds. (https://alohamedicinals.com, accessed 13 July 2019). However, other supplement manufacturing companies reportedly formulate their supplements from Ganoderma polysaccharides and triterpenoids extracted from the fruit bodies and mycelia and then tableted or encapsulated separately or integrated together in some proportions (Wachtel-Galor et al., 2011). 1.5. Identification of Ganoderma Species In view of the growing interest in Ganoderma mushroom as health promoting biomedical fungus, it is very important to establish correctly the identity of the various species used in formulating health supplements. The key morphological features used traditionally in identification of Ganoderma mushroom species include (i) shape and size of basidiospores, (ii) inner core context color and consistency and (iii) microanatomy of fruit body pilear crust (Wachtel-Galor et al., 2011). However, these morphological features vary widely a result of differences in geographical locations, climatic conditions and natural genetic influences such as mutations and recombination of University of Ghana http://ugspace.ug.edu.gh https://alohamedicinals.com/ 7 individual species. This has imposed serious limitation on Ganoderma identification (Hapuarachchi et al., 2018). Furthermore, fungal identification by traditional taxonomy is an experience-based science and most researchers in Africa lack expertise in this field. As a result, researchers have recommended nuclear based molecular approach as one of the most reliable methods for identifying fungi such as Ganoderma (Richter et al., 2015; Welti et al., 2015). The nuclear based molecular approach, which involves DNA sequence analysis, is one of the reliable practical methods for molecular identification of Ganoderma muhrooms. Recently, Ghana Food Research Institute collaborated with with University of Minnesota to establish the molecular identity of Ganoderma from Ghana using nuclear based molecular approach. This collaborative study led to naming of two Ganoderma species, Ganoderma wiiroense (Otto et al., 2015) from Sisala district of Northern Ghana and Ganoderma mbrekobenum from Brong Ahafo and Greater Accra Regions of Ghana (Otto et al., 2016). The Lower Volta River Bank (LVRB) of Ghana, undulating land covered with extensive water bodies and high vegetation, has a widespread distribution of polypore mushrooms resembling Ganoderma but no well-structured studies have been conducted on Ganoderma species from this part of Ghana. The extensive water bodies and high vegetation may explain why there is a widespread distribution of polypore mushrooms in the area. 1.6. Research Question ● What is the molecular identity of the Ganoderma mushrooms collected from the riverine Lower Volta Basin using sequence analysis of nuclear ribosomal regions (ITS2, ITS and LSU)? ● What are the major active components (lanostane triterpenoids) present in the mycelia biomass of these native Ganoderma mushrooms uing LC-MS-MS metabolomic approach? ● How would the extracts and fractions of the mycelial biomass influence survival or vibility of human prostate carcinoma (PC-3), human T lymphoblastic leukemia (Jurkat) and human plasmacytoid dendritic cell (pDC) deived from acute leukemia, in comparison with Chang (normal) liver cell? University of Ghana http://ugspace.ug.edu.gh 8 1.7. Aims and Objectives 17.1. Aim of Study: To study molecular identity, metabolomics and biological activity of polypore mushroom resembling Ganoderma collected in the present study from the Lower Volta River Basin 1.7.1.1 Specific Objectives: The specific objectives of this study were to: 1.7.1.1.1 To establish molecular identity and phylogenetic position of Ganoderma mushrooms collected from LVRB by analyzing the nuclear ribosomal ITS2, ITS and LSU regions 1.7.1.1.2. To characterize the major active components (lanostane triterpenoids) present in the native Ganoderma mushroom from Lower Volta Basin by LC-MS-MS metabolomic analysis. 1.7.1.1.3 To elucidate the effect of extracts, fractions and subfractions of mycelial biomass on human prostatic tumor cell line (PC-3) and human lymphoma cell lines; Jurkat, derived from a T cell leukemia and plasmacytoid dendritic cell (pDC) derived from acute leukemia, in comparison with Chang liver cell (normal liver cell) 1.8. Justification for the Study The study would help to establish the molecular identity and phylogenetic status of the collected Ganoderma mushrooms from the riverine Volta Basin of Ghana, which would be helpful in future studies relating to molecular evolution and medical implications of Ganoderma isolates from Ghana and other parts of the world. The UPLC- Q-TOF-MS-MS metabolomic study would not only help to clarify the differences and similarities in the metabolites present in the Ganoderma mushrooms from this area but would also provide insight into the major secondary metabolites (lanostane triterpenoids) produced by these local Ganoderma muhrooms. The evaluation University of Ghana http://ugspace.ug.edu.gh 9 of effect of extracts, fractions and subfractions of mycelial biomass on human carcinoma cell lines (PC-3) and two human lymphoma cell lines; Jurkat, derived from a T cell leukemia, and PMDC05, a plasmacytoid dendritic cell (pDC) derived from acute leukemia, may help to unlock biopharmaceutical potentials of native LVRB Ganoderma isolates University of Ghana http://ugspace.ug.edu.gh 10 CHAPTER 2 LITERATURE REVIEW University of Ghana http://ugspace.ug.edu.gh 11 2.1 Ganoderma and Historical Account Li et al. (2014) described Ganoderma as a medicinally famous mushroom belonging to the family Ganodermataceae, order Polyporales, class Agaricomycetes and phylum Basidiomycota The name gan is a Latin word which mens “shiny” referring to the surface appearance and derm, which means “skin,” denoting the glossy exterior and woody texture of this biomedical fungus (Wachtel-Galor et al., 2011). Ganoderma mushrooms has been classified into six main categories (red, black, yellow, white, blue and purple) based on the fruit body colour. However, at the macroscopic or morphological level, Zheng et al. (2007) reported Ganoderma mushrooms have been divided into two distinctive subgenera namely, G. lucidum complex and G. applanatum complex. While in Japan, the medicinal mushroom Ganoderma is called Reishi, which means ‘divine mushroom’ or Mannetake, meaning ‘10,000-year mushroom’, in China, it is variously called by names, including Ling Chu, Ling Chih and Ling Zhi, denoting mushroom of immortality (Halpern, 2007). Ganoderma is known to have a reputation as a medicinal material and referred to as a “herb of spiritual potency,” particularly in most Asian countries (Halpern, 2007). The consumption of this ancient medical fungus, prior to its artificial cultivation, was the preserve of only the rich and privileged in society (Wachtel-Galor et al., 2011). Currently, Ganoderma mushroom gained popularity as functional food supplement in China, Japan, USA (Lindequis et al., 2005) and now can be found in health shops in several other countries including Ghana. Although the medicinal attributes of Ganoderma is