Isolation and Characterization of Extracts from Wild Edible and Non-Edible Mushrooms in Zimbabwe

Isolation and Characterization of Extracts from Wild Edible and Non-edible Mushrooms in Zimbabwe

Tsungai Reid

Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry

Department of Biochemistry Faculty of Science University of Zimbabwe

July 2019

Supervisor: Professor Takafira Mduluza Co-supervisor: Doctor Chenjerayi Kashangura

DECLARATION

I hereby declare that the material contained in this thesis is my own original work and has not been submitted for a degree in any other university.

Tsungai Reid

ABSTRACT Poor nutrition and an increasing emergence of infectious diseases, particularly in developing countries, represent major threats to human health. Mushrooms are known to possess numerous bioactivities such as antioxidant and antimicrobial activities. However, the role of local Zimbabwean mushrooms in human health remains largely unexplored. In this study, ten local mushrooms, namely; Amanita zambiana, Boletus edulis, Cantharellus heinemannianus, C. miomboensis, C. symoensii, Lactarius kabansus, Amanita species, Coprinus species, Ganoderma lucidum and Trametes strumosa, collected from selected areas of Zimbabwe, were characterised for nutritional, secondary metabolites and biological activity. The main research hypothesis was that the extracts of different wild edible and non-edible mushrooms of Zimbabwe contained nutritional, antibacterial and anti-HIV components. Determination of protein content of mushroom powder was carried out using the Kjeldhal method while the qualitative analysis of carbohydrates was done using Molisch’s and Benedict’s tests. The lectin content was detected by the haemagglutination activity of mushrooms against sheep and goat erythrocytes. The total protein content ranged from 9.3% ± 0.99 to 30.8% ± 1.27. Amanita sp., B. edulis and L. kabansus were able to agglutinate both sheep and goat erythrocytes. Amanita zambiana showed high levels of both carbohydrates and reducing sugars. Crude extracts from 50 mushrooms,were obtained using hot water, cold water, methanol, ethanol or acetone were used to determine the protein and total phenol contents using the Folin Ciocalteu (FC) and Lowry C methods, respectively. Boletus edulis had high protein and total phenolic content (2.02 mg ± 0.1 and 503.70 mg ± 20.7, respectively). The antibacterial effect of the crude extracts against Escherichia coli, Salmonella typhi, Staphylococcus aureus and Streptococcus pneumoniae was determined using the agar disc diffusion method. The extracts exhibited antibacterial properties against the four bacteria tested. Sixteen of the extracts that showed high levels of bacterial growth inhibition were selected for further characterization. A total of 131 compounds (CP1 – CP131) were isolated using Preparative Thin Layer Chromatography from the 16 extracts. Thirteen of the isolated compounds exhibited high inhibitory activity against the growth of S. typhi (82 to 99.8%). One of the compounds (CP50) inhibited S. aureus growth (87.5%). Identification of compounds responsible for the high antibacterial activity was carried out using LC-MS. The tepernoids (boviquinone 4, cavipetin D, goshonoside, lucidenic acid M, 26-methyl nigranoate and notoginsenoside); phospholipid (C16 sphinganine) and fatty acid derivatives (11-amino- undecanoic acid, z-13-oxo-9-octadecanoic acid, palmitic amide, sorbitan oleate and stearamide) were identified as compounds partly responsible for the antibacterial activity observed. The effect of the crude extracts against HIV replication was determined using the anti-HIV-1c reverse transcriptase (RT) and HIV-1c p24 ELISA assays. The cold water extract of L. kabansus demonstrated the highest level of HIV-1 RT inhibitory activity (92.6%), whilst the hot water extracts from Coprinus species and C. heinemannianus exhibited high potent levels of HIV-1c p24 inhibitory activity, with IC50 values of 24.3 µg/ml and 33.8 µg/ml, respectively. This study revealed for the first time the presence of bioactive compounds in the local Zimbabwean mushrooms studied. The hypothesis that the extracts of different wild edible and non-edible mushrooms of Zimbabwe contained nutritional, antibacterial and anti- HIV components was proven to be true. The information obtained will potentially enable development of more efficient foods as medicine in the country, based on the wealth of information generated on the health-promoting properties of the ten mushrooms studied. From this study, the development of anti-bacterial and anti-HIV therapeutic agents from the local mushrooms is recommended, due to the presence of antimicrobial compounds identified in the mushroom extracts. The study also recommends the development of edible local mushrooms such as A. zambiana, B. edulis and L. kabansus into functional food products due to their high nutritive value. iii

Dedicated with love to my dear husband, Andrew John Cheyne Reid and our lovely children, Simbarashe Michael, Tinashe Anesu, Rumbidzwaiishe Esther, Joshua Munashe and Nyashadzaishe Mercy

ACKNOWLEDGEMENTS

I am deeply grateful to my Heavenly Father, the Almighty God, for the gift of life, His unfailing love to me and in whose strength l could do all things (Phillipians 4:13). I love you LORD and without You l am nothing.

Many people have helped me along the journey, for this work to be a reality and although l may not name everyone here, l am so thankful. My sincere appreciation goes to my mentor and supervisor, Professor Takafira Mduluza. Thank you Prof. for your guidance, expert advice and support throughout this project and for constantly pushing me to achieve my career goals. Thank you to my co-supervisor, Dr Chenjerayi Kashangura, for your advice, support and expertise in the mushroom field that l could tap into anytime. To my project mentors, Professor Mudadi Albert Benhura and Dr Catherine Chidewe, l am most grateful for your valuable input, especially during the early stages of my research.

To Dr Sikhulile Moyo, Dr Simani Gaseitsiwe and Mr Terrence Mohammed at the Botswana Harvard Partnership HIV Reference Laboratory (BHP), thank you so much for all your support, for hosting me and generously affording me the opportunity to do part of my research work at your institution. I also extend my gratitude to Dr Lucy Mupfumira and Mr Wonderful Choga and many hospitable staff members at BHP for your great help and support. I am thankful to Dr Melvin Leteane and Boingotlo Raphane from the University of Botswana for all your assistance and laboratory skills on HIV research that you taught me while at BHP.

I acknowledge the support of Professor James Hakim, Dr Tariro Makadzange, Professor Val Robertson and Professor Dexter Tagwireyi, through your wisdom and mentorship. To the Chairman of the Biochemistry Department, Professor Stanely Mukanganyama, the Chief Technician, Mrs Elizabeth Chinyanga, my work colleagues, Dr Farisai Chidzwondo, Dr Fiona Robertson, and the entire staff in the Department, l am very thankful to you all for your input and support. To Professor Christopher Chetsanga and Professor Idah Sithole-Niang, thank you for the molecular techniques and skills you taught me during my earlier academic studies and research. Thank you again Professor Chetsanga for your unwavering support, wisdom and mentorship.

I acknowledge and greatly appreciate the financial support l received from the Letten Foundation and University of Zimbabwe Research Grant to enable this study to become a reality. Thank you so much Professor Babill Stray-Pederson for believing in me. To Mrs Auxillia Mazhambe, thank you for your support and facilitation. Thank you to the Letten Foundation family for all your input.

I want to acknowledge the impact that my spiritual parents, Pastors Tom (my dear dad) and Bonnie Deuschle (my dear mum), have had on my life. Your teachings, your message of reformation over the years and how you and your family model every aspect of what you teach us, keeps inspiring me to strive to achieve more and never give up. Pastor Bonnie, your prophetic voice and actions have been a source of encouragement to me all the way. I love you and thank God for you and family. To my dear mum Refiloe, siblings, family (Reid family) in New Zealand and grandmums Esther and Eva, thank you so much for all your support and encouragement.

Last but certainly not least I would like to express my heartfelt gratitude to my dear husband, Andrew John Cheyne for your kindness, patience, love, encouragement and unwavering support. Thank you for being my number one cheer leader. Thank you for releasing me to do my research work even at odd hours and in distant lands, while you minded our family and the hectic schedules, with your own busy schedule. You are amazing! To my lovely sons and daughters: Simbarashe, Tinashe, Rumbidzwaiishe, Joshua and Nyashadzaishe; thank you so much for your kindness, love and support. Your keen interest in what l was doing and cheering me on meant a lot and l truly appreciate. You and your dad are truly a great blessing to me. I treasure and love you and thank God for the precious gift of having all of you in my life.

TABLE OF CONTENTS

Declaration……………………………………………………………………………... (ii) Abstract………………………………………………………………………………… (iii) Dedication……………………………………………………………………………… (iv) Acknowledgements…………………………………………………………………….. (v) Table of Contents………………………………………………………………………. (vii) List of Figures………………………………………………………………………….. (xii) List of Tables…………………………………………………………………………… (xiv) List of Abbreviations………………….……………………………………………….. (xvi) List of Appendices...………………………………………………………...………...... (xix)

CHAPTER ONE: INTRODUCTION

1.1 BACKGROUND INFORMATION……………………………………………. 2 1.2 PROBLEM STATEMENT AND JUSTIFICATION…………………………… 3 1.3 OBJECTIVES OF THE STUDY……………………………………………….. 5 1.3.1 Main Objective of the Study…………………………………………….. 5 1.3.2 Specific Objectives of the Study………………………………………… 5 1.4 RESEARCH QUESTIONS……………………………………………………… 6 1.4.1 Other Research Questions……………………………………………….. 6 1.5 RESEARCH HYPOTHESES …………………………………………………… 6 1.5.1 Other Research Hypotheses……………………………………………… 6

CHAPTER TWO: LITERATURE REVIEW

2.1 MUSHROOM BIOLOGY AND CLASSIFICATION………………………….. 8 2.1.1 Classification of Mushrooms……………………………………………. 9 2.1.2 Types of Mushrooms ……………………………………………………. 12 2.1.2.1 Edible Mushrooms……………………………………………….. 12 2.1.2.2 Non-edible Mushrooms………………………………………….. 13 2.2 HUMAN HEALTH BENEFITS OF MUSHROOMS AND THEIR EXTRACTS 14 2.2.1 Mushrooms as Food and Medicine………………………………………. 14

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2.2.1.1 Mushrooms as Source of Proteins………………………………… 15 2.2.1.2 Mushrooms as Source of Carbohydrates…………………………. 16 2.2.1.3 Phenolic Compounds and Antioxidant Properties of Mushrooms… 17 2.2.2 Mushroom Extracts as Medicine……………………………………...... 18 2.2.2.1 Antibacterial Properties of Mushrooms………………………….. 19 2.2.2.2 Antiviral Properties of Mushrooms………………………………. 21 2.2.2.3 Mushroom Lectins and their Role in Human Health.……………. 22 2.3 CHARACTERIZATION OF MUSHROOM EXTRACTS……………………… 23 2.3.1 Methods of Extraction……………………………………………………. 23 2.3.1.1 Choice and Effect of the Extracting Solvent……………………… 24 2.3.2 Methods of Seperation of Constituents from Mushroom Extracts……….. 26 2.3.2.1 Thin Layer Chromatography (TLC)……………………………... 26 2.3.2.2 High Performance Liquid Chromatography (HPLC)……………… 27 2.3.3 Methods of Identification of Compounds………………………………... 27 2.3.3.1 Ultra Violet (UV)-Visible Spectroscopy………………………….. 28 2.3.3.2 Liquid Chromatography- Mass Spectroscopy (LC-MS)…………... 28 2.3.4 Classes of Secondary Metabolites in Mushrooms……………………….. 29 2.3.4.1 Alkaloids…………………………………………………………. 29 2.3.4.2 Glycosides………………………………………………………… 30 2.3.4.3 Flavonoids………………………………………………………… 30 2.3.4.4 Phenolics………………………………………………………….. 31 2.3.4.5 Terpenoids………………………………………………………… 32 2.3.4.6 Steroids and Sterols……………………………………………….. 32 2.3.5 Methods of Detecting Antibacterial Activity…………………………….. 32 2.3.5.1 Agar Disc and Well Diffusion Methods…………………………. 33 2.3.6 Methods of Detecting Anti-HIV Activity…………………………..…….. 34 2.3.6.1 MTT or XTT Assay………………………………….……………. 35 2.3.6.2 HIV-1 p24 Expression Assay..………………………..……...... 36 2.3.6.3 HIV-1 Reverse Transcriptase (RT) Inhibition Assay…………….. 36 2.4 MUSHROOMS FOUND IN ZIMBABWE…………………………………………... 37

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CHAPTER THREE: MATERIALS AND METHODS

3.1 COLLECTION, IDENTIFICATION AND PREPARATION OF MUSHROOMS…………………………………………………………………... 42 3.2 CHARACTERIZATION OF MUSHROOMS…………………………………… 42 3.2.1 Quantitative Determination of the Total Protein Content………………… 42 3.2.2 Determination of Carbohydrates………………………………………….. 43 3.2.3 Determination of Lectins…………………………………………………. 44 3.2.3.1 Extraction of Mushroom Crude Protein…………………………… 44 3.2.3.2 Preparation of the Red Blood Cells……………………………… 44 3.2.3.3 Haemaggutination Assay………………………………………….. 44 3.3 CHARACTERIZATION OF MUSHROOM CRUDE EXTRACTS…………….. 45 3.3.1 Preparation of Crude Mushrooms Extracts……………………………….. 45 3.3.2 Determination of Protein Content………………………………………… 46 3.3.3 Determination of Total Phenolic Content……………………………...... 46 3.3.4 Determination of Antibacterial Activity of Crude Extracts….…………… 46 3.3.4.1 Analysis of Mushroom Crude Extracts Showing High Antibacterial Activity by Spectrophotometry………………………...... 47 3.3.4.2 Analysis of Crude Extracts by TLC……………………………….. 47 3.3.4.3 Selection of Mobile Phase for Preparative Thin Layer Chromatography (PTLC) ………………………………….………….…... 48 3.3.4.4 Isolation of Compounds from Mushroom extracts by PTLC……… 48 3.3.4.5 Screening of Isolated Fractions for Antibacterial Activity………. 49 3.3.4.6 Identification of Antibacterial Compounds of Mushroom Extracts by Non-targeted LC - MS………………………………………… 50 3.3.5 Determination of Anti-HIV Activity……………………………………… 51 3.3.5.1 Determination of Anti-HIV-1 Reverse Transcriptase Activity…..... 51 3.3.5.2 Cytotoxicity Assay of the Mushroom Extracts…………………….. 52 3.3.5.3 Determination of the in vitro Anti-HIV-1 Activity Using HIV p24 Expression Assay………………………………………… 53 3.3.5.4 Assay for HIV-1c Induced Cytopathic Effect…………………. ….. 54 3.3.5.5 Analysis of the Anti-HIV Mushroom Crude Extracts by Non-targeted LC – MS…………………………………………….. 55