based largely on hearsay rather than hard facts, traditional uses as well as ritual and cultural practices, several modern biopharmacological studies, to some extent now, support many of the ancient therapeutic claims (Halpern, 2007, Wachtel-Galor et al., 2011, Radwan et al, 2015, Cheng et al., 2010, Jiang et al.2004 ). Ganoderma has a long and memorable history. It has been reported that Taoist priests were the first to experience the medicinal effects of Ganoderma mushrooms. Halpern (2007) reported that Chinese Taoist priests were using Ganoderma to prepare special spiritual or magic food that would enable them to attain higher spiritual state. Professor Dr. Georges M. Halpern disclosed in his book “Healing Mushrooms” an interesting poem regarding the spiritual use of Ganoderma mushroom written by Wang Chung of the Han Dynasty as follows: University of Ghana http://ugspace.ug.edu.gh 12 “They dose themselves with the germ of gold and jade And eat the finest fruit of the purple polypore fungus By eating what is germinal, their bodies are lightened And they are capable of spiritual transcendence” The poem suggested the Chinese priests were using purple polypore fungus (purple Ganoderma) which might contain metabolites that were helping them to build energy, increase stamina and calm cells of the brain (Halpern, 2007). In the Chinese oldest materia medica called Herbal Classic, Ganoderma was assigned the first place in the superior grade ahead of ginseng, a popular Korean herbal drug. Halpern (2007) reported that Ganoderma muhrooms were attributed with numerous medicinal properties. These attributes include spleen tonifying, energy enhancing, strengthening of cardiac function, memory enhancing and anti-aging. The famous State Pharmacopoeia of China (2000) stated that Ganoderma mushrooms can relx the mind. This may explain why this medical fungus may be used to attain a higher state of consciousness during meditation. 2.2. Ganoderma Systematics and Phylogenetics Richter et al. (2015) reported Ganoderma mushroom species in the world ranged from 250 to more than 400. The origin of Ganoderma is currently in doubt and several authors argued this biomedical fungus originates from the tropical regions, because they can survive under hot and humid conditions, before spreading to the temperate zones of the world (Pilotti et al., 2004; Jargalmaa et al., 2017). The reproductive structure of Ganoderma, which is the form that grows from a living or dead wood trunk which may influence the metabolites they are likely to produce, is characterized by a shiny pileus surface and a two-layered basidiospore wall with a truncated apex (Moncalvo, 2000). Although these structural features are used in traditional fungal systematics, they have very limited value for species identification. University of Ghana http://ugspace.ug.edu.gh https://www.ncbi.nlm.nih.gov/books/NBK92757/ https://www.ncbi.nlm.nih.gov/books/NBK92757/ 13 2.2.1. Traditional Systematics Morphological features such as (i) basidiospore shape and size, (ii) context colour and consistency and (iii) microstructure of pilear crust seem to be more reliable for Ganoderma mushroom identification. However, it has been observed that a typical spore is similar for dozens of Ganoderma species. This has made accurate identification of Ganoderma based on basidiospore shape and size very difficult (Jargalmaa et al., 2017). Although colour and consistency of context and pilear crust microstructure are used by some researchers in Ganoderma mushroom identification, they are known to change with age and exposure to different environmental conditions, leading to variation in these macroscopic and microscopic features (Hong et al., 2001). As a result, identifying Ganoderma mushrooms based on macroscopic and microscopic features only is very difficult (Zheng et al., 2007). Although chlamydospore production and shape as well as optimum growth temperature are reported to be useful cultural characters for differentiating similar species, they have also been found to be limited in addressing phylogenetic relationships between taxa (Moncalvo 2000), thereby affecting the development of a natural classification system. As a result, the traditional taxonomic methods for identifying Ganoderma are not only confusing but inconclusive (Hong et al., 2002) and Ganoderma is said to be in a state of “taxonomic chaos” (Jargalmaa et al., 2017). 2.2.2. Molecular Systematics Molecular systematics is now being considered as one of the most practical methods for identifying fungal species. This is because genetic composition of every fungus is not only unique but is also not affected by factors such as age and environmental conditions (Chan, 2003). Lee (2006) reported that molecular method for identifying fungal species has a number of advantages such as being simple, rapid, accurate and doe not require large amount of samples. Above all, the molecular method can be employed easily for Ganoderma mushroom samples in the powdered forms or drug formulation combintions (Lee, 2006). Although molecular systematic is a valuable tool University of Ghana http://ugspace.ug.edu.gh https://www.ncbi.nlm.nih.gov/books/NBK92757/ 14 for identifying fungal species, it does not directly reflect the pharmacological activity. Nevertheless, it can provide relevant information regarding pharmaceutical product quality necessary for public health protection. Currently, a number of PCR based molecular markers are used in identifying various Ganoderma species. The most popular among these molecular markers include nuclear large subunit (Lee et al., 2006), nuclear small subunit (Latiffah et al., 2002), internal transcribed spacer (Moncalvo, 2000; Gottlieb et al., 2000; Latiffah et al., 2002) and some other specific genes (Zhou et al., 2008). Several authors including Kim and Lee, 2000; Park et al., 2000 and Chen et al., 2004 reported that the nLSU and nSSU genes are conserved at the genus or species levels. As a result, most researchers focussed rather on ITS for establishing the natural relationship of mushroom species such as Ganoderma. Hong et al. (2000) and Wesselink et al. (2002) interestingly established that the D1, D2 and D3 regions of the nLSU have enough divergency. These regions of the nLSU are, therefore, used in some studies for inferring phylogenetic relationships between fungal species Since the nLSU region has more diversity than the nSSU, it is used by some researchers alongside ITS (Hong et al., 2000; Wesselink et al., 2002) for establishing the natural relationship within Ganoderma species. Indeed, the ITS region has been shown to have greater sequence variation between closely related species and have a high rate of evolution for fungal species identification (Monard et al., 2013). Although the ITS has been used to characterize severl fungal species, it failed to reolve the molecular identity of some other fungal species (Gazis et al. 2011), therefore, calling for need for additional markers. Recently, the internal transcribed spacer 2 (ITS2) region, for several reasons, has been proposed as one of the most suitable DNA