3.4 STATISTICAL ANAYLSIS……………………………………………………… 55

CHAPTER FOUR: RESULTS

4.1 COLLECTION, IDENTIFICATION AND PREPARATION OF MUSHROOMS…………………………………………………………………… 57 4.2 CHARACTERIZATION OF MUSHROOMS…………..……………………….. 58 4.2.1 Quantitative Determination of the Total Protein Content…………………... 58 4.2.2 Qualitative Determination of Carbohydrates……………..………………… 59 4.2.3 Determination of Lectins…………………………………………………… 61 4.3 CHARACTERIZATION OF MUSHROOM EXTRACTS…………………….. 63 4.3.1 Determination of Protein Content of the Crude Extracts…………………. 63 4.3.2 Determination of Total Phenolic Content………………………………… 64 4.3.3 Determination of the Antibacterial Activity of Mushroom Crude Extracts…………...... 66 4.4 CHARACTERIZATION OF MUSHROOM EXTRACTS SHOWING HIGH ANTIBACTERIAL ACTIVITY…...... 74 4.4.1 Analysis of the Extracts using Absorption Spectroscopy and TLC………. 74 4.4.2 Separation of Mushroom Crude Extracts by TLC………………………… 78 4.4.3 Isolation of Components of Mushroom Crude Extracts by PTLC……….. 84 4.4.4 Determination of the Antibacterial Activity of the Isolated Fractions……. 84 4.4.5 Identification of the Most Potent Antibacterial Compounds of Mushroom Extracts by LC – MS…………………………………………………...... 86 4.5 DETERMINATION OF THE ANTI-HIV ACTIVITY OF MUSHROOM CRUDE EXTRACTS…….………………………………………………………. 88 4.5.1 Determination of Anti-HIV-1 Reverse Transcriptase Activity…………… 88 4.5.2 Cytotoxicity Assay of Mushroom Extracts …….………………………… 90 4.5.3 Determination of the In Vitro Anti-HIV-1 Activity using HIV p24 Antigen Expression Assay………………………………………………... 92 4.5.4 Analysis of the Anti-HIV Mushroom Crude Extracts by Non-Targeted LC – MS………………………………………………………………….. 94

CHAPTER FIVE: DISCUSSION

5.1 CHARACTERIZATION OF MUSHROOM FRUITING BODIES……………... 97 5.1.1 Quantitative Determination of the Total Protein Content………………… 97 5.1.2 Qualitative Determination of Carbohydrates……………………………... 98 5.1.3 Determination of Lectins…………………………………………………. 99 5.2 CHARACTERIZATION OF MUSHROOM CRUDE EXTRACTS…………….. 99 5.2.1 Determination of Protein Content of Crude Extracts……………………... 99 5.2.2 Determination of Total Phenolic Content of Crude Extracts …………...... 100 5.2.3 Determination of Antibacterial Activity of Crude Extracts ……………… 101 5.2.4 Characterization of Mushroom Extracts Showing High Antibacterial Activity……………………………………………………………………. 105 5.2.4.1 Analysis of the Extracts Using Absorption Spectroscopy and TLC……………………………………………………………….. 105 5.2.4.2 Separation of Mushroom Compounds by TLC and Isolation of Components of Mushroom Extracts by PTLC……………….. 106 5.2.4.3 Determination of Antibacterial Activity of the Isolated Compounds………………………………………..……… 107 5.2.4.4 Analysis of Antibacterial Components of Mushroom Extracts by LC - MS ………………………………………………………...... 108 5.2.5 Determination of the Anti-HIV Activity of Mushroom Crude Extracts…. 112 5.2.5.1 Determination of Anti-HIV-1 Reverse Transcriptase Activity……. 112 5.2.5.2 Cytotoxicity Assay of Mushroom Extracts………………………… 113 5.2.5.3 Determination of the in vitro Anti-HIV-1 Activity Using p24 Antigen Expression Assay……………………………………….. 113 5.2.5.4 Analysis of the Anti-HIV Mushroom Crude Extracts by LC – MS…………………………………………………………... 115

6.0 CONCLUSION ……………………………………………………………….. 117 6.1 RECOMMENDATIONS…………………………………………………………. 119 7.0 REFERENCES………………………………………………………………….. 121 8.0 APPENDICES…………………………………………………………………… 137

LIST OF FIGURES

Figure No. Title Page No. Figure 2.1: Agaricus bisporus 8 Figure 2.2: Amanita fulvoalba 9 Figure 2.3: Overview of the six main classes of secondary metabolites with representative examples 30 Figure 2.4: Agar disc diffusion assay showing zones of inhibitions of bacterial growth 34 Figure 2.5: Antiretroviral targets in HIV Life Cycle 35 Figure 2.6: HIV-1 ELISA p24 antigen assay 36 Figure 2.7: Different types of mushrooms commonly found in Zimbabwe and used in this study 39 Figure 4.1: Sliced pieces of Cantharellus miomboensis during the drying process 58 Figure 4.2: Percentage total protein content of nine different mushrooms 59 Figure 4.3: Analysis of carbohydrates showing positive tests for (A) Benedict’s test for reducing sugars and (B) Molisch’s test for carbohydrates 60 Figure 4.4: Haemagglutination assay of three of the ten mushroom species with sheep erythrocytes 62 Figure 4.5: Protein content of mushrooms extracted by methanol, ethanol, acetone, cold water and hot water 64 Figure 4.6: Total phenolic content of mushrooms extracted by methanol, ethanol, acetone, cold water and hot water 66

Figure 4.7: Representative UV spectra obtained from acetone (Ac) and water (H2O) extracts of mushrooms 75 Figure 4.8: Representative UV spectra obtained from ethanol (Eth) extracts of mushrooms 75 Figure 4.9: Representative UV spectra obtained from methanol (Meth) extracts of mushrooms 76 Figure 4.10: Representative UV spectra obtained from different solvent extracts of the same mushroom 77 Figure 4.11: Representative chromatograms of mushrooms extracts developed in TEM solvent and sprayed with vanillin - sulphuric acid 78

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Figure 4.12: Representative assay for anti-HIV RT activity of mushroom crude extracts 89 Figure 4.13: HIV-1 Reverse transcriptase inhibitory activity of crude extracts from edible and non-edible mushrooms 90

Figure 4.14: Representative curve used to derive the CC50 values of the different mushroom extracts 91 Figure 4.15: Anti-HIV activity of the hot water extracts of Coprinus sp. and C. heinemannianus, showing a dose dependent inhibitory activity 94 Figure 5.1: Structure of lucidenic acid M 109 Figure 5.2: Structure of cavipetin D 110 Figure 5.3: Structure of palmitic amide 110 Figure 5.4: Structure of phytosphingosine 111

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LIST OF TABLES

Table No. Title Page No. Table 2.1: Simplified classification of the major groups of fungi according to Ainsworth, 1971 11 Table 2.2: Effect of solvents in extracting different components from biological sources 25 Table 4.1: Different types of mushrooms collected from the woodlands of Zimbabwe 57 Table 4.2: Qualitative analysis of carbohydrate content in mushrooms using Molisch’s test and Benedict’s tests 61 Table 4.3: Haemagglutination assay of the ten mushroom species with sheep and goat erythrocytes 62 Table 4.4: Specificity activity of crude extracts of mushrooms showing haemagglutination activity 63 Table 4.5: Antibacterial activities of methanol extracts of mushrooms on test organisms 67 Table 4.6: Antibacterial activities of ethanol extracts of mushrooms on test organisms 68 Table 4.7: Antibacterial activities of acetone extracts of mushrooms on test organisms 69 Table 4.8: Antibacterial activities of cold water extracts of mushrooms on test organisms 70 Table 4.9: Antibacterial activities of hot water extracts of mushrooms on test organisms 71 Table 4.10: Extracts exhibiting high antibacterial activity (9 – 14 mm zones of inhibition) which were selected for further study 73 Table 4.11: Absorption spectrum peaks obtained from the selected crude extracts of mushrooms that showed high antibacterial activity 74

Table 4.12: The Rf values of fractions separated from selected mushroom extracts using different TLC mobile phases 80 Table 4.13: Number of fractions obtained after scrapping bands from each of the separated crude extract on the PTLC plate 84

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Table 4.14: Percentage inhibition of growth of S. typhi and S. aureus by the fourteen potent compounds isolated from different mushroom crude extracts using PTLC 86 Table 4.15: Compounds identified from seven of the most potent components isolated from the antibacterial crude extracts of mushrooms 87

Table 4.16: The CC50 values of mushroom extracts showing varying levels of inhibitory effects to the growth of PBMCs 92 Table 4.17: Inhibitory effects of different mushroom extracts on HIV-1 replication 93 Table 4.18: Cytotoxicity and anti-HIV-1 activity of the hot water extracts of

Coprinus species and C. heinemannianus using HIV-1c (MJ4) in PBMC cells 94

LIST OF ABBREVIATIONS A. bisporus Agaricus bisporus ANOVA Analysis of variance AZT Azidothymidine A. zambiana Amanita zambiana ABTS 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) Amanita sp. Amanita species Anti-DIG-POD Anti-digoxigenin peroxidase B. edulis Boletus edulis BSA Bovine serum albumin

CC50 50% cytotoxic concentration CD4 Cluster of differentiation 4 CFU Colony forming units CM Chloroform: methanol

CO2 Carbon dioxide C. heinemannianus Cantharellus heinemannianus C. miomboensis Cantharellus miomboensis C. symoensii Cantharellus symoensii DNA Deoxyribonucleic acid DMSO Dimethyl sulphoxide DDDP DNA-dependant-DNA polymerase DW Dry weight EEW Ethyl acetate:ethanol:water EMW Ethyl acetate:methanol:water E. coli Escherichia coli

EC50 Half maximal effective concentration ELISA Enzyme linked immunosorbent assay EDTA Ethylenediaminetetraacetic acid FBS Fetal bovine serum FC Folin – Ciocalteu G. lucidum Ganoderma lucidum GAE Gallic acid equivalent GC – MS Gas chromatography – mass spectroscopy

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HAU Hemagglutination unit HIV-1c Human immunodeficiency virus type 1 subtype C HIV-1 gp120 HIV-1 surface glycoprotein 120 HPLC High performance liquid chromatography HRP Horse radish peroxidase IL-1 Interleukin 1 IL-2 Interleukin 2 IR Infra-red LC–DAD Liquid chromatography – diode array detection LC – MS Liquid chromatography – mass spectroscopy LC – NMR Liquid chromatography – nuclear magnetic resonance spectroscopy L. edodes Lentinus edodes L. kabansus Lactarius kabansus MAE Microwave assisted extraction Mg Milligram m/z Mass-to-charge ratio MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide Oligo-dT Oligonucleotide-deoxythymine PBMCs Peripheral blood mononuclear cells PBS Phosphate buffered saline PHA Phytohaemagglutinin Poly A Poly adenine Psig Per square inch gauge PTLC Preparative thin layer chromatography Q-TOF Quadrupole-time of flight mass spectrometer RPMI-1640 Roswell Park Memorial Institute-1640 medium RNA Ribonucleic acid RNase H Ribonleclease H

Rf Retention factor S. aureus Staphylococcus aureus S. pneumoniae Streptococcus pneumoniae SD Standard deviation SI Selectivity index

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TEM Toluene:ethylacetate TLC Thin layer chromatography TLC-DB Thin layer chromatography – direct bioautography TMB 3,3',5,5'-Tetramethylbenzidine T. strumosa Trametes strumosa UAE Ultrasound assisted extraction UV Ultra violet V Voltage v/v Volume per volume w/v Weight per volume XTT Sodium 3-[1-(phenylamino)-carbonyl]- 3,4-tetrazoliumbis (4-methoxy- 6-nitro) benzene-sulfonic acid hydrate

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LIST OF APPENDICES

Appendix No. Title Page No. Appendix 8.1: Publications arising from the work in this thesis 137 Appendix 8.2: Preparation of reagents 138 Appendix 8.3: Percentage inhibition of bacterial growth by components isolated from different mushroom extracts using PTLC 139 Appendix 8.4: LC-MS Chromatograms of isolated components of mushroom samples that exhibited high anti-bacterial activity analyzed in positive mode with a column 142 Appendix 8.5: LC-MS profiles of some of the mushroom crude extracts that exhibited high anti-HIV activity analyzed in positive mode with a column 143

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CHAPTER ONE:

INTRODUCTION

1.0 INTRODUCTION

1.1 BACKGROUND INFORMATION

Mushrooms are a group of fungi that belong to the higher phyla Ascomycota and

Basidiomycota, with distinctive fruiting bodies and reproductive structures. The fruiting bodies especially basidiomes are usually umbrella shaped structures that produce spores in large numbers (Camassola, 2013; Florence and Balasundaran, 2000). Mushrooms lack chlorophyll, unlike green plants and obtain nutrition from non-living organinc matter or living plants in their surroundings. Some mushrooms are edible, such as Cantharellus miomboensis while other mushrooms are extremely poisonous, such as Amanita phalloides. A number of mushroom varieties have global economic importance, through being all year round cultivated delicacies considered as food and medicine. These include button (Agaricus bisporus) and shiitake (Lentinus edodes) mushrooms which are considered as the two most cultivated mushrooms worldwide (Reagile, 2011, Stamets, 2000).