barcodes for fungal species identification. Firstly, the ITS2 region is short and the sequences are easy to amplify with one pair of universal primers, secondly, the region has high inter-specific divergence and thirdly its identification accuracies have been shown to be very high (Chen et al., 2010). Although ITS2 has advantages over other nuclear genomic regions including the ITS, many researchers are not using this nuclear ribosoml region for identification of fungal species. This is partly because some past studies suggested that ITS1 and ITS display higher species diversity relative to ITS2 (Kress et al., 2005). Nevertheless, so far no universal University of Ghana http://ugspace.ug.edu.gh 15 primers for ITS1 and ITS have been developed for general taxonomic use, which has led to low DNA amplification, thus calling for need for specific PCR additives and conditions (Chase et al, 2007; Kress et al., 2007). Based on a recent available evidence and findings, some researchers indicated the ITS2 region could be used as a universal barcode for the identification, especially in closely related species (Müller et al., 2007 and Chen et al., 2010) Although Ganoderma lucidum is the most cited Ganoderma species in scientific literature, current cumulative evidence suggests that many of the Ganoderma species have been cited wrongly (Moncalvo, 2005). In Ghana, most identified Ganoderma species lack supporting molecular data (Obodai et al., 2017); making phylogenetic position of Ganoderma isolates reported from Ghana previously published in research doubtful. This development calls for well-structured research into phylogenetic of Ganoderma from Ghana. This study would pave the way not only for monitoring but also managing diseases caused by Ganoderma mushrooms to cash crops such as cocoa, coffee, cashew in Ghana. This is achievable because the vegetative Ganoderma mycelia isolated, for example, from cash crops and the entire forest ecosystem could easily be identified using modern DNA molecular techniques. Currently, molecular phylogenetic evidence available suggests Africa habours the highest diversity of Ganoderma mushrooms. However, many of these Ganoderma mushroom from Africa have not been reported in published research and the few reported Ganoderma mushroom species were identified based on morphological features (Moncalvo, 2005). Considering the difficulties associated with Ganoderma mushroom identification based on traditional taxonomic approach, the nuclear sequence-based method may be a more practical tool for identifying the poorly sampled different Ganoderma mushrooms from most African countries, including Ghana. In fact, this position is strongly supported by the current ease, low cost of PCR amplification and rapid expansion of molecular databases for Ganoderma at the GenBank. University of Ghana http://ugspace.ug.edu.gh 16 2.2.3. Ganoderma Phylogenetics The systematics of the Ganodermataceae is known to have been carried out hundreds of years. Ganodermataceae family include Ganoderma with a laccate pileal surface and truncated basidiospores and Amauroderma with globose to ellipsoid basidiospores without a truncated apex (Sun et al., 2022). Although Ganoderma mushrooms can easily be recognized in the field based on the macro-morphological character of the sporocarp, a number of researchers recommended that species discrimination should be supported with molecular phylogenetic analysis in order to attain a more stable taxonomic identification. This may explain why, in recent times, numerous studies have been carried on phylogenetic relationships of Ganoderma mushrooms from different geographical regions of the world. Moncalvo et al. (1995), for example, sequenced the 25S ribosomal RNA gene and the internal transcribed spacers of Ganoderma species. The results of the phylogenetic analysis showed tnucleotide sequences of the internal transcribed spacers could discriminate between most Ganoderma species except G. tsugae group which was misnamed. Based on combined data of the D2 region of the 25S ribosomal RNA gene and the internal transcribed spacers, the subgenus Elfvingia was shown to be monophyletic. Furthermore, the results of the phylogenetic analysis of the D2 region alone supported Amauroderma as a sister taxon of Ganoderma. Moncalvo et al. (1995), therefore, concluded that D2 region could be suitable for Ganodermataceae systematics at higher taxonomic levels. In another interesting development, the nuclear ribosomal DNA ITS sequences were used to study the phylogenetic relationships between 34 Ganoderma isolates cultivated in China and the results of the study revealed the 34 isolates clustered into five distinct groups, namely the subgenus Elfvingia, the sect. Phaeonema, and three groups within the sect. Ganoderma. It was also observed in the study that 85.7% of the Ganoderma isolates formed a single group within the sect. Ganoderma (Su et al., 2007) and the genetic diversity between the subgenus Elfvingia and the sects Phaeonema and Ganoderma was to distinctly clear. This observation made the authors to conclude that phylogenetic analysis is a more effective and useful approach not only for studying the taxonomy of Ganoderma but also for establishing phylogenetic relationships within the genus, compared to methods based on morphological analysis (Su et al., 2007). University of Ghana http://ugspace.ug.edu.gh 17 In a recent field trip, an interesting Ganoderma specimen characterized by perennial, sessile fruiting body, fuscous to black pileal surface with laccate crust, was collected from South Africa (Xing et al., 2016). To establish the phylogenetic relationships, the nuclear internal transcribed spacer regions (ITS) and the translation elongation factor 1-α gene (EF1-α) sequences were analyzed. The results of the phylogenetic analysis based on combined ITS and EF1-α sequences showed the collected specimen clustered with G. enigmaticum, but forming a distinct lineage and, therefore, proposed as a new G. aridicola species within G. lucidum complex (Xing et al., 2016). In a similar study, seven specimens of Ganoderma were collected from Yunnan Province of China and the results of phylogenetic analysis of the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α) and the second subunit of RNA polymerase II (RPB2) sequences showed five out of the seven collections clustered together with high bootstrap support, forming a clade sister to G. shanxiense. The remaining two other collected specimens clustered with G. aridicola, G. bambusicola, G. casuarinicola, G. calidohilum, G. enigmaticum and G. thailandicum, but formed distinct lineages and, therefore, proposed as new Ganoderma species, namely G. dianzhongense and G. esculentum. In a more recent study, Gunnels et al. (2020) amplified and sequenced the nuclear ribosomal internal transcribed spacer regions (ITS) of Ganoderma mushrooms commonly used in developing food supplements. The results of phylogenetic analyses of this interesting study revealed the presence of G. lingzhi DNA in all seven herbal supplements. The authors concluded that ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication of fungal and plant species that are otherwise difficult to identify by morphological or biochemical methods (Gunnels et al., 2020). In an earlier study, Liao et al. (2015) studied Ganoderma containing crude drugs, mycelia, spores, and authentic extracts and spore oils using DNA barcoding and the results revealed that G. lucidum cultivated in China was different from those cultivated in Europe. The study also revealed that G. lucidum and G. sinense clustered into two clades that were separated from the other Ganoderma species, strongly supporting the hypothesis that G. lucidum species originating from Europe and East Asia are not the same species. By comparing the ITS2 sequences and RNA secondary structures, the fruiting bodies and commercial products of G. lucidum and G. sinense were successfully distinguished from those of other University of Ghana http://ugspace.ug.edu.gh 18 in this interesting study (Liao et al., 2015). The researchers concluded that DNA barcoding method is applicable to the authentication of commercial products containing Ganoderma species. In a more recent study, Zhang et al (2017) sequenced seven internal transcribed spacer (ITS) sequences of Ganoderma lucidum strains. Phylogenetic analysis of the ITS1 sequences differentiated the strains into three geographic groups while the ITS2 could only differentiate the strains into two groups. It was further observed that the secondary structures of the ITS1 sequences exhibited similar structures with a conserved central core and differed helices but the ITS2 sequences shared similar structures with the difference in helix 4. Thus, compared to ITS2 region, ITS1 region could differentiated Ganoderma lucidum into three geographic originations based on phylogenetic analysis and secondary structure prediction but it is not clear whether the ITS 1 would successfully delineate other Ganoderma mushroom strains at the intraspecific level. Back in Africa, two Ganoderma species were collected from Ficus and Citrus trees from the North East Nile Delta, Egypt. To establish the taxonomic positions, phylogenetic analysis of the ribosomal 5.8S rRNA gene including the flanking internal transcribed spacers (ITS) was performed. The results of this study confirmed the status of the collected ganodeerma mushrooms as G. resinaceum EGM and Ganoderma sp EGDA (El-Fallal et al., 2015). In India, molecular taxonomy of Ganoderma was studied by analyzing the ITS rDNA sequences (Malarvizhi Kaliyaperumal, 2013) because the authors believed identification based on macro-microscopic features could lead to a large number of synonyms resulting in several taxonomic names. The authors found that the Indian isolate coded MYC1 as Ganoderma cupreum clustered with Malaysian and Australian ‘cupreum’. This finding according to the author represented the first molecular evidence of G. cupreum from Asian origin. Kinge et al. (2012) studied the phylogenetic relationships among species of Ganoderma from Cameroon using molecular techniques. Analysis of the internal transcribed spacer and mitochondria small subunit of 28 isolates revealed the isolates belong to eight species, which only G. ryvardense was previously described from Cameroon while G. cupreum and G. weberianum are new records. The remaining five species did not match with any previously described species and have been designated as Ganoderma with different species affinities (Kinge et al., 2012). Recently, Du et al. (2023) performed a phylogenetic studies on the type materials of G. sichuanense (holotype, epitype, and topotype) and G. University of Ghana http://ugspace.ug.edu.gh 19 lingzhi (holotype) and found that G. lucidum is a name mistakenly applied to the widely cultivated Ganoderma species in China, that the scientific binomial for Lingzhi is G. sichuanense and G. lingzhi is the later synonym of G. sichuanense. In another novel phylogenetic studies on Ganodermataceae using six gene loci including the internal transcribed spacer regions (ITS), the large subunit of nuclear ribosomal RNA gene (nLSU), the second largest subunit of RNA polymerase II gene ( rpb2 ), the translation elongation factor 1-αgene (tef1), the small subunit mitochondrial rRNA gene (mtSSU) and the small subunit nuclear ribosomal RNA gene (nSSU), 14 genera, namely Amauroderma, Amaurodermellus, Cristataspora, Foraminispora, Furtadoella, Ganoderma, Haddowia, Humphreya, Magoderna, NeoGanoderma, Sanguinoderma, SinoGanoderma, Tomophagus and Trachydermella were confirmed (Sun et al., 2022). These authors recommended that a combined multi-gene dataset with ITS, nLSU, rpb2, tef1, rpb1 and tub is better for phylogenetic analyses of Ganodermataceae since the internal transcribed spacer region (ITS) considered as the universal barcode of fungi may be limited in identifying complex groups or potential new species. 2.3. Bioactive Molecules of Ganoderma Boh et al. (2007) reported that the main bioactive metabolites responsible for these pharmacological activities of Ganoderma mushrooms include triterpenoids, polysaccharides, steroids, proteins, fatty acids, amino acids, nucleosides and alkaloids. 2.3.1 Ganoderma Polysaccharides Huie and Di (2004) and Wasser (2010) reported that over 200 polysaccharides have been isolated from Ganoderma mushrooms. It has been reported by several authors that the Ganoderma polysaccharides (Figure 1) belong to three main classes, namely β-D-glucans, heteropolysaccharides and glycoproteins (Wasser, 2011; Chang et al., 2012, Mizuno et al., 2013). University of Ghana http://ugspace.ug.edu.gh 20 Figure 1. Typical Ganoderma polysaccharide structure (Zeng, et al., 2019) 2.3.1.1. Ganoderma β-D-glucans Structurally, Ganoderma β-D-glucans contain β-(1→3) D-glucopyranosyl as main chain and glucosyl residue side chains at C-6 position of the main chain (Benkeblia, 2015). Several authors have documented that the degree of substitution of the backbone chain and the length of chain play a role in the biological activities of Ganoderma polysaccharides (Bao et al., 2002; Lin et al., 2005). Zhu et al. (2005), however, argued that biological activities of Ganoderma polysaccharides are rather dependent on the molecular mass, solubility in water and triple-helical structure formation. Other researchers, however, disagreed and insisted β -D-(1→3)-glucosidic linkage is the essential structural feature exclusively responsible for the biological activities of Ganoderma polysaccharides and this may explain why polysaccharides such as starch, consisting of α -D-(1→4)-glucosidic linkages so far, has no known biological activity. Although some authors reported helical structures are neither essential nor advantageous for biological activities, Bao et al. (2002) is of the view that triple-helical structure of β -D-1,3- linked glucans is favourable for T-lymphocyte proliferation. University of Ghana http://ugspace.ug.edu.gh 21 2.3.1.2. Ganoderma Heteropolysaccharides Several heteropolysaccharides, containing different combinations of sugars, have been isolated from different species of Ganoderma mushrooms. Li et al. (2007), for example, isolated a polysaccharide, consisting of galactose, mannose, glucose, arabinose and rhamnose, from submerged mycelial culture of Ganoderma lucidium. Pana et al. (2012) isolated a neutral heteropolysaccharide, which consists of galactose, rhamnose and glucose in the molar ratio of 1.00:1.15:3.22. Wang et al. (2011) similarly isolated 5 water soluble heteropolysaccharides (GL-I to GL- V) from artificially cultured fruit body of the same mushroom species. 