Since ancient times, mushrooms have been recognized as functional foods (foods containing health-giving additives) and as a source for the development of medicines and nutraceuticals throughout the world, including being prescribed for the treatment of various human microbial diseases (Alves et al., 2012; Gbolagade and Fasidi, 2005; Halpern, 2007; Prasad et al., 2015; Ziarati and Ghasemynezhad – Shanderman, 2015). Medicinal mushroom application can be through concentrates or powdered forms on hot water extracts and essences, which are applied as alternative medicine regularly in Korea, China, Japan and eastern Russia (Prasad et al., 2015). Species like Inonotus obliquus, Coprinus comatus,

Ganoderma lucidum, Fomitopsis officinalis, Piptoporus betulinus and Fomes fomentarius have been applied in the treatment of gastrointestinal disorders, diabetes, haemorroids, bronchial asthma and different types of cancers (Ardigo, 2016; Nowacka et al., 2015).

Furthermore, many varieties of mushrooms such as Agaricus bisporus, A. brasiliensis, A. subrufescens, Lentinus edodes, Pleurotus florida and Tricholoma giganteum, contain biologically active compounds that have been reported to exert immunomodulating, hepatoprotective (Moukha et al., 2011), antifibrotic, anti-inflammatory, antidiabetic (Moon and Lo, 2013), antioxidant (Chowdhury et al., 2015; Gan et al., 2013), antiviral (Wang and

Ng, 2004), antimicrobial (Padmavathy et al., 2014; Tehrani et al., 2012) and anticancer properties (Durgo et al., 2013; Geethangili et al., 2013; Moukha et al., 2011). These compounds include phenolic compounds, terpenes, flavonoids (Ramesh and Pattar, 2010), polysaccharides, triterpenoids (Chang and Wasser, 2012; Reis et al., 2012), glycopeptides, ribonucleases and lectins (Moukha et al., 2011).

The number of mushroom species identified all over the world is estimated at about 140 000 and of these only 22 000 have been investigated (Faridur et al., 2010; Hawksworth, 2001;

Nowacka et al., 2015). Despite their potential and enormous diversity in tropical ecosystems, many species of mushrooms have not been tapped, particularly in the field of medicine

(Prasad et al., 2015). Thus, considering that many varieties of mushrooms are already a valued source of active ingredients, this study embarked on assessing the nutritional and chemical composition, as well as the biological potential, of ten mushroom species growing in the wild in Zimbabwe. The mushrooms used in this study included both edible and non-edible species.

1.2 PROBLEM STATEMENT AND JUSTIFICATION

In recent years, there has been an upsurge of interest in mushrooms in various countries not only as a health food but also due to the presence of biologically active compounds with potential therapeutic properties (Prasad et al., 2015). Poor nutrition and an increasing

emergence of infectious diseases, caused by pathogenic bacteria, fungi and viruses, represent major threats to human health (Lindahl and Grace, 2015; Shridhar et al., 2015). There is a general agreement that many chronic health problems worldwide, relate mainly to unhealthy food choices. Obesity and dehydration and ailments like cardiac problems, diabetes mellitus and arthritis are on the increase, especially in developing countries (Shridhar et al., 2015).

Drug resistance continues to present a large and growing problem in treatment of infections that account for most of Africa’s disease burden, including Human Immunodefficiency Virus

(HIV) infections, respiratory and diarrheal diseases (Avert Newsletter, 2018; Kharsany and

Karim, 2016; Okon et al., 2013; Padmavathy et al., 2014; Sangeeth et al., 2014). Sub-Saharan

Africa continues to have the highest burden of HIV/Acquired Immune Deficiency Syndrome

(AIDS) worldwide and although the clinical management of HIV infection has greatly improved, resistance to antiretroviral drugs has emerged (Hamers et al., 2013; Wainberg et al., 2011; WHO Report, 2012). Hence, there is a need for continuous search and development of novel antimicrobial/antiviral substances from different biological sources to minimize the threat of further antimicrobial/antiretroviral resistance (Padmavathy et al., 2014; Shah et al.,

2014). Due to the production of a large variety of secondary metabolites with interesting biological actions, mushrooms are reservoirs of valuable chemical resources and can be used as a source for nutritional supplements and biotherapeutics (Prasad et al., 2015). However, despite the enormous therapeutic potential of mushrooms, there is very little research and awareness on local mushrooms as a healthy food and as an important source of biologically active substances with medicinal value in most of the African countries. Although Zimbabwe is rich in mushroom diversity, very little work has been carried out on profiling the nutritional, secondary metabolite composition and therapeutic value of mushrooms in

Zimbabwe. In addition, the potential of mushrooms as a source of new drugs is still largely unexplored. Nowadays, it is highly desirable to characterize and search for natural

constituents with health benefits due to the burden of civilization diseases (including HIV) affecting humans. Thus, the main goal of this study was to determine the nutritional and chemical composition of mushrooms, as well as the antibacterial and anti-HIV potential of the aqueous and organic extracts from selected wild mushrooms found in Zimbabwe. The chemical composition and intensity of the therapeutic effect of the extracts has been reported to be dependent upon mushroom species, extracting solvent, concentration of mushroom extract and the organism being tested (Alves et al., 2012; Pushpa and Purushothama, 2010).

In this study, different extracting media were also employed. The information obtained in this study will inform further studies needed for a better understanding of the health-promoting properties of mushroom constituents. These properties will enhance the use mushrooms in preventing and treating human diseases.

1.3 OBJECTIVES OF THE STUDY

1.3.1 Main Objective of the Study

The main objective of the study was to characterize mushrooms and their extracts through analysis of the nutritional and secondary metabolite content, as well as the antibacterial and anti-HIV properties of selected wild edible and non-edible mushrooms found in Zimbabwe.

1.3.2 Specific Objectives of the Study

The specific objectives of the study were to:

1.3.2.1 determine the nutritional and secondary metabolite composition of wild edible and

non-edible mushrooms found in Zimbabwe.

1.3.2.2 determine the antibacterial and anti-HIV effects of extracts of wild edible and non-

edible mushrooms.

1.3.2.3 isolate, characterize and identify signature compounds from mushroom extracts

exhibiting high antimicrobial activity.

1.3.2.4 investigate the effect of various solvents in extracting bioactive compounds from

mushrooms.

1.4 MAIN RESEARCH QUESTION

Do extracts of different wild edible and non-edible mushrooms of Zimbabwe possess nutritional, antibacterial and anti-HIV properties?

1.4.1 OTHER RESEARCH QUESTIONS

1.4.1.1 Do different solvents affect the composition and bioactivity of crude extracts and

compounds obtained from different mushrooms?

1.4.1.2 Do wild edible and non-edible mushrooms found in Zimbabwe contain considerable

levels of chemicals and secondary metabolites?

1.5 MAIN RESEARCH HYPOTHESIS

Extracts of different wild edible and non-edible mushrooms of Zimbabwe contain nutritional, antibacterial and anti-HIV properties.

1.5.1 OTHER RESEARCH HYPOTHESES

1.5.1.1 Different solvents affect the composition and bioactivity of crude extracts and

compounds obtained from different mushrooms.

1.5.1.2 Wild edible and non-edible mushrooms found in Zimbabwe contain considerable

levels of chemical and secondary metabolites.

CHAPTER TWO:

LITERATURE REVIEW

2.0 LITERATURE REVIEW

2.1 MUSHROOM BIOLOGY AND CLASSIFICATION

Mushrooms are broadly defined as macrofungi with distinctive spore-bearing fruiting bodies that can be either epigeous (grow above ground) or hypogeous (below ground) and are large enough to be seen with the naked eye and to be picked by hand (Chang and Miles, 1992;

Stamets, 2000). Most types of mushrooms are commonly found in the form of umbrella- shaped fruiting body with pileus (cap) under which spores are produced and have a stipe

(stem), for example Lentinus edodes. In addition, some species possess an annulus (ring), for example Agaricus bisporus (Figure 2.1), or a volva (cup), for example Amanita fulvoalba

(Figure 2.2), or have both the annulus and the volva. The annulus may disappear with age while the volva is usually buried in the ground (Chang and Miles, 1992). However, mushrooms can also be in a wide variety of forms where some of them look like pliable cups, golf balls, or small clubs, with some resembling corals while others are yellow or orange jellylike globes (Chang, 2008). Usually, the forms that deviate from the usual umbrella-shape have more specific common names, such as puffballs, stinkhorn and morels.

pileus

gills annulus

stipe

Figure 2.1: Agaricus bisporus (Source: Gry and Andresson, 2014)

volva

Figure 2.2: Amanita fulvoalba (Source: Mighell et al., 2019)

Mushrooms have two phases of growth: the reproductive phase (fruit bodies) and the vegetative phase (mycelia). Only the fruiting body of the mushroom can be seen whereas the rest of the mushroom remains in the substrate as mycelium (Wani et al., 2010). The mycelium comprises a system of branching threads and cordlike strands that branch out through the soil, compost, wood log or other lignocellulosic material on which the fungus is growing. After a period of growth, and when the conditions such as moisture and temperature are right, the established mycelium produces the fruiting structure, which is the mushroom (Chang and

Miles, 1992; Sanchez, 2010; Stamets, 2005).

2.1.1 Classification of Mushrooms

Historically, mushrooms were classified among the so-called lower plants in the division

Thallophyta by Linnaeus. This was largely due to the relatively simple, anatomically uncomplicated structural attributes (lack of true roots, stems, leaves, flowers and seeds) of the mushrooms. The presence of a cell wall related them to plants rather than to animals. Modern studies have established that mushrooms, together with other fungi, have features of their own, which are sufficiently and significantly distinct to place them in a separate fungal kingdom. Unlike green plants, mushrooms do not contain chlorophyll and so they cannot

manufacture their own nutrients from simple inorganic materials like water and carbon dioxide. They exploit foods from complex organic materials stored in dead or living tissues of plants and animals (Ren, 2014; Sharp, 2011).

Mushrooms belong to two subdivisions in the fungal kingdom (Mycota) which belongs to the

Domain Eucarya, namely, Basidiomycetes and Ascomycetes (Table 2.1). The spores for these two groups are located in a special structure or cell. In Ascomycetes, the sexual spores

(ascospores) are produced inside a club-shaped or cylindrical cell known as the ascus, while in Basidiomycetes, the sexual spores (basidiospores) are borne externally on a club-shaped cell known as a basidium (Dickinson, 1979). Basidiomycetes include gilled fungi (cultivated button mushroom, Agaricus), bracket fungi, boletes and the brightly coloured Cantharellus mushroom species (Sharp, 2011; 2014).

Table 2.1: Simplified classification of the major groups of fungi according to Ainsworth, 1971. Subdivision Class Order Family Ascomycotina Discomycetes Helotiales Geoglossaceae Helvellaceae

Pezizales Pezizaceae Sarcosomataceae

Pyrenomycetes Clavicipitales Clavicipitaceae Hypocreales Hypocreaceae Sphaeriaes Xylariaceae

Basidiomycotina Hymenomycetes Auriculariales Auriculariaceae Dacrymycetales Dacrymycetaceae Tremellales Tremellaceae Agaricales Agaricaceae Amanitaceae Coprinaceae Cortinariaceae Hygrophoraceae Tricholomataceae

Aphyllophorales Clavariaceae (Polyporales) Coniophoraceae Ganodermataceae Polyporaceae Schizophyllaceae

Boletales Boletaceae Cantharellales Cantharellaceae Russulales Russulaceae Gasteromycetes Lycoperdales Broomeiaceae Lycoperdeaceae

Phyllales Clathraceae Phallaceae

Sclerodermatales Astraeaceae Sclerodermataceae

The mushrooms used for this study belong to the family of mushrooms highlighted in bold under Class Hymenomycetes. Thus, for example, according to the Ainsworth classification, the genus Ganoderma belongs to the Family Ganodermataceae, of the Order Aphyllophorales under the Class Basidiomycetes or Hymenomycetes which belong to the Subdivision Basidiomycotina under the Kingdom Mycota or Myceteae which belongs to the Domain Eucarya.

2.1.2 Types of Mushrooms

Mushrooms can be roughly divided into four categories: (i) those which are fleshy and edible, for example Agaricus bisporus; (ii) mushrooms which are considered to have medicinal applications, for example, Ganoderma lucidum; (iii) those which are proven to be, or suspected to be poisonous, for example Amanita phalloides and (iv) those in a miscellaneous category which includes a large number of mushrooms whose properties remain less well- defined. However, many types of mushrooms are not only edible, but also possess tonic and medicinal properties such as Lentinus edodes and Agaricus species (Miles and Chang, 2004).