2.4. Ganoderma Proteins 2.4.1. Ganoderma Glycoproteins In one interesting study, Wang et al. (2002) isolated a fucose-containing glycoprotein fraction which has the ability to stimulate proliferation of spleen cell and expression of cytokines, including IL-1, IL-2 and IFN-γ from water- soluble extract of G. lucidum. Wu and Wang (2009) in another study purified a water-soluble glycopeptide (PGY), consisting of two moieties of carbohydrate and peptide, from G. lucidum fruit bodies. The glycopeptide (PGY) has been shown to have low DPPH (1, 1diphenyl-2-picryl hydrazyl) radical-scavenging activity but exhibited strong superoxide radical-quenching effect, suggesting this glycopeptide may be a good of source of natural antioxidants. 2.4 2. Ganoderma Lectins Thakur et al. (2007) isolated a 114 kDa hexametric lectin from the fruiting bodies of G. lucidum. Biochemical characterization revealed the lectin has neutral sugar (9.3%) by composition. This 114 kDa hexametric lectin has shown to have hemagglutinating activity against protease treated human erythrocytes. Girjal et al (2011), on the University of Ghana http://ugspace.ug.edu.gh 22 other hand, isolated a smaller (15 kDa) lectin, which has shown biological activity against a number of microorganisms. 2.4 3. Ganoderma Protease Inhibitors Currently, there is a substantially great interest in protease inhibitors. Dunaevsky et al. (2013) reported protease inhibitors are becoming useful for treating diseases such as cancer, malaria, autoimmune and neurodegenerative diseases. El Zawawy et al. (2016) studied antiproteolytic effect of methanol extract of G. lucidum on Pseudomonas aeruginosa, host tissue damaging bacteria. The result revealed methanol extract of G. lucidum could serve as promising approach for treating skin burn infections caused by protease-producing extended spectrum β- lactamase and multidrug resistant Pseudomonas aeruginosa (ESβLMDRPA). 2.5. Ganoderma Triterpenes The triterpenes derived from Ganoderma possess lanostane structure (Figure 2), consisting of 30 or 27 or 24 carbon atoms. Figure 2. Typical lanostane structure (Xia et al., 2014) University of Ghana http://ugspace.ug.edu.gh 23 The majority of triterpenes derived from Ganoderma mushrooms possess a double bond at C-8 (9) on the ring, however, a double bond can also be found at C-7(8), C-9(11), C-20(22), C-22 (23) and C-24(25). In addition to a double bond, some of the triterpenes may have substituent groups at C-3, 7, 11, 12, 15, 22, 23, 24 and 25 positions of the parent nucleus (Xia et al., 2014). On the basis of position of double bond and substituent group, Ganoderma triterpenes can be classified into different structural types. These structural types include ganoderic acid, lucidenic acid, ganoderenic acids, ganoderiols, epoxyganoderiols and lucialdehydes among others. Kubota et al. (1982) isolated the first two lanostane triterpenes, namely ganoderic acid B and ganoderic acid A. Nishitoba et al. (1984) two years after isolated lucidenic acids A, were B and C, possessing C27 carbon atoms, from cultured mycelium of Ganoderma mushrooms. One year later, another structurally similar group, ganoderenic acids A, B, C and D, were isolated G. lucidum Komoda et al., 1985). The same researchers isolated ganoderic acid E, F, G and lucidenic acid D2 from the same Ganoderma mushroom. Over time, several oxygenated compounds, including ganoderic acid C1 to O, P to T, U, W, X, Y, Z and lucidenic acids D1, D2, E1, E2, F, G to M, N, O, P, were isolated from the fruit bodies and cultured mycelia of Ganoderma mushrooms (Baby et al., 2015). Several terpenes, including ganoderiol A, B, C, D, E, F, G, H, I and J as well as epoxyganoderiol A, B and C were isolated from Ganoderma mushrooms such as G. lucidum, G. concinna, G. sinense, G. hainanense and G. amboinense (Baby et al., 2015). Similarly, lucialdehydes A, B, C and lucidal D and E, possessing an aldehydic group in their side-chains, were isolated from G. lucidum, G. pfeifferi and G. concinna (Gao et al., 2002; Niedermeyer et al., 2005; Ma et al., 2012). 2.6. Ganoderma Steroids A number of steroids have been isolated from Ganoderma mushrooms. Structurally, these Ganoderma steroids (Figure 3) have at least one double at C5–C6, C6–C7, and C7–C8, two double bonds at C5–C6/C7–C8, C4– C5/C7–C8, C6–C7/C9–C11, C7–C8/C16–C17 or three double bonds at C4–C5/C6–C7/C8–C14 in the ring University of Ghana http://ugspace.ug.edu.gh 24 systems. Although most of these Ganoderma steroids have C22–C23 double bonds in their side-chains, some have no side-chain double bonds (Baby et al., 2015). Figure 3. Examples of Ganoderma steroids: (1) ergosterol peroxide and (2) ergosterol (Seo et al., 2009) The popular Ganoderma steroids include ergosterol, ergosterol peroxide and stella sterol (Seo et al., 2009) among several other steroidal compounds, including β-sitosterol (Joseph et al., 2011) and daucosterol (Lee et al., 2005). 2.7. Ganoderma Alkaloids Zhao et al. (2015) isolated four novel polycyclic alkaloids (lucidimine A, B, C and D (Figure 4) from the methanol extract of G. lucidumfruit bodies. University of Ghana http://ugspace.ug.edu.gh 25 Figure 4. Structures of lucidimine A, B, C and D (Chen et al., 2018) Huang et al. (2016) similarly isolated lucidimines A and B from G. calidophilum, but they were named as ganocalicines A and B. 2.8. Ganoderma Biopharmacological Activities Numerous authors observed that a number of biopharmacological activities have been attributed to Ganoderma mushrooms. These include anticancer, immunomodulation, antiinflammatory, radioprotective, antiviral, antioxidative, cholesterol synthesis inhibitory, hypoglycemic, hepatoprotective, inhibition of lipid peroxidation/oxidative DNA damage, antimicrobial and anti-aging properties (Smith et al., 2002; Xia et al, 2014; Baby et al., 2015; Kao et al., 2016) University of Ghana http://ugspace.ug.edu.gh 26 2.8.1. Anticancer Activities of Ganoderma Normal cells are known to divide at a self regulated rate which controls their cell cycle. Nevertheless, when the controls fail, the cell cycle is deregulated and it causes abnormal cell reproduction, which eventually leads to cancer (Williams et al., 2012). Cancer has been recognized as a tremendous threat to human health in most nations of the world, including Ghana. As a result, several efforts have been directed to finding antitumour drugs with low toxicity but high efficacy. Several authors reported that Ganoderma species have inhibitory effect on a number of cancer cell lines (Silva, 2003; Xia et al., 2014). This may partly explain why Ganoderma mushrooms are used for treating cancers. The human cancer cell lines that