2.1.2.1 Edible Mushrooms

Edible mushrooms are the fleshy and edible fruit bodies of several species of macrofungi. The fruiting bodies of edible mushrooms are mainly consumed in their fresh or dried form

(Cheung, 2013). The edibility of mushrooms may be defined by criteria that includes absence of poisonous effects on humans and palatability. Edible mushrooms include many fungal species that are either harvested wild or cultivated (Cheung, 2013; Ching et al., 2011). Some mushrooms that are toxic when raw are said to be edible when cooked. For example, Amanita muscaria is edible if parboiled to leach out toxins. However, because there is no known test by which to tell if a mushroom is edible or not, a mushroom should never be eaten unless it has been accurately identified and the edibility of the species is known (Moukha et al., 2011).

In Southern Africa, roadside sellers only offer "safe species" and most market places are a reliable means of obtaining known, edible wild mushrooms (Boa, 2004).

Mushrooms are not easy to separate into different categories of beng medicinal and even being edible because many of the common edible species are also beneficial in the prevention and treatment of various human diseases. The medicinal properties of these mushrooms is

linked to their richness in bioactive compounds, such as phenolic compounds, polyketides, terpenes, steroids, beta-carotenes, and vitamins A and C (Buruleanu et al., 2018; Rai et al.,

2005; Zhang et al., 2016). The edible class of mushrooms that show potential medicinal and functional properties includes Cantharellus, Lentinus, Auricularia, Hericium, Grifola and

Pleurotus species (Prasad et al., 2015).

2.1.2.2 Non-edible Mushrooms

Non-edible mushrooms include species that are not palatable while some from this category are extremely poisonous. The Amanita phalloides group is an example of mushrooms that cause the most dangerous type of mushroom poisoning. The toxins involved belong to the phallotoxin and amatoxin complexes (Duffy, 2008). Several Coprinus species, such as C. micaceus and C. atramentarius, when consumed with an alcoholic drink, produce unpleasant symptoms which include reddening of the face, increased rate of heartbeat and, in some cases, vomiting and diarrhoea (Chang and Miles, 1992). Several non-edible mushrooms such as

Ganoderma and Trametes, have been studied and used for their medicinal properties (Cheung,

2013; Ching et al., 2011; Prasad et al., 2015). Ganoderma mushroom lacks gills on its underside and releases its spores through pores, leading to its morphological classification as a Polypore. Ganoderma is documented to show strong potency, not only as an immune response booster but also as an anti-bacterial, anti-parasitic, anti-tumour and anti- inflammation mushroom (Haoses-Gorases and Goraseb, 2013; Kamra and Bhatt, 2012; Prasad and Wesely, 2008). Bioactive molecules have also been isolated from non-edible species such as the ones belonging to the Polyporaceae, Xylariaceae, Thelephoraceae and Paxillaceae families (Reis et al., 2011).

2.2 HUMAN HEALTH BENEFITS OF MUSHROOMS AND THEIR EXTRACTS

2.2.1 Mushrooms as Food and Medicine

Wild edible mushrooms have been collected and consumed by humans worldwide for thousands of years, with the Chinese growing Auricularia auricular, the wood ear mushroom, around AD 600 (Boa, 2004; Kues and Liu, 2000; Moon and Lo, 2013; Wang and Xu, 2014).

During the early days of civilization, mushrooms were consumed mainly for their palatability and unique flavors. However, the current use of mushrooms has now changed because of a lot of research that has been done on the chemical composition of mushrooms, which revealed that mushrooms can be used as a diet to combat diseases (Wani et al., 2010). Thus, interest in consumption of edible mushrooms, both wild and cultivated, has increased remarkably over the past few decades in many countries, due to the increasing awareness of their nutritional value (Wani et al., 2010). Mushrooms are high in protein, low in fat and provide low energy content, factors that make them an excellent food for low-caloric diets. Some mushrooms are even consumed for medicinal purposes as they contain valuable bioactive components, for example, Ganoderma lucidum (Buruleanu et al., 2018; Ching et al., 2011; Moon and Lo,

2013; Wang and Xu, 2014; Ziarati and Rabizadeh, 2013).

When in season, the mushrooms provide a notable contribution to diets in Central and

Southern Africa (Cheung, 2008). They are a significant source of nutrition for rural people in particular, as well as a delicacy for some people (Boa, 2004). Mushrooms as functional foods are used as nutrient supplements to enhance immunity in the form of tablets. Due to low starch content and low cholesterol, they suit diabetic and heart patients. The food value of mushrooms has been reported to compare favourably with meat, egg and milk food sources

(Wani et al., 2010). Recently, various studies have been conducted to increase the application

of mushrooms in processed foods. For example, mushrooms can be added to the products directly as functional ingredients in various baked products (Moon and Lo, 2013).

2.2.1.1 Mushrooms as Source of Proteins

Proteins are the most critical component contributing to the nutritional value of food and is an important constituent of dry matter of mushrooms. Mushrooms have good nutritional value, particularly as a source of protein that can enrich human diets, especially in some developing countries where animal protein may not be available or is expensive (Boda et al., 2012; Wani et al., 2010). The protein content of fresh mushrooms is 3.7% while the crude protein content in percent dry weight (% DW) of edible mushrooms range from 15 to 35 % as compared to

7.3 % in rice, 12.7 % in wheat, 38.1 % in soybean, 1.4 % in cabbage, 9.4 % in corn, 12 - 14% in poultry meat (Soriano, 2010) and 20 – 25% in red meat (Williams, 2007). Thus, in terms of the amount of crude protein, mushrooms rank well above most other foods, including milk

(2.9 – 3.3 %), which is an animal product. In addition, mushroom protein contains all the essential amino acids required by humans. The crude protein content varies greatly among the mushroom species, depending on the size of the pileus and their stage of development (Boa,

2004; Boda et al., 2012; Chang, 2008; Cheung, 2008; Mattila, 2000; Wani et al., 2010).

Mushrooms produce many kinds of proteins with biological activities, including lectins, antifungal, antiviral and antibacterial proteins, polyphenol oxidase and ribonucleases (Tehrani et al., 2012; Xu et al., 2014).

Due to their high amount of proteins, mushrooms can be used to bridge the protein malnutrition gap (Boda et al., 2012). In underdeveloped countries where protein malnutrition has taken epidemic proportions, Food and Agricultural Organization (FAO) has recommended the intake of mushroom foods to address the condition (Boda et al., 2012; Wani et al., 2010).

The digestibility of mushroom protein has been reported to be as high as 72 to 83 %.

Mushrooms are regarded as an ideal protein source for vegetarian diets, since they contain some essential amino acids which are found in animal proteins, as well as for old age people who are unable to chew meat (Wani et al., 2010).

2.2.1.2 Mushrooms as Source of Carbohydrates

Mushrooms contain different amounts of carbohydrates ranging from 51 – 88 % on dry weight basis (Cheung, 2008). Free sugars amount to about 11 % while mannitol, also referred to as mushroom sugar, constitutes about 80 % of the total free sugars. Fresh mushrooms are reported to contain 0.9 % mannitol, 0.28 % reducing sugar, 0.59 % glycogen and 0.91 % hemicellulose (Waktola and Temesgen, 2018; Wani et al., 2010). Raffinose, sucrose, glucose, fructose and xylose are reportedly dominant in some mushrooms (Wani et al., 2010).

Several polysaccharides and protein-bound polysaccharides with immunomodulatory and antitumor activities have been isolated from a variety of mushrooms (Linderquist et al.,

2005). The β-glucans, pleuron from Pleurotus ostreatus and lentinan from Lentinus edodes, have been reported to increase intestinal mucosal resistance to inflammation and decrease the occurrence of ulcers in the intestine (Linderquist et al., 2005). Lentinan and another polysaccharide, schizophyllan from mushroom Schizophyllum, may activate lymphocytes (T and B cells), macrophages and natural killer cells (Wiater et al., 2011). Polysaccharide-K

(PSK) and polysaccharopeptide (PSP) are polysaccharide-proteins extracted from the mushroom Corilus versicolor. KrestinTM, which is the trade name for PSK, displays biological activities that include stimulation of functional maturation of macrophages, inhibition of the cytophatic effect of HIV infection and an ability to scavenge reactive oxygen species (Chang and Buswell, 1996).

2.2.1.3 Phenolic Compounds and Antioxidant Properties of Mushrooms

Mushrooms are considered to be a natural and good source of antioxidants, chemical compounds that protect cells from the damage caused by unstable molecules known as reactive oxygen species. Reactive oxygen species generated during oxidative phosphorylation by NADPH oxidase, are normal components of healthy cells and also mediators of the first defensive actions of cells (Kozarski, 2015). However, overproduction of reactive oxygen species and oxygen-derived free radicals creates oxidative stress, which may induce many diseases, such as rheumatoid arthritis, atherosclerosis, diabetes, cancer and aging. The antioxidants are an important defense of the body against free radicals. Fruit bodies and mycelia of several mushrooms have been reported to show high levels of antioxidant activity

(Buruleanu et al., 2018; Wani et al., 2010).

Different studies have revealed a positive correlation between the total phenolic content in the mushroom extracts and their antioxidative properties, such as, inhibition of lipid peroxidation by L. edodes and the radical scavenging and chelating effect on ferrous ions by methanolic extracts of mushrooms Dictyophora indusiata and Grifola frondosa (Cheung, 2008; Gan et al., 2013; Ramesh and Pattar, 2010). Phenolic compounds represent a large group of secondary plant metabolites which now attract great interest due to their benefits for human health. There are studies that have shown that the antioxidant activity exhibited by phenolic compounds offers protection against chronic degenerative diseases, cardiovascular diseases, diabetes mellitus, and neurodegenerative diseases (Buruleanu et al., 2018). Phenolic acids, such as trans-cinnamic acid, hydroxbenzoic acid, protocatechuic acid and caffeic acid have been reported in A. bisporus and L. edodes (Wani et al., 2010).

The alcoholic extracts of Coprinus comatus were shown to be more effective in scavenging activity on hydroxyl radicals than hot water extracts (Li et al., 2010). The naturally occurring antioxidant components included total phenols, tocopherols, flavonoids and polysaccharides.

Tyrosinase from A. bisporus has reportedly shown antioxidant activity. The high potential of mushrooms as a dietary source of phenolic antioxidants can be used to enhance the low antioxidant status in the human body (Cheung, 2008), a condition prevailing during certain infections like HIV. Furthermore, the presence of antioxidant and anti-inflammatory compounds in mushrooms might be clinically relevant in the management of heart and circulation health complications (Moon and Lo, 2013).

2.2.2 Mushroom Extracts as Medicine

Scientific studies confirmed recently that bioactive compounds from many edible mushrooms are involved in lowering the cholesterol levels and protecting against various disorders including tumors (Ruiz-Rodriguez, 2009; Valverde, 2015). Some medicinal mushrooms exhibit cardiovascular, anticancer, antiviral, antibacterial, antiparasitic, anti-inflammatory and antidiabetic properties (Buruleanu et al., 2018; Feng et al., 2016). The potential of wild mushrooms as sources of antibiotics was reported in 1941 (Sudirman, 2010; Moukha et al.,

2011). In the reported study, extracts of fruiting bodies and mycelia culture from over 200 species were tested. Several compounds that inhibit the growth of a large spectrum of saprophytic and phytopathogenic fungi and bacteria were isolated from Basidiomycetes. The study on polypores, such as several species of Ganoderma, Trametes versicolor, T. marianna,

T. cingulata, and Laetiporus sulphureus and gilled mushrooms, such as P. ostreatus, Lentinus connatus, and Lentinus edodes showed either the antibacterial, anti-Candida, antiviral or cytotoxic activities (Sudirman, 2010; Moukha et al., 2011).

Several mushroom species belonging to the Polyporaceae family are now being regarded as potential sources of valuable medicines (Prasad and Wesely, 2008). Nortriterpenoids isolated from Ganoderma showed a wide range of biological activities such as, anti-tumor, anti- inflammatory, neurotrophic, hepatoprotective and anti-HIV-1 protease activities (Chen et al.,

2017). Aqueous extracts from Pleurotus pulmonarius var sajor caju proved effective in renal failure while the hot water extracts from several mushrooms exhibited antitumor effects. An antitumor polysaccharide, named lentinan, was isolated from the shiitake fruiting bodies. It was reported that mushrooms cure epilepsy, wounds, skin diseases, heart ailments, rheumatoid arthritis, diarrhea, dysentery, cold, anesthesia, liver and gall bladder diseases

(Afiukwa et al., 2013; Pala and Wani, 2011; Valverde et al., 2015). Most of the mushroom extracts are now available as therapeutic drugs in China (Ganeshpurkar et al., 2010; Wani et al., 2010). Puffballs have been used in urinary infections while Pleurotus tuberregium mushroom has been used for curing headache, high blood pressure, smallpox, asthma, colds and stomach ailments. Mushroom health supplements can be marketed in the form of powders, capsules or tablets made of dried fruiting bodies, extracts of mycelium with substrate, or extracts from liquid fermentation (Wani et al., 2010).

2.2.2.1 Antibacterial Properties of Mushrooms

Both the edible and non-edible wild mushrooms have antibacterial properties. Inhibition of microbial growth by mushroom extracts is due to the presence of bioactive components in the mushrooms (Ramesh and Pattar, 2010). Crude organic and aqueous extracts from Ganoderma have been reported to inhibit in vitro growth of Escherichia coli, Staphylococcus aureus,

Bacillus cereus, Neisseria meningitides, Alcaligenes faecalis and Proteus vulgaris, bacteria known to cause wound infections, intestinal and urinary-genital tract infections and skin infections (Kamra and Bhatt, 2012; Prasad and Wesely, 2008; Shikongo et al., 2013).