Ganoderma mushrooms demonstrated biological activity against include prostate (Wang et al., 2015; Qu et al., 2017), breast (Rios-Fuller et al., 2018; Yang et al., 2018), leukemia (Müller et al., 2006, Calviño et al., 2010; Yang et al., 2016;), cervix (Liu et al., 2015), ovarian (Dai et al., 2014), colonic (Hong et al., 2004), bladder MTC-11 (Lu et al., 2004) and uroepithelial (Lu et al., 2004). Currently, the mechanism by which Ganoderma mushrooms exert anticancer activities is not completely understood. Wu et al. (2013), however, reported that anticancer activity of Ganoderma mushrooms is exerted, for exmples, through cell cycle arrest and programmed cell death among others. G. lucidium, for example, has been shown to induce cell cycle arrest in estrogen-independent breast cancer cells at G0/G1 phase (Jiang et al., 2006) and at G1 phase in lung cancer cells (Tang et al., 2006). Wachtel-Galor et al (2011) observed that G. lucidumca used cell cycle arrest at G2 phase in human cancer cells such as bladder, prostate, and leukemia cells. In another study, Jiang et al. (2004) investigated the effects of G. lucidum extracts on apoptosis of human prostate cancer cells (PC-3). The results of this study revealed that the mushroom decreased the expression of NF-κB regulated B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL), two important anti-apoptotic proteins that inhibit apoptosis and therefore play a critical role in cancer development and resistance to treatment. The result of this study further revealed that G. lucidum markedly unregulated expression of Bcl-2-associated X Protein (Bax), a proapoptotic protein, leading to the enhancement of the ratio of Bax/Bcl-2 and Bax/Bcl-xL, indicating G. lucidum hold promise as a natural anticancer source. Smina et al. (2017) reported that Ganoderma total triterpene can induce apoptosis University of Ghana http://ugspace.ug.edu.gh 27 in human breast adenocarcinoma (MCF-7) cells by down-regulating the levels of cyclin D1, Bcl-2, Bcl-xL but by up-regulating the levels of Bax and caspase-9. In addition to the total triterpenes, some isolated triterpenes have shown significant cytotoxic effect against several carcinoma cell lines (Cheng et al., 2010). Radwan et al (2015), for example, reported that ganoderic acid A can induce apoptosis in lymphoma cells through caspase-3 and caspase-9. Ganoderic acid A has also been shown to enhance HLA-II mediated antigen presentation and CD4+ T-cell recognition, indicating ganoderic acid A is a candidate for future drug design for the treatment of lymphoma. In another study, ganoderic acid DM demonstrated a number of interesting anticancer effects, including inhibition of cell proliferation, induction of DNA damage, cell cycle arrest at the G1 phase, and apoptosis in human breast cancer cells (Wu et al., 2012). Ganoderic acid DM, similarly, has been shown to improve CD4+ T-cell recognition in melanoma cells (Hossain et al., 2012). This indicates ganoderic acid DM has a chemo-immunotherapeutic potential for inducing a cross-talk between autophagy and apoptosis, as well as improving immune recognition for sustained melanoma tumor clearance (Hossain et al., 2012). On the other hand, ganoderenic acid D has been shown to have cytotoxicity against human hepatocellular carcinoma (hep G2), colorectal adenocarcinoma (caco-2) and cervical adenocarcinoma cells growth (Ruan et al., 2014). Similarly. Ganodermanontriol, a lanostanoid triterpene alcohol, has been shown to suppress the proliferation of human breast cancer cells MDA-MB-231 by expressing the regulatory protein CDC20, which is overexpressed in tumours compared to normal mammary epithelial cells from breast cancer patients (Jiang et al., 2011). Ganodermanontriol has also been shown to inhibit breast cancer cell lines. The inhibition is chieved through urokinase-plasminogen activator (uPA suppression and expression of uPA receptor inhibitiob. This finding suggests Ganodermanontriol as a natural agent has potential for breast cancer treatment (Jiang et al., 2011). In another recent investigation, a new triterpenoid (ethyl lucidenate A) obtained from G. lucidum ethyl acetate fraction has been shown to be cytotoxic human leukemia cells and Burkitt’s lymphoma cells with IC50 values of 25.98 and 20.42 µg /mL, respectively. (Li et al., 2013). In one other interesting study, lucidenic acid B has been shown to suppress phorbol-12-myristate-13-acetate (PMA)-induced HepG2 cells by suppressing the matrix metalloproteinase (MMP)-9 activity in a dose-dependent manner at the transcriptional level. The study further University of Ghana http://ugspace.ug.edu.gh 28 revealed that lucidenic acid B inhibit nuclear factor-kappa B (NF-kB) and activator protein-1 (AP-1) DNA-binding effects of HepG2 liver cells, which lead to downreguling of Matrix metalloproteinase-9 (MMP-9) expression (Weng et al., 2008). Besides triterpenes, Ganoderma polysaccharides have been shown to suppress tumorigenesis and tumor growth (Shang et al., 2011). The polysaccharide designated SeGLP-2B-1, a nutritionally available organic seleno- compound purified and characterized from G. lucidum, for example, has been shown to inhibit growth of the breast cancer cell line MCF-7 by disrupting the mitochondrial membrane potential, and increasing the activities of caspase-9, -3 and poly (ADP-ribose) polymerase (Shang et al., 2011). The combination of ling-zhi polysaccharide fraction 3 (LZP-F3) and arsenic trioxide have been shown to have a significant synergistic growth inhibition on human urothelial carcinoma (UC) cell and arsenic-resistant cell through proapoptotic pathway (Huang et al., 2010). Two water-soluble derivatives, sulfated and carboxymethylated G. lucidumpolysaccharides, coded S-GL and CM- GL, have been demonstrated to inhibit proliferation of Sarcoma 180 (S-180) tumor cells with an IC50 value of 26 and 38 μg/mL, respectively. The Ganoderma polysaccharides S-GL and CM-GL have also been shown to inhibit the growth of S-180 solid tumors implanted in BALB/c mice, with low toxicity (Wang et al., 2009). The antitumor enhancement effects of S-GL and CM-GL indicate they could be developed into anticancer drugs. 