Aqueous, ethanol, methanol and xylene extracts of A. bisporus and Pleurotus pulmonarius var sajor caju have been reported to exhibit antibacterial activity against E. coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Klebsiella pneumoniae. Most of the species of

Agrocybe perfecta, Hexagonia hydnoides, Irpex lacteus and Tyromyces duracinus showed antimicrobial activity against bacteria and yeasts (Chaudhary and Tripathy, 2015). Pleurotus ostreatus, commonly known as white oyster mushroom, has been reported to possess compounds that inhibit the growth of E. coli, Bacillus megaterium, S. aureus, K. pneumoniae isolates and species of Streptococcus and Enterococcus (Sala Uddin et al., 2015).

Some selected mushroom metabolites were reported to have high inhibitory activity against

Gram-positive organisms, including acid fast bacterium Mycobacterium smegnatis

(Gbolagade and Fasidi, 2005). Crude extracts that were obtained from Portuguese wild mushrooms including Cantharellus cibarius, Hypholoma fasciculare and Ramaria botrytis showed antibacterial activity against Gram-positive bacteria (Barros et al., 2008). Two triterpenes, trichomycins A and B, separated from Tricholoma species exhibited antibacterial activity against S. aureus and S. pneumoniae. A glucosylceramide isolated from Pleurotus citrinopileatus was found to be active against E. coli and S. aureus (Chomcheon et al., 2013).

Several species in the genera Cantharellus, Lentinus, Russula, Agaricus and Pleurotus have shown antimicrobial properties against Bacillus, Enterococcus, Streptococcus,

Staphylococcus and Micrococcus species (Alves et al., 2012; Khan and Tania, 2012; Pushpa and Purushothama, 2010). The European Ganoderma has been reported to inhibit growth of most bacteria especially methillicin-resistant S. aureas (Linderquist et al., 2005). Different mushroom species vary in their antimicrobial activity. The intensity of the antimicrobial effect is dependent upon mushroom species, concentration of the mushroom extract and the organism being tested against (Ramesh and Pattar, 2010).

2.2.2.2 Antiviral Properties of Mushrooms

The HIV type 1 pandemic afflicts approximately 34 million people worldwide (Jadaun et al.,

2016; Tran et al., 2011; WHO, 2018). Side effects such as hypersensitivity, lactic acidosis, bleeding and anaemia, and uneven access to anti-retroviral drugs remain considerable therapeutic challenges (Leteane et al., 2012; Mataftsi et al., 2010; Tran et al., 2011;

Yunihastuti et al., 2014). A variety of mushrooms have been reported to possess strong anti-

HIV properties. Proteins, peptides and polysaccharopeptides from mushrooms have been reported to be capable of inhibiting human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease, the two key enzymes in the life cycle of the HIV (Roupae et al.,

2012). The laccase enzyme, produced by fungi of the genera Ganoderma and Lentinus, was reported to inhibit the reverse transcriptase (RT) of HIV-1 in in vitro cell-free models (Orozco et al., 2016). The melanin-glucan complex obtained from Fomes fomentarius mushroom showed higher anti-HIV activity in comparison with the drug zidovudine in vitro (Friedman,

2016).

Medicinal mushrooms such as Tricholoma giganteum, Hericium erinaceum, Russula paludosa, Pleurotus eryngii, G. lucidum and L. edodes have shown to contain in their extracts, ribosome inactivating proteins, lectins, ubiquitin-like proteins and laccases with strong antiviral effects (Orozco et al., 2016). Lectins from A. bisporus have shown inhibitory activity against HIV-1 reverse transcriptase. Some triterpenes from G. lucidum are effective as antiviral agents against HIV-1 (Linderquist et al., 2005). Flammulina velutipes contains a ribosome inactivating protein that inhibits HIV-1 reverse transcriptase. Some types of mushrooms such as maitake mushrooms increase CD4+ cell counts, enhancing the activity of

T-helper cells and reducing symptoms and secondary illnesses caused by HIV (Linderquist et al., 2005; Wani et al., 2010). A methyl gallate compound with anti-HIV activities has been

isolated from the mushroom Pholiota adiposa (Wang et al., 2014). In studies conducted in

Zambia, Tanzania and Namibia, the effectiveness of Ganoderma in the therapy of HIV/AIDS patients and opportunistic infections was reported. The overall health of the patients was significantly improved compared to the control groups, while significant increases were noted for body weight, appetite, as well as the CD4+ cell count (Haoses-Gorases and Goraseb,

2013). Lentinan sulphate obtained from Lentinus species has also reportedly inhibited HIV

(Wani et al., 2010).

2.2.2.3 Mushroom Lectins and their Role in Human Health

Lectins are proteins or glycoproteins of non-immune origin which have the ability to bind specifically and reversibly to complex carbohydrates that are abundant on cell surfaces, resulting in agglutination of cells or precipitation of glycoconjugates. The detection of lectins relies on the ability of lectins to agglutinate red blood cells and lectins inhibition by a specific sugar, which is a major attribute of these proteins (Dhamodharan and Mirunalini, 2011; Sun et al., 2014; Zhang et al., 2014). Accumulation of lectins in crude extracts of mushrooms can be detected by hemagglutination assay using human (A, B and O blood groups) and animal

(goose, rabbit, rat and sheep) red blood cells. At least 60 mushroom lectins have been identified (Santhiya and Jansi, 2013). The high content of lectins in mushrooms has been detected in diverse species of genera Lactarius, Russula, Boletus, Phallus, Amanita and

Hygrophorus. Mushroom lectins are highly affected by the environment, such as time of harvest, geographic location and part of mushroom where the lectin was isolated from. The same mushroom species can have different types of lectins depending on the environment where the mushrooms were collected (Dhamodharan and Mirunalini, 2011; Zhang et al.,

2014).

In recent years, mushroom lectins have drawn the attention of many researchers, mainly due to the discovery of some of these lectins displaying an array of functions such as antimicrobial, antitumor, immune-enhancing, anti-insect, antiviral, mitogenic and anti-HIV-1 reverse transcriptase activities (Dhamodharan and Mirunalini, 2011; Eghianruwa et al., 2011;

Koyama et al., 2002; Santhiya and Jansi, 2013; Zhang et al., 2014;). Agaricus bisporus lectin has exhibited antiproliferative action against human colon cancer and breast cell lines (Patel and Goyal, 2012). Mushroom lectins that are specific to mannose have antiviral activity. The

Pholiota adiposa lectin exhibited HIV inhibitory activity by targeting the reverse transcriptase and also antiproliferative activity towards hepatoma Hep G2 cells (Zhang et al., 2009).

Volvariella volvacea lectin possesses antitumor activity to sarcoma S-180 cells while Boletus lectins have been found to have mitogenic activity towards tumour cells (Bovi et al., 2011;

Sun et al., 2014; Zheng et al., 2007), antimicrobial activity as well as inducing IL-1 and IL-2

(Licastro et al., 1993). Mushroom lectins specific to mucin have been found to have antimicrobial activity and antiproliferative activity (Lutsik-Kordovsky et al., 2001). These findings clearly indicate that mushrooms are a valuable source of lectins for drug discovery.

2.3 CHARACTERIZATION OF MUSHROOM EXTRACTS

2.3.1 Methods of Extraction

Characterization of mushroom extracts begins with the pre-extraction and extraction procedures. The basic pre-extraction steps include washing and drying of mushrooms or freeze drying and grinding to powder to obtain a homogenous product which increases the contact of sample surface with the solvent system. Proper action must be taken to ensure that potential active constituents are not lost, distorted or destroyed during the preparation of the extracts (Altemimi et al., 2017; Sasidharan et al., 2011). Extraction is a crucial step in the analysis of mushrooms, because it is necessary to extract the desired chemical constituents

from the mushroom samples for further separation and characterization. Solvent extraction has been the most widely used method for the recovery of active compounds from natural sources, particularly phytochemical constituents, although other technologies such as microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and supercritical fluid extraction are gaining recognition (Zhang et al., 2018). The methods are aimed at increasing the extract yields at lower cost. In addition, modifications on the methods are continuously being developed (Altemimi et al., 2017; K l a u s et al., 2009; Shen et al., 2017).

However, the yields and bioactive efficacy of the extracts obtained is strongly affected by the polarity of the solvent as well as the chemical nature of the isolated compounds (Anwar and

Przybylski, 2012).

2.3.1.1 Choice and Effect of the Extracting Solvent

One of the most important factors affecting the efficient extraction of bioactive compounds from natural sources is the extraction solvent, since the compounds can range from very polar to non-polar. The nature of the extraction solvent and varying chemical characteristics and polarities of chemical compounds can result in different extraction yields and biological activities of the mushroom (Azwanida, 2015; Doughari, 2012; Gan et al., 2013; Ngo et al.,2017). Polar compounds are easily extracted using polar solvents while non-polar compounds will be easily extracted by the non-polar solvents. Thus, extraction of the mushroom samples can be carried out using different solvents because of the diversity of the chemical nature of their components and the different solubilities in different solvents

(Altemimi et al., 2017; Anwar and Przybylski, 2012; Nur Syukriah et al., 2014).

Water and other organic solvents such as methanol, ethanol, acetonitrile, acetone, hexane and diethyl ether have usually been applied in the extraction of bioactive compounds from plants

and mushrooms (Table 2.2). Polar solvents such as water, methanol, ethanol, acetone and their aqueous mixtures, are mostly recommended for the extraction of polyphenols (Anwar and Przybylski, 2012; Dailey and Vuong, 2015; Tatiya et al., 2011). Water is a common medium for biochemical reactions and has been shown to be capable of extracting different classes of active compounds depending on the temperature used (Askin et al., 2007; Shen et al., 2017). However, although a higher temperature may increase extraction efficiency, it may also result in degradation of temperature-sensitive antimicrobials. Thus, the chemical profile of the extracts obtained with different solvents can vary and result in variations of their antimicrobial properties (Shen et al., 2017).

Table 2.2: Effect of solvents in extracting different components from biological sources

Water Ethanol Methanol Chloroform Ether Acetone

Anthocyanins Tannins Anthocyanins Terpenoids Alkaloids Phenols

Starches Polyphenols Terpenoids Flavonoids Terpenoids Flavonols

Tannins Polyacetylenes Saponins Coumarins

Saponins Flavonol Tannins Fatty acids

Terpenoids Terpenoids Xanthoxyllines

Polypeptides Sterols Totarol

Lectins Alkaloids Quassinoids

Lactones

Flavones

Phenones

Polyphenols

Although ethanol and methanol have similar polarities, methanol extracted more secondary metabolites than ethanol. Chloroform and acetone extracted the least compounds (Source: Tiwari et al., 2011)

2.3.2 Methods of Separation of Constituents from Mushroom Extracts

Once extracted from the source, the bioactive components have to be separated (Doughari,

2012). Separation of biologically active constituents from crude extracts for the process of identification and characterization still remains a big challenge due to the fact that mushroom and plant extracts usually occur as a combination of various types of bioactive compounds or phytochemicals with different polarities. Common techniques used in the isolation of these bioactive compounds include Thin Layer Chromatography (TLC), Paper Chromatography,

Column Chromatography, Flash Chromatography and High Performance Liquid

Chromatography (HPLC). The pure compounds are then used for their identification and biological activity (Altemimi et al., 2017; Ingle et al., 2017).

2.3.2.1 Thin Layer Chromatography (TLC)

Although there is a wide range of chromatographic methods for isolation of crude extracts, thin layer chromatography remains a valid and simple analytical procedure for qualitative detection and quantitative determination of components of mushroom extracts (Masoko,

2007; Ingle et al., 2017). The TLC method is an adsorption chromatography where samples are separated based on the interaction between a thin layer of adsorbent attached on the plate.

The technique is mostly employed for the separation of low molecular weight compounds

(Ingle et al., 2017). The method does not require expensive instrumentation, is easy to run, quick, reproducible and samples do not require extensive purification prior to analysis. The adsorbent layer of silica gel-G has been used with a variety of mobile phase solvent systems

(Pyka, 2014). Preparative or semi-preparative TLC techniques can be used to obtain larger amounts of the fraction or compound of interest. Preparative TLC plates with a thickness of 1 mm can be prepared using the same stationary and mobile phases, to isolate the bioactive components (Altemimi et al., 2017). The sample is applied as a wide band and developed

under selected chromatographic conditions. After solvent evaporation, bands of desired bioactive compounds are scraped off together with silica gel, and then they are eluted with appropriate solvent (Choma and Jesionek, 2015).

2.3.2.2 High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC) is a versatile, robust, and widely used technique for the isolation of natural products. The technique is gaining popularity among various analytical techniques as the main choice for fingerprinting study for the quality control of biological sources (Sasidharan et al., 2011). The resolving power of HPLC is ideally suited to the rapid processing of such multicomponent samples on both an analytical and preparative scale. Chemical separations can be accomplished using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase. The extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase. Generally the identification and separation of phytochemicals can be accomplished using isocratic system (using single unchanging mobile phase system) (Sasidharan et al., 2011). However, gradient elution, in which the proportion of organic solvent to water is altered with time, is used when more than one sample component is being studied (Altemimi et al., 2017; Sasidharan et al., 2011)

2.3.3 Methods of Identification of Compounds

The identification of the bioactive compounds isolated from crude extracts can be obtained using chromatographic techniques such as HPLC and TLC as well as different varieties of spectroscopic techniques such as UV-visible, Infrared (IR), Nuclear Magnetic Resonance

(NMR), and mass spectroscopy (Altemimi et al., 2017; Buruleanu et al., 2018). Spectroscopy is based on passing electromagnetic radiation through an organic molecule that absorbs some

of the radiation. By measuring the amount of absorption of electromagnetic radiation, a spectrum, specific to certain bonds in a molecule, can be produced. Depending on these spectra, the structure of the organic molecule can be identified (Altemimi et al., 2017).