2.8.2. Immunomodulatory Activities The development of tumours is known to be supported by immune evasion. This is why natural bioactive compounds with immunomodulatory capabilities are critically needed against immune evasion by cancer cells. Ganoderma is reported by several researchers as one of the natural sources of immunomodulatory compounds. Lin et al. (2004) reported G. lucidum modulates the immune system through immune system enhancement. Liu et al. (2018) has demonstrated that administration of β-D-glucan in phosphate buffered saline (PBS) stimulates lymphocyte proliferation, promotes macrophages to form pseudopodia, and enhances the levels of inflammatory cytokines IL-6 and TNF-α. Structural analysis revealed that the β-D-glucan fractions with molecular weight higher University of Ghana http://ugspace.ug.edu.gh 29 than 1.82 × 106 g·mol-1 exhibit better activity in enhancing the release of inflammatory cytokines, suggesting the bioactivity of Ganoderma polysaccharides is largely influenced by molecular weight. Wang and others also investigated the effects of G. atrum polysaccharide (PSG-1) on dendritic cells (DCs) and found that PSG-1 induce activation and maturation of murine myeloid-derived DCs through mitogen-activated protein kinase (MAPK) pathways (Wang et al., 2017). In an earlier molecular mechanistic study, PSG-1 was found to induce TNF-α secretion through phosphoinositide 3-kinase (PI3K)/Akt, MAPK and NF-κB signaling pathways in RAW264.7 macrophage cells (Yu et al., 2012), thus providing a theoretical basis for the potential of PSG-1 as a novel immunomodulating agent. Zhang et al. (2010) investigated the immunoactivity capacities of proteoglycan fraction (GLIS) of Ganoderma mushroom fruit body on spleen-derived B lymphocytes and bone marrow-derived macrophages. It was found in the study that GLIS exerted anticancer effect by increasing humoral and cellular immune responses. In an earlier study, GLIS treatment was found to enhance proliferation of bone marrow macrophages (BMMs), increase significantly nitric oxide (NO) production, induce cellular respiratory burst, and increase levels of interleukins (IL- 1β), IL-6, IL-12p35, IL-12p40, IL-18, and TNF-α gene expression and levels of TNF-α, IL-1β, and IL-12 secretion. Rubel et al. (2010) studied effect of G. lucidum supplemented diet, formulated from Ganoderma mycelium grown by solid-state culture on wheat grain, and found that the diet produced a significant decrease in T lymphocytes (CD3+ and CD8+) population in spleen cells for three months. Although, in the study, the IFN-γ concentration was significantly increased in both plasma and supernatant of the adherent peritoneal cell cultures from mice fed with this supplement, the adherent peritoneal cells showed a significant increase in IL-10 production, decrease in TNF- α production and decrease in nitric oxide production (Rubel et al., 2010). The study suggests that the G, lucidum mycelium supplemented diet used in the study did not only enhance immunity against cancer cells or pathogenic microorganisms, but also alleviated adverse effects associated with immune system dysfunction. Meng and his research team conducted a comparative tudy on immunomodulatory activity of polysaccharides from two official species of Ganoderma: namely G. lucidum and G. sinense. The finding revealed both GSPS and GLPS potently University of Ghana http://ugspace.ug.edu.gh 30 promote macrophage phagocytosis. The polysaccharides are also known to increase release of nitric oxide and cytokines such as IL-1α, IL-6, IL-1 Fungal immunomodulatory proteins, a special class of glycoproteins have been isolated from Ganoderma mushrooms. Four such glycoproteins, LZ-8 from G. lucidum, FIP-gts from G. tsugae, GMI from G. microsporum and FIP-gsi from G. sinensis, have been shown to have immunomodulatory and cancer prevention effects. LZ-8, the first FIPs isolated from G. lucidium, is regarded as a potential candidate for treating and preventing autoimmune diseases (Bao et al., 2018) while the second, FIP-gts, isolated from G. tsugae, have been shown to possess antitumor activity against lung and urothelial cancer cells (Bao et al., 2018). Hsin et al. (2016) investigated combination treatment of FIP-gts and chloroquine and found that chloroquine increased FIP-gts-induced cytotoxicity in parental and cisplatin-resistant urothelial cancer cell lines, showing the combination treatment may provide an intereting strategy for urothelial cancer treatment (Hsin et al., 2016). Chen and others studied the antiinflammatory and neuroprotective effects of FIPs extracted from G. microsporum (GMI). These researchers found GMI reduced LPS/IFN-γ-induced inflammatory mediator production. Chen et al. (2018) reported antiinflammatory and neuroprotective effects of GMI was achieved through suppression of NO, TNF-α, IL-1β, and PGE2 production. The authors suggested GMI may have potential for treating neuroinflammation and neurodegenerative diseases such as Parkinson’s, Alzheimer’s and stroke. In a related study, Lu et al. (2018) demonstrated that GMI combined with chidamide induced apoptosis and suppressed distal tumor metastasis in melanoma cells, indicating the combination has potential as an immunotherapeutic adjuvant for metastatic melanoma. 2.8.3. Anti-Oxidative Activities It is well known that reactive oxygen species or free radicals seriously harm cells in the body through oxidative processes. There is, therefore, a great interest in antioxidative molecules that could prevent or treat free radical University of Ghana http://ugspace.ug.edu.gh 31 and reactive oxygen species-mediated diseases. Rani et al. (2015) evaluated the free radical scavenging activity of aqueous and methanol extracts of G. lucidum fruiting bodies cultivated on bread fruit (Artocarpus heterophyllus). The study results revealed the methanol extract has stronger scavenging activity for 1,1-diphenyl- 2-picrylhydrazyl (DPPH) with IC50 value of 290 μg/ml and 2,2'-azino-bis (3-ethylbenzothiazoline- 6-sulphonic acid (ABTS), IC50 value of 580 μg/ml) compared to the substrate, whereas the aqueous extract had better scavenging activity for ferric reducing antioxidant power with IC50 value of 5 μg/ml. In a similar study, Wong et al. (2004) investigated the antioxidative effect of G. lucidum against ethanol-induced heart toxicity in the mouse model and found that G. lucidum exhibits a dose-dependent antioxidative effect on the mouse heart homogenate. These researchers attributed the observed antioxidative activity to the cardioprotective effect of G. lucidum and suggest the mushroom may be helpful in protecting the heart from superoxide induced damage (Wong et al. (2004). In another closely related study, Sun et al. (2004) studied antioxidant activity of G. lucidum peptide (GLP) using different oxidation systems and found that GLP has remarkable antioxidant activity in the rat liver tissue homogenates and mitochondrial membrane peroxidation systems and block hemolysis of rat red blood cells in a dose-dependent manner. On the basis of this result, Sun and his co-researchers suggested GLP could play an important role in the inhibition of lipid peroxidation in biological systems through its antioxidant and free radical scavenging activities (Sun et al., 2004). Mahendran et al. (2012) studied the antioxidant capacity of crude exopolysaccharide (EPS) extracts of Ganoderma lucidum by hydrogen peroxide scavenging, 1-1-Diphemy1- 2picryl-hydrazyl (DPPH) radical scavenging and ABTS (2,2 azino bis (3-etheylbenzothiazoline- 6-sulphonicacid) diammonium salt) assays and found that crude exopolysaccharides of the fruiting bodies of G. lucidum has potent antioxidant activity. Park and Kim (2018) cultured G. lucidum mycelium on black bean (Rhynchosia nulubilis) to verify if the mycelium could be used as a functional health ingredient for formulation of dietary supplements. The biological activity of the mycelium ethanol extract was evaluated by DPPH and ABTS assays. The results revealed that G. lucidum mycelium cultivated on Rhynchosia nulubilis has significantly higher radical scavenging activity compared to only the ethanol extract from Rhynchosia nulubilis (Park and Kim, 2018). This finding suggests G. lucidum mycelium University of Ghana http://ugspace.ug.edu.gh 32 cultivated on Rhynchosia nulubilis can significantly enhance antioxidant activity better compared to raw Rhynchosia nulubilis which G. lucidum mycelium was not cultivated on. In another study, the antioxidant activities of four polysaccharides obtained from fermented soybean curd residue by G. lucidum was investigated by hydroxyl radical, reducing power, DPPH free radical, chelating activity, ABTS radical-scavenging and SOD-like activity. The results showed four polysaccharides exhibit antioxidant activities in a concentration-dependent manner. Among the four polysaccharides, GLP-III and GLP-IV exhibited the higher scavenging effects on hydroxyl radicals, ABTS radical, DPPH free radical, and stronger reducing power and SOD- like activity than GLP-I and GLP-I. Shi et al. (2013), therefore, reported that GLP from Ganoderma fermented soybean could have applications in medical and food industries. Liu and others studied antioxidant activity of two low-molecular-weight polysaccharide, GLP(L)1 and GLP(L)2, from fruit body of G. lucidum and found that both GLP(L)1 and GLP(L)2 displayed antioxidant activities but GLP(L)1 was more effective in free radicals scavenging and Fe (2+) chelating. These authors therefore concluded that these two low-molecular-weight polysaccharides may play an important role in the exploration of natural antioxidants in food industry and pharmaceuticals (Liu et al., 2010). Peng et al. (2016) in a phytochemical study isolated 8 aromatic terpenoids from fruiting bodies of G. capense and the isolated compounds showed potent the DPPH radical scavenging antioxidant activity with IC50 values ranging from 6.00±0.11 to 8.20±0.30μg/ml. 2.8.4. Anti-viral Activity Several reports indicate Ganoderma produces different substances with demonstrable antiviral activity (Eo et al., 2000). Recently, Stamets et al. (2018) demonstrated that cultured mycelium extracts of G. resinaceum reduce the levels of honey bee deformed wing virus (DWV) and Lake Sinai virus (LSV), in a dose-dependent manner, indicating honey bees may gain health benefits from this polypore mushroom because of its antimicrobial compounds. Enterovirus 71 (EV71), one of the main causative pathogens of hand, foot and mouth disease (HFMD), has emerged as a major concern among pediatric infectious diseases. Zhang et al. (2014) evaluated the University of Ghana http://ugspace.ug.edu.gh 33 antiviral activities of two G. lucidum triterpenoids (GLTs), lanosta-7,9(11),24-trien-3-one,15;26-dihydroxy (GLTA) and ganoderic acid Y (GLTB), against Enterovirus 71 (EV71 and found Ganoderma triterpenoids prevent EV71 infection. In this interesting study, GLTA and GLTB also demonstrated the ability to significantly inhibit the replication of the viral RNA (vRNA) of EV71. The findings indicate that these two G. lucidum triterpenoids may be potential therapeutic agents to control and treat EV71 infection (Zhang et al., 2014). Niedermeyer et al. (2005) isolated three bioactive compounds (ganoderone A, lucialdehyde B and ergosta-7,22- dien-3beta-ol) from G. pfeifferi and reported that they have potent inhibitory activity against herpes simplex virus. In a similar study, Kim et al. (2000) showed that the antiviral activity of acidic protein bound polysaccharide (APBP) isolated from carpophores of G. lucidum in combination with IFN alpha showed more potent effect on herpes simplex virus than synergistic effects of APBP with IFN gamma, suggesting APBP may be good candidate for developing antiherpetic agent. Several other substances isolated from Ganoderma mushrooms showed biological activity against influenza and HIV virus. El-Mekkawy et al. (1988), for example, reported that ganoderiol F and Ganodermanontriol demonstrated activity against HIV while Ganodermadiol showed in vitro antiviral activity against influenza virus type A. Similarly, ganoderic acid GS-2, 20-hydroxylucidenic acid N, 20(21)-dehydrolucidenic acid N and ganoderiol F have been shown to have inhibitory properties against human immunodeficiency virus-1 protease (Sato et al., 2009). 2.8.5. Anti-inflammatory Chen et al. (2019) investigated the anti-inflammatory activity of G. lucidum polysaccharides (GLPS) on carbon tetrachloride (CCl4)-induced acute liver injury in mice and found GLPS have potential for the prevention and treatment of liver inflammation. In an earlier study, Chen et al. (2018) investigated the anti-inflammatory potential of fungal immunomodulatory protein extracted from G. microsporum (GMI) in an in vitro rodent model of primary University of Ghana http://ugspace.ug.edu.gh 34 neuron/glia cultures. The study results revealed that GMI has the ability to suppress NO, TNF-α, IL-1β, and PGE2 production, indicating GMI may have a potential towards the treatment of neuroinflammation, responsible for the pathogenesis of a number of neurodegenerative diseases. The anti-inflammatory activity evaluation of two new farnesyl phenolic compounds namely, ganoduriporols A and B from G. duripora showed the two compounds exhibit antiinflammatory effects in RAW 264.7 cells by inhibiting the production of TNF-α, IL-1β, IL-6 and PGE2 through the suppression of COX-2, MAPK and NF-κB signaling pathway in LPS-induced macrophage cells. These findings suggest the two new farnesyl phenolic compounds could serve as interesting antiinflammatory agents (Liu et al., 2018). In another development, Zhang et al. (2018) investigated the antiinflammatory properties of GLPss58, a sulfated polysaccharide from G. lucidum formed by chemical sulfation and found that GLPss58 inhibits L-selectin/sTyr-sLeX binding significantly, blocks binding of anti-l-selectin antibodies to L-selectin on the surface of human peripheral blood lymphocytes (HPBLs) and inhibit the secondary lymphoid tissue chemokine-induced chemotactic invasion of HPBLs. This study further revealed that GLPss58 has the ability to inhibit the complement system and cytokines mediated inflammation; suggesting GLPss58 is a favorable potential antiinflammatory agent (Zhang et al., 2018). 2.9. Principles and Theories of Metabolomics 2.9.1. What metabolomics? Metabolomics refers to the systematic and comprehensive study of small molecules present in a biological system including cells, tissues, biological fluids and organisms at a specific point in time. Although the analysis of complete set of metabolites also known as metabolome has been present in biological research for decades, according to the Patti et al. (2012) the term “metabolomics” was only recently coined. As an emerging field of ‘omics’, metabolomics aims principally to compare the endogenous metabolites present in a biological system or a specific physiological state by applying a combination of analytical chemistry, bioinformatics, statistics and University of Ghana http://ugspace.ug.edu.gh 35 biochemistry; explaining why metabolomics is considered an interdisciplinary field of science (