2.3.3.1 Ultra Violet (UV) - Visible Spectroscopy

The isolated compounds can be visualized on a TLC plate by physical (colour or fluorescence of a compound in Ultra Violet [UV] light) and chemical (coloured reactions of separated compounds with visualizing reagents) methods (Pyka, 2014). After spraying the TLC plates with reagents such as vanillin-sulphuric acid, many different compounds could be observed.

Analysis of the compounds is done by comparing the distance traveled relative to the solvent front called retention factor (Rf value) on the TLC against a reference value of a standard

(Altemimi et al., 2017; Pyka, 2014; Sasidharan et al., 2011). In TLC fingerprinting, the data that can be recorded using a high performance TLC (HPTLC) scanner includes the recording of the chromatogram, retention time of individual peaks, the colour of the separated bands and their absorption spectra. The information generated can be used in the identification of a compound. The UV-visible spectroscopy can be performed for qualitative analysis and for identification of certain classes of compounds in both pure and biological mixtures, due to aromatic molecules that are strong chromophores in the UV range. The technique was reportedly used to determine the total phenolic extract (280 nm), flavones (320 nm), phenolic acids (360 nm) and total anthocyanins (520 nm) from a plant (Altemimi et al., 2017).

2.3.3.2 Liquid Chromatography - Mass Spectroscopy (LC-MS)

Recent approaches of applying a combination of chromatography and spectrometry techniques such as Liquid Chromatography with Photo Diode Array Detection (LC–DAD),

Gas Chromatography - Mass Spectrometry (GC-MS), LC-MS and LC- NMR are increasingly

providing the additional spectral information, which is very helpful for the qualitative analysis and structure determination of novel compounds. In mass spectrometry, organic molecules are bombarded with either electrons or lasers and thereby converted to charged ions, which are highly energetic (Altemimi et al., 2017). A mass spectrum gives a plot of the relative abundance of a fragmented ion against the ratio of mass/charge of these ions. Using mass spectrometry, the relative molecular mass (molecular weight) of a compound can be determined with high accuracy and an exact molecular formula can be determined (Altemimi et al., 2017). Liquid chromatography coupled with mass spectrometry (LC-MS) facilitates rapid and accurate identification of chemical compounds in extracts, especially when a pure standard is not available. Recently, LC-MS has been extensively used for the analysis of phenolic compounds (Altemimi et al., 2017; Sasidharan et al., 2011).

2.3.4 Classes of Secondary Metabolites Found in Mushrooms

2.3.4.1 Alkaloids

Alkaloids are a group of naturally occurring chemical compounds that mostly contain basic nitrogen atoms. The compounds have basic properties and are alkaline in reaction. Most alkaloids are readily soluble in alcohol and though they are sparingly soluble in water, their salts are usually soluble (Kumar, 2014). These nitrogenous compounds function in the defence of plants against herbivores and pathogens, and are widely exploited as pharmaceuticals, stimulants, narcotics, and poisons due to their potent biological activities.

Alkaloids are found in certain types of fungi, such as psilocybin which occurs in the fungus of the genus Psilocybe (De Geyter, 2012; Doughari, 2012).

2.3.4.2 Glycosides

A glycoside (Figure 2.3) is a molecule in which a sugar (including polysaccharides) is bound to a non-carbohydrate part, usually a small organic molecule. The sugar group is referred to as the glycone and the non-sugar group as the aglycone (De Geyter, 2012). Alcohol, glycerol or phenol represents aglycones. Glycosides are classified on the basis of the type of sugar component, chemical nature of the aglycone or pharmacological action. Chemically, the bitter principles contain the lactone group that may be diterpene lactones or triterpenoids. Extracts of biological sources that contain cyanogenic glycosides are reportedly used as flavouring agents in many pharmaceutical preparations (Doughari, 2012).

Alkaloids Fatty acids Glycosides

Example: nicotine Example: butyric acid Example: salicin

Peptides Phenols Terpenes

Example: glycylglycine Example: phenol Example: isoprene

Figure 2.3: Overview of the six main classes of secondary metabolites with representative examples (De Geyter, 2012)

2.3.4.3 Flavonoids

Flavonoids constitute the largest group of polyphenolic substances, exhibiting extraordinary diversity and variation. Their chemical structure is built upon a C6-C3-C6 skeleton and the 30

three-carbon bridge is usually cyclised with oxygen. Flavonoids are known to be synthesized in response to microbial infection. Naturally occurring flavonoids and their glycosides have been reported to possess various biological properties including anti-inﬂammatory, antimicrobial, antiallergic, antioxidant, cytotoxic, antitumour, antidiabetic antiviral and anticancer activity (Abugria and McElhenney, 2013; Bylka et al., 2004; Cushnie and Lamb,

2005; Djouossi et al., 2015; Kumar and Pandey, 2013). Studies on flavonoids by spectroscopy have revealed that most flavones and flavonols exhibit two major absorption bands: band I

(320–385 nm) representing the B ring absorption, while band II (250–285 nm) corresponds to the A ring absorption (Kumar and Pandey, 2013). The antimicrobial activity of total flavonoid compounds and the antibacterial activities of 2,4-dihydroxybenzoic, protocatechuic, vanillic, and p-coumaric acids from different wild mushrooms have been reported (Alves et al., 2012;

Chowdhury et al., 2015). Flavonoids occur as aglycones, glycosides, and methylated derivatives (Kumar and Pandey, 2013). Some flavonoides exhibit anti-HIV-1 effect in vitro and can interact at different steps in the life cycle of HIV-1, including viral entry, reverse transcriptase, integrase, and viral protease (Sudsai et al., 2017).

2.3.4.4 Phenolics

Phenolics, phenols or polyphenolics are characterized by at least one aromatic ring (C6) and one or more hydroxyl groups. Phenols range from simple tannins to more complex flavonoids that give plants much of their red, blue, yellow, and white pigments (De Geyter, 2012).

Different studies have reported the bioactive potential of phenolic acids, including their application in the control of human pathogenic infections. Phenolics are classified into (i) phenolic acids, (ii) flavonoid polyphenolics (flavonones, flavones, xanthones and catechins) and (iii) non-flavonoid polyphenolics (Doughari, 2012; De Geyter, 2012; Taofiq et al., 2015;

2016).

2.3.4.5 Terpenoids

Terpenoids are highly flammable unsaturated hydrocarbons, found in plants, animals and macrofungi (Duru and Cayan, 2015). They are named based on the number of isoprene units

(from which they are built), for example, monoterpenes (10 carbons), sesquiterpenes (15 carbons), diterpenes (20 carbons), sesterterpenes (25 carbons), triterpenes (30 carbons), and tetraterpenes (40 carbons). Terpenoids isolated from mushrooms have been associated with various pharmacological activities like anticancer, antimalarial, antiviral, antibacterial and anti-inflammatory activities (De Geyter, 2012; Duru and Cayan, 2015; Taofiq et al., 2016).

2.3.4.6 Steroids and Sterols

Steroids and sterols are produced from terpenoid precursors and include cholesterol, vitamin

D and (steroidal) saponins. A steroid is a type of terpenoid compound that contains a specific arrangement of four rings that are joined to each other. Sterols are special forms of steroids, with a hydroxyl group at position-3 and a skeleton derived from cholestane (De Geyter,

2012). Steroids are derived either from the sterol lanosterol (animals and fungi) or from cycloartenol (plants). Steroids have been reported to play several biological functions such as anti-tumor, antioxidant, immune function as well as prevention of certain diseases (De Geyter,

2012; Taofiq et al., 2016).

2.3.5 Methods of Detecting Antibacterial Activity

A number of antimicrobial test methods are available to determine the susceptibility of bacteria to antimicrobial agents, but they vary in their sensitivity and efficacy. The method selected should ideally be simple, rapid, reproducible, flexible, cost-effective and maximize high sample throughput in order to cope with a varied number of extracts and fractions

(Bailey, 2013; OIE Terrestrial, 2012). The currently available screening methods for the

detection of antimicrobial activity of natural products include bioautographic, diffusion and dilution methods. The bioautographic and diffusion methods are referred to as qualitative techniques since these methods will only give an idea of the presence or absence of compounds with antimicrobial activity. On the other hand, dilution methods are regarded as quantitative assays when they are used to determine the minimal inhibitory concentration

(Valgas et al., 2007).

2.3.5.1 Agar Disc and Well Diffusion Methods

The disk diffusion method is regarded as the gold standard for confirming the susceptibility of bacteria (Khan et al., 2019). In this method, the isolated colony of the target organism is suspended into growth media, and standardized through a turbidity test. The standardized suspension is then inoculated onto the solidified Muller Hinton agar plate. Paper discs impregnated with specific antibiotics or the test substances are placed on the surface of the inoculated agar plate (Hudzicki, 2009; Khan et al., 2019). The plates are incubated overnight at 37 °C as the disc containing the antibiotic or test substance is allowed to diffuse through the solidified agar, resulting in the formation of an inhibition zone. Thereafter, the zones of inhibition around each paper disc are measured (Hudzicki, 2009; Khan et al., 2019). In agar well diffusion method, the antimicrobials present in the test extract are allowed to diffuse out into the medium and interact in a plate, freshly seeded with the test organisms. The resulting zones of inhibition will be uniformly circular as there will be a confluent lawn of growth. The diameter of zone of inhibition can be measured in millimeters (Valgas et al., 2007).

zone of inhibition

Figure 2.4: Agar disc diffusion assay showing zones of inhibition of bacterial growth (Photo taken by T. Reid)

2.3.6 Methods of Detecting Anti-HIV Activity

In the life cycle of HIV, there are various steps that can be targeted for therapeutic intervention (Figure 2.5). Preliminary screening of crude extracts and/or isolated compounds for anti-HIV potential can be carried out with simple cell-based assays such as MTT (3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) or XTT (Sodium 3-[1-

(phenylamino)-carbonyl]- 3,4-tetrazoliumbis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate) assay and HIV p24 expression using human T-cell lines. Target specific studies can be achieved through evaluating the effect of the test samples on HIV-1 gp120/CD4 interactions and enzymes involved in the replication of HIV-1 using techniques such as

ELISA, flourometric and spectrophotometric methods (Narayan and Rai, 2016; Rege et al.,

2015).

Figure 2.5: Antiretroviral targets in HIV life cycle (Adapted from Rege et al., 2015). Steps in the life cycle of HIV, that can be targeted by antiretroviral drugs, include the entry of the HIV virus into the host genome cell (entry inhibitors), reverse transcription of viral RNA (reverse transcriptase inhibitors), integration of the double stranded DNA into the host cellular DNA (integrase inhibitors), protein synthesis and viral assembly (protease inhibitors).

2.3.6.1 MTT or XTT assays

Anti-HIV and cytotoxic effects of natural products or isolated compounds can be evaluated simultaneously with Human T-cell lines such as Jurkat, CEM-SS, MT4, H9 and PBMCs using

MTT or XTT assays. The results can be expressed as 50 % cytotoxic concentration (CC50), 50

% effective concentration (EC50) and the selectivity index (SI) can be calculated as CC50/EC50 ratio (Chen et al., 2013). Thus, SI reflects both antiviral activity and eventual toxicity of the test material. A high SI value indicates low toxicity of the test compound and high activity against the virus. The MTT or XTT assays do not provide detailed information on the mechanism of action of anti-HIV compounds but allows an estimation of the in vitro therapeutic index for compounds being considered for further development studies.

2.3.6.2 HIV-1 p24 Expression Assay

The effect of crude extracts or isolated compounds on HIV-1 replication can be tested by viral core protein p24 expression in cell-free supernatants using commercial ELISA kit, according to the manufacturers’ instructions. The culture supernatant is incubated in microtitre wells pre-coated with anti-p24 antibody and assayed for p24 antigen by biotin-labelled anti-p24 antibody followed by streptavidin-peroxidase conjugate (Figure 2.6). Horse Radish

Peroxidase (HRP) in the presence of peroxide converts tetramethylbenzidine (TMB) substrate to a bluish reaction product. Upon acidification by HCl or H2SO4 a bright yellowish color is generated enabling detection and quantification of p24 present in the supernatant by absorbance at 450 nm.

HRP TMB

conjugate-biotin

p24 antigen

p24 A b

Figure 2.6: HIV-1 ELISA p24 antigen assay (Adapted from http://i- base.info/guides/testing/appendix-3-how-hiv-tests-work). The p24 viral antigen in the test sample is captured onto the immobilized antibody specific for the p24 gene product of HIV-1, during incubation. The captured antigen is then reacted with human anti-HIV-1 antibody conjugated with biotin and then streptavidin-peroxidase is added. When TMB is added, colour develops as the bound enzyme reacts with the substrate.

2.3.6.3 HIV-1 reverse transcriptase (RT) inhibition assay

The HIV-1 reverse transcriptase (RT) is of tremendous medical interest as it is the target enzyme for one of the best known anti-HIV drug, AZT, which acts by causing chain termination of the polymerase reaction (University of Pretoria, https://repository.up.ac.za/). It is a multifunctional enzyme with 3 enzymatic activities. Firstly, the polymerase domain

transcribes viral RNA to viral DNA. Secondly, in the course of reverse transcription, an intermediary RNA/ DNA hybrid is formed and RT through its ribonuclease H (RNase H) domain degrades the RNA component of the hybrid. Thirdly, RT carries out DNA-dependent-

DNA-polymerase (DDDP) activity, producing complementary DNA strands. The completion of each of these processes is required for the formation of competent viral DNA capable of integrating into the genome of the infected cell. Hence, RT enzyme is considered as one of the most important targets for antiretroviral substances (Bessong et al., 2005, Li et al., 2008). The effect of natural products on RT enzyme can be evaluated by using non-radioactive ELISA and/or by ribonuclease H activity (Rege et al., 2015).

2.4 Mushrooms Found in Zimbabwe

In Zimbabwe, a wide range of mushroom species have colonized almost every available niche in the environment, from the hot, dry Lowveld and Zambezi Valley to the cool, wet Eastern parts of the country. The country’s Miombo woodlands of Msasa (Brachystegia spiciformis) and Mnondo (Julbernardia globiflora) are particularly rich in mushrooms (Sharp, 2011).

During the rainy season, which is generally between November and March, spores that have been released by mushrooms from the previous season begin to germinate and produce hyphae, which develops into a mycelium mat and eventually fruiting bodies. Over 120 wild edible and non-edible mushroom species have been described in Zimbabwe out of the estimated several hundreds of mushroom species (Sharp, 2011; 2014). The most common wild mushrooms include: Termite fungi (Termitomyces), Chanterelles (Cantharellus),

Lactarius, Russula, Boletes (Boletus) or ‘sponge fungi’and Amanita species (Sharp, 2011).

The largest mushroom found in Zimbabwe so far is a bracket fungi (Lenzites elegans) measuring almost 1.60 m in diameter. Termitomyces titanicus, Zimbabwe’s largest gilled

fungus, is according to the Guinness Book of Records, the largest mushroom in the world.

The cap of the mushroom has a diameter of up to one metre. The largest tubed fungus (bolete) in Zimbabwe is Phlebopus colossus, known as “Dindindi” in Shona and can weigh up to 3.5 kg and measure more than 35 cm in diamenter (Sharp, 2011). Non-edible mushrooms such as

Ganoderma are also found in Zimbabwe, even after the rainy season (Mabveni, 2004;

Ryvarden et al., 1994). Other mushroom species common in Zimbabwe and generally in

Southern Africa include Coprinus micaceus, Podaxis pistillaris, Coriolopsis polyzona,

Leucoagaricus bisporus, Phlebopus sudanicus, Coprinus micaceus, Coprinus plicatilis,

Pycnoporus sanguineus, Laccaria placate, Lactarius deliciosus, Lactarius hepaticus,

Panaeolus papilionaceus, Scleroderma citrinum as well as Laccaria amethystine

(Branch, 2001; Gryzenhout, 2010; Reagile, 2011).

Many wild mushrooms are used as a seasonal domestic food source contributing significantly to Zimbabwean household food security, with sustainance of livelihoods achieved through roadside selling of edible mushrooms (Ryvarden et al., 1994). Amanita zambiana (Figure

2.7), commonly known as the “Zambian Slender Caesar”, is an edible basidiomycete fungus in the genus Amanita which is commonly sold in markets and plays a major role in the achievement of healthy diets during the rainy season. Amanita zambiana was first described scientifically by British mycologists Pegler and Piearce, 1980 from Zambia. Piearce had published an illustration of the species three years earlier, but without a description (Branch,

2001; Ryvarden et al., 1994). The Chanterelles (Figure 2.7) are fairly common mushrooms and also one of the most highly prized edible ones. The mushrooms are a good source of potassium and vitamins C and D, so adding these to one’s diet is very useful (Sharp, 2011).

The Boletes, Chanterelles and edible Amanita species, have a ready market for export

(Chiroro, 2004).

A B

C D

E F Figure 2.7: Different types of mushrooms commonly found in Zimbabwe and used in this study. The mushrooms were collected from Harare under the Miombo woodlands. A: Amanita zambiana, B: Boletus edulis, C: Cantharellus heinemannianus, D: Cantharellus miomboensis, E: Cantharellus symoensii, F: Ganoderma lucidum (Photos taken by T. Reid)

In this study different mushroom genera from Zimbabwe were investigated to determine their bioactive capabilities against bacteria and HIV infection. The results obtained will to aid in the development of an integrated balanced diet that includes mushroom food with medicinal 39

role. Information for further development of effective anti-infective therapies based on mushooms will be obtained as well.

CHAPTER THREE:

MATERIALS AND

METHODS

3.0 MATERIALS AND METHODS

3.1 COLLECTION, IDENTIFICATION AND PREPARATION OF MUSHROOMS

A total of ten different mushrooms, namely; Amanita zambiana, Boletus edulis, Cantharellus heinemannianus, Cantharellus miomboensis, Cantharellus symoensii, Lactarius kabansus

(edible mushrooms), Amanita species, Coprinus species, Ganoderma lucidum and Trametes strumosa (non-edible mushrooms), were collected from the woodlands of Zimbabwe. The mushrooms, with the exception of G. lucidum, were collected during the rainy season, between January and March. The edible mushrooms selected for this study, are the most common mushrooms consumed in Zimbabwe, when they are available during the rainy season. Although Ganoderma lucidum and Trametes strumosa have been widely studied elsewhere, not much research has been done on the local mushroom species. Identification of the mushrooms was done on the basis of morphological characteristics, including colour of the mushroom cap and spore print. Final identification was done by comparing the visual appearance and the recorded characters of mushroom species with standard mushroom collection guides by Sharp (2011; 2014) and Ryvarden et al., (1994). The fresh mushrooms were sliced into thin strips and sun dried for seven days. The dried mushrooms were then ground to powder using an electrical grinder (Siebtechnik Steel Pulverizer 2, GmbH,

Germany).

3.2 CHARACTERIZATION OF MUSHROOMS

3.2.1 Quantitative Determination of the Total Protein Content

Total protein content of the mushrooms was determined using Kjeldhal method (Rana, 2016).

One and half grams each of the dried mushroom samples were digested in a Kjeldahl digestion flask by boiling with 25 ml of concentrated sulfuric acid and 10 g of Kjeldahl catalyst tablet until the mixture was clear. After cooling the flasks to ambient temperature,

400 ml of cold water and 100 ml of 40 % sodium hydroxide were added. The contents were distilled until 200 ml of solution was collected, and mixed with 50 ml of boric acid with indicator. The solution was titrated with 0.1 mol/L HCl, which had been standardized with sodium carbonate. The protein content was calculated using Formulae 1 and 2:

% Nitrogen = (A × [HCl] × 0.014× 100) ∕ weight of sample in grams,

Formula 1 where: A is the titration volume of 0.1 mol/L hydrochloric acid minus volume of blank.

% Crude Protein (CP) = % Nitrogen × Protein Factor (6.25)

Formula 2

The experiment was carried out once, in duplicate.

3.2.2 Determination of Carbohydrates

Analysis of carbohydrates in the mushrooms was done using Benedict’s test for reducing sugars and Molisch’s test for carbohydrates (Carbohydrate Lab Report, 2016). For Benedict’s test, 2 ml of Benedict’s solution was mixed with 0.5 ml of an aqueous mushroom extract in a boiling tube. The tube was placed in boiling water bath for 5 minutes and then set aside to cool to ambient temperature. A red or yellow precipitate indicated a positive reaction. For

Molisch’s test, 2 drops of ethanolic α-naphthol solution were added to 1 ml of an aqueous mushroom extract. After mixing, 3 ml of concentrated sulphuric acid was carefully added, a purple colour at the interface indicated a positive result for carbohydrates. Glucose was used as a positive test for both the tests while sucrose and starch were used as positive tests for the

Molisch’s test. Distilled water was used in place of the mushroom extract for the negative control.

3.2.3 Determination of Lectins

3.2.3.1 Extraction of mushroom crude protein

Half a gram of the powdered form of the mushrooms under study were each mixed with freshly prepared 0.15 M NaCl in the ratio 1:10 w/v. The mixture was incubated at 4 ℃ for 48 hours in the cold room. After incubation, the mixture was centrifuged at 5 200 rpm using a

Japson centrifuge (Centrifuge Machine Digital, Jambu Pershad & Sons, Ambala, India) for 35 minutes. The supernatant was collected and used in haemagglutination assay.

3.2.3.2 Preparation of the red blood cells

Preparation of red blood cells was carried out according to the methods by Albores et al.,

2014 and Singh et al., 2013, with modifications. A sterile syringe was used to draw two sets of blood from the sheep and goat (5 ml each per set). One set of blood was mixed with 0.7 ml of Alsever solution in a 10 ml falcon tube and the other set was poured into falcon tubes containing EDTA. A volume of 2 ml of blood was drawn out from each tube and poured into a 50 ml falcon tube. The volume of each tube was made up to 30 ml by adding 0.9 % saline azide. The blood was centrifuged at 3 500 rpm for 15 minutes using a Japson centrifuge

(Centrifuge Machine Digital, Jambu Pershad & Sons, Ambala, India) kept at 4 ℃ in the cold room. The supernatant was discarded and 0.9 % saline azide was added up to the 30 ml mark.

The washing process was repeated twice and the resultant pellet was resuspended in 20 ml of

0.9 % saline azide to give a 5 % red blood cell suspension.

3.2.3.3 Haemaggutination assay

Lectins in the crude mushroom extracts were detected by their haemagglutination activity against sheep and goat erythrocytes (Albores et al., 2012; Singh et al., 2013). The haemagglutination assay was carried out in 96-well, U-bottom Sarstedt microtiter plates. A

serial two-fold dilution of the mushroom extract in microtiter (25 µl) was mixed with 50 µl of a 5 % suspension of sheep and goat red blood cells in 0.9 % saline azide solution at room temperature. The first two rows containing 0.9 % saline azide and erythrocytes in a plate were the negative control. The results were recorded after one hour (60 minutes) when the erythrocytes in the negative control had fully sedimented. The hemagglutination titer, defined as the reciprocal of the highest dilution exhibiting agglutination, represented one hemagglutination unit. Specific activity was determined as the number of hemagglutination units/mg mushroom. The assays were carried out in duplicate.

3.3 CHARACTERIZATION OF MUSHROOM EXTRACTS

3.3.1 Preparation of Crude Mushroom Extracts

The ten different mushrooms were sliced into thin strips prior to sun drying for seven days and ground to powder using an electrical grinder (Siebtechnik Steel Pulverizer 2, GmbH,

Germany). Dried mushroom powder (ranging from a tenth of a gram to a gram, depending on the available quantity of each mushroom type) was extracted with 15 ml of sterile cold water, absolute methanol, ethanol or acetone at 25 ℃ and 150 rpm for 24 hours using an Orbital floor incubator shaker (Gallenkamp, UK). Hot water extracts were obtained by boiling the same amounts of mushrooms in 15 ml of distilled water for 10 minutes and then allowing the suspension to cool to room temperature. All the suspensions were then filtered using

Whatman no. 1 filter paper, dried under a stream of non-sterile air and reconstituted to 10 mg/ml in sterile water for water extracts or 100 % dimethyl sulfoxide for the rest of the extracts. The reconstituted crude extracts were filter sterilized using sterile syringe filters

(Filter-Bio, 0.22 µm, Filter Bioscience Membrane Technology Co., Ltd, China). A total of fifty different extracts were obtained and coded CME 1-50 which were then stored at 4 ℃ for further use.

3.3.2 Determination of Protein Content

Protein content in each mushroom extract was determined using the Folin Ciocalteu (FC) and

Lowry C reagents method with bovine serum albumin as the standard (Owusu-Apenten,

2002). Briefly, 50 µl of each sample was diluted to 250 µl using sterile water and mixed with

250 µl of 0.5 M NaOH. Two and a half millilitres of freshly prepared Lowry C reagent was added, after which 250 µl of the FC reagent was added. After 30 minutes incubation, absorbance was measured at 720 nm using a spectrophotometer (SpectronicR 20 GenesysTM,

Spectronic Instruments, USA).

3.3.3 Determination of Total Phenolic Content

Total phenolic content in each mushroom extract was determined using the Folin and

Ciocalteu (FC) reagent method with gallic acid as the standard (Gan et al., 2013). Briefly,

40 µl of each sample was diluted to 200 µl using sterile water or 100 % dimethyl sulfoxide and mixed with 200 µl of Folin and Ciocalteu’s phenol reagent, diluted 1 : 9 ml in distilled water. After 6 minutes incubation, 200 µl of 7.5 % sodium carbonate was added to the mixture and adjusted to 2 ml with distilled water. The reaction was kept in the dark for 60 minutes, after which the absorbance was measured at 725 nm using a spectrophotometer

(SpectronicR 20 GenesysTM, Spectronic Instruments).

3.3.4 Determination of Antibacterial Activity of Crude Extracts

Antibacterial effect of the mushroom extracts against E. coli, S. typhi, S. aureus and S. pneumoniae was determined using the agar disc diffusion method. E. coli and S. aureus were obtained from Cimas Medical Aid Society Laboratory, S. pneumoniae from Lancet

Laboratory and S. typhi from the University of Zimbabwe. The bacterial strains were sub- cultured on Muller Hinton agar to obtain pure cultures. Briefly a suspension containing

1 x 106 cfu/ml of bacteria, adjusted using McFarland turbidity standard assay (Hudzicki,

2009), was inoculated into Mueller Hinton Agar (Mast Group Ltd., Merseyside, U.K.). The discs (6 mm) were dipped in 200 µg/ml of mushroom extract for one hour, dried and placed on the inoculated agar. Negative controls were prepared with the same solvents used to dissolve the sample extracts. Kanamycin (50 μg/ml) and vancomycin (30 μg/ml) were used as positive controls for the tested bacteria. After 2 hours incubation at 4 ℃, inoculated plates were incubated at 37 ℃ for 18 hours in an incubator (Gallenkamp, UK). At the end of the incubation period, the zones of inhibition around the discs were measured.

3.3.4.1 Further analysis of mushroom crude extracts showing high antibacterial activity by spectrophotometry

Based on the results obtained from the antibacterial assays (Section 3.3.4), sixteen extracts, that showed high potency against the tested bacteria were selected for further characterization.

The absorption spectra, employing wavelengths from 200 nm to 800 nm for the mushroom extracts, were obtained using the UV-Visible spectrophotometer (UV-1601, Shimadzu,

Japan).

3.3.4.2 Analysis of crude extracts by TLC

The sixteen extracts selected for further study were analysed by TLC, using toluene : ethyl acetate: methanol (40 : 0.5 : 0.5 v/v) as the mobile phase. Five microliters of the mushroom extract was spotted onto a TLC plate (Silica Gel 60 F254 plate, Analtech, USA) and left to dry for five minutes. The TLC plates were dipped in 100 ml of the mobile phase in a developing tank and left to run for I hour. Visualization of the spots was done by viewing the plates under

UV lamp (P.W. Allen and Co., United Kingdom). Staining with vanillin sulphuric acid

solution, a universal staining reagent, and heating the plates at 110 ºC for five minutes was carried out to determine the compound profile of the selected extracts.

3.3.4.3 Selection of mobile phase for preparative thin layer chromatography (PTLC)

Six different types of mobile phases were used to separate the components of the selected sixteen mushroom extracts and to analyse the components using TLC. The data obtained was used to determine the best solvent to use for preparative TLC for each extract. The mobile phases used were: (i) chloroform : methanol (CM1) 9.5 : 0.5 v/v, (ii) chloroform : methanol

(CM2) 9 : 1 v/v, (iii) ethyl acetate : ethanol : water (EEW) 8 : 2 : 0.2 v/v, (iv) toluene : ethyl acetate (TE) 9.5 : 0.5 v/v, (v) toluene : ethyl acetate : methanol (TEM) 4 : 0.5 : 0.5 v/v and

(vi) ethyl acetate : methanol : water (EMW) 100 : 13.5 : 10 v/v. Seven microliters of mushroom extract was spotted onto a TLC plate (Silica Gel 60 F254 plate, Analtech, USA) and left to dry for five minutes. The TLC plates were dipped in 100 ml of the mobile phase in a developing tank and left to run for 30 minutes. Visualization of the spots was done by viewing the plates under UV lamp (P.W. Allen and Co., United Kingdom) and staining with vapour from iodine crystals.

3.3.4.4 Isolation of compounds from mushroom extracts by PTLC

Two mobile phases, namely, TEM and CM1, were selected for separation of components of the sixteen mushroom extracts by PTLC. The selection was based on the number of bands and good resolution from TLC plates. Two hundred and fifty microliters of each mushroom extract was carefully spread onto a preparative TLC plate (Silica Gel GF F254, 1 000 micron,

Analtech, Inc., USA) and left to dry. The plates were dipped in 100 ml of the mobile phase in a developing tank and left to run for 50 minutes. Visualization of the spots was done by viewing the plates under UV lamp (P.W. Allen and Co., United Kingdom) and staining with

iodine crystals vapour. The separated bands were each scrapped off the silica plates and extracted with 15 ml of 100 % ethyl acetate, filtered through Whatman no. 1 filter paper and dried under a stream of sterile air. The dried extracts were re-suspended in methanol or dimethyl sulphoxide at a concentration of 10 mg/ml for fraction yields that were 5 mg and above, and 1 - 8 mg/ml of the same solvents for yields that were below 5 mg. A total of one hundred and thirty one samples were obtained and coded CP1 to CP131.

3.3.4.5 Screening of isolated fractions for antibacterial activity

The isolated samples were screened for their antimicrobial activity against the same bacteria previously inhibited by the crude exracts, namely, S. typhi, S. aureus and S. pneumoniae, using the MTT cell viability assay. Briefly, a suspension containing 1 x 106 cfu/ml of bacteria, adjusted using McFarland turbidity standard assay (Hudzicki, 2009), was mixed with four dilutions of each mushroom fraction in wells of a 96-well microtitre plate and made up to 250

µl using Muller Hinton nutrient broth (Sigma-Aldrich, Germany). The final concentrations of the diluted mushroom fractions were 200, 400, 600 and 800 µg/ml. Negative controls were prepared with the same solvents used to dissolve the fractions as well as the media alone with the cells. Ampicillin (50 µg/ml) was used as a positive control for the tested bacteria. The plates were then incubated at 37 ℃ for 18 hours. Twenty-five microlitres of 5 mg/ml MTT was then added into each well and the plates were incubated at 37 ℃ for one hour, after which absorbance was read at 560 nm using an ELISA plate analyser (Micro Plate Read, Global

Diagnostics and Medical Solutions, Belgium). Percentage inhibition of each fraction was calculated using formula 3:

% inhibition = [(A560 control – A560 sample)/A560 control] x 100

Formula 3

3.3.4.6 Identification of antibacterial compounds of mushroom extracts by non-targeted

LC – MS

Seven of the isolated fractions that exhibited high percentage inhibition against the growth of

S. typhi and S. aureus were used for further analysis using an Agilent HPLC 1260 System

(Agilent, USA) coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-

TOF) mass spectrometer (Agilent, USA). The HPLC 1260 System was equipped with a binary pump, autosampler and thermostated column compartment. The separation of samples was performed on an Agilent Poroshell 120 C18 column (100 x 2.1 mm, 2.7 µm, Agilent

Technologies) at a column temperature of 40 ℃. The flow rate was 0.25 ml/min and the mobile phase consisted of 0.1 % (v/v) formic acid in sterile water (A) and 0.1 % (v/v) formic acid in acetonitrile (B). The following gradient program was used: 0 – 3 min, 80 – 20 % B; 3

– 7 min, 70 – 30 % B; 7 –10 min, 65 – 35 % B; 10 – 13 min, 60 – 40 % B; 13 – 17 min, 40 –

60 % B; 17 – 20 min, 100 % B; 20 – 25 min, 80 –20 % B. The sample injection volume was 5

µl and the mass detection was operated in positive and negative ion modes with parameters set as follows: drying gas flow rate, 8 L/min; gas temperature, 350 °C; pressure of nebulizer,

35 psig; nozzle voltage, 1 000 V; fragmentor voltage, 175 V; skimmer voltage, 65 V and a scan range of m/z 100 – 1 000. The mushroom fractions were also analysed in the positive mode and negative mode without a column.

Mass spectrometry analysis was performed on an Agilent 6530 Q-TOF spectrometer (Agilent,

USA) fitted with a jet stream electrospray ionisation source. Data was analysed using the

Mass Hunter Qualitative Analysis software package (Agilent Technologies). Blanks using each of the solvent extraction systems were analysed using the Find by Molecular Feature algorithm in the software package to generate a compound list of molecules with abundance greater than 500 counts. This was used as an exclusion list to eliminate background

contaminant compounds from the analysis of the fractions. Each fraction was then analysed using the Find the Molecular Feature function to generate a putative list of compounds in the extracts. Compound lists were then screened against three accurate mass databases; a database of known plant compounds of therapeutic importance generated specifically for this study

(800 compounds); the Metlin metabolomics database (24,768 compounds); and the Forensic

Toxicology Database by Agilent Technologies (7,509 compounds). Empirical formula for unidentified compounds was determined using the Find Formula function in the software package.

3.3.5 Determination of Anti-HIV Activity

3.3.5.1 Determination of anti-HIV-1 reverse transcriptase activity

The dried powder from ten different wild mushrooms (Section 3.1) were extracted with either sterile cold water, distilled boiling water, methanol, ethanol or acetone as in Section 3.3.1.

The suspensions were filtered, dried under a stream of non-sterile air and reconstituted to 10 mg/ml in sterile distilled water. The reconstituted crude extracts were filter sterilized using sterile syringe filters (Filter-Bio, 0.22 µm, Filter Bioscience Membrane Technology Co., Ltd).

The effect of the mushroom extracts against HIV-1 reverse transcriptase (RT) activity was carried out as instructed in the protocol supplied with the assay kit (HIV-1 RT assay kit,

Roche, Germany). Briefly, a 60 µl reaction mixture, consisting of RT solution (20 µl or 4 ng) diluted in lysis buffer, mushroom test sample (20 µl of 300 µg/ml) and the template/primer hybrid poly (A) x oligo (dT)15, was prepared in a micro-centrifuge tube. After one hour incubation at 37 ℃, the reaction mixture was transferred into each well of a 96-well plate of the assay kit. The plate was incubated at 37 ℃ for one hour. Washing buffer (250 µl) was added to each well and the wells were washed five times. Antibody (anti-DIG-POD)(200 µl) was added to each well and the plate was incubated at 37 ℃ for one hour, after which the

wells were washed. In the final step, 200 µl of peroxidase substrate, ABTS substrate solution, was added and the plate was left at room temperature for 20 minutes. The absorbance of the sample was measured at 405 nm using an ELISA plate analyser (Micro Plate Read, Global

Diagnostics and Medical Solutions, Belgium). Two control samples, one with no RT solution and the other with sterile distilled water in place of mushroom sample, were included. The resulting signal intensity was directly proportional to the actual RT activity. The inhibitory activity of the mushroom extracts was calculated as percent inhibition compared to a control sample without the mushroom extracts. The assays were carried out in duplicate.

3.3.5.2 Cytotoxicity assay of the mushroom extracts

The general cytotoxicity (CC50) of the fifty different mushroom extracts was determined by a tetrazolium (MTT) viability assay. The human peripheral blood mononuclear cells (PBMCs) were adjusted to 1 x 105 cells/ ml in RPMI-1640 medium supplemented with 10 % FBS. A volume of 100 µl of PBMC suspension was transferred into each well of a 96-well plate and left to adhere for 24 hours at 37 ℃/5 % CO2. Cells were then exposed for 48 hours to serial dilutions (0.001 µg/ml to 1 000 µg/ml) of mushroom extracts in RPMI-1640 at 37 ℃/5 %

CO2. After incubation, 25 µl of MTT was added into each well and the plates were left at 37

℃/5 % CO2 for 4 hours. The medium was then carefully removed and the MTT crystals were dissolved in 100 µl of 100 % dimethyl sulphoxide. Two independent experiments were carried out in triplicate. The plates were read at 450 nm in an ELISA plate reader (Labsystems

Multiskan, USA). The CC50 values were calculated with Graph pad Prism (Version 5.03,

GraphPad Software Inc.).

3.3.5.3 Determination of the in vitro anti-HIV-1 activity using HIV p24 expression assay

Twenty extracts, exhibiting low or no cytotoxic effects to the PBMCs, were selected for the anti-HIV assay. The PBMCs were obtained from HIV-1 seronegative donors of blood donated to the Botswana/Harvard Partnership Laboratory, Gaborone. The HIV-1c (MJ4) molecular clone was used to infect the PBMCs. The MJ4 is an infectious HIV-1 subtype C molecular clone from a Southern African (Botswana) isolate (Ndung’u et al., 2001). Five millilitre vials containing PBMCs were removed from the liquid nitrogen tank and immediately placed on dry ice. Ten millilitres of RPMI growth media, warmed at 37 ℃ was mixed with 10 % fetal bovine serum (FBS), 2 µl of benzoate and streptomycin. One millilitre of the media was pipetted out and added dropwise into the thawed PBMC vial. All the contents in the vial were then drawn out and poured into the tube containing 10 ml of growth media. The tube was spun at 400 rpm for 10 minutes and the supernatant was carefully decanted. Another 10 of growth media, without benzoate, was added and the pocess was repeated. The pellet was resuspended in 5 ml growth media and incubated at 37 ℃ and 5 % CO2 overnight.

The determination of the in vitro anti-HIV activity of mushroom extracts was carried out according to the method by Leteane et al., 2012, with slight modifications. PBMCs cultured overnight, at 37 ℃ and 5 % CO2, were stimulated with 1 µg/ml of phytohemagglutinin (PHA) and 100 U/ml of interleukin 2. The PHA stimulated cells were infected for 3 hours with HIV-

1c (MJ4) molecular clone, at a multiplicity of infection (M.O.I.) of 0.1, in the presence or absence of the selected mushroom extracts at concentrations of 50, 100 and 150 µg/ml. The cells were then washed 3 times with PBS and cultured for seven days in 1x AIM-V (Gibco,

USA) growth medium containing PHA. After 7 days of incubation, the HIV-1c (MJ4) p24 antigen from PBMC culture supernatant was quantified by enzyme-linked immunosorbent assay (ExpressBio Life Sciences, USA) according to the manufacturer’s instructions.

Briefly, 100 µl of the supernatant was transferred into the wells of an HIV p24 antigen plate containing 100 µl of 1x AIM-V (Gibco, USA) growth medium and 20 µl of lysis buffer and incubated at 37 ℃/5 % CO2 for an hour. The supernatant was removed and the plates were washed five times with wash buffer. One hundred microlitres of detector antibody was added into each well and incubated at 37 ℃ for one hour. After aspiration of the detector antibody, the plate was rinsed again five times. Streptavidin HRP (horse radish peroxidase)(100 µl) conjugate was added into each well and the plate was incubated at room temperature for 30 minutes. The conjugate was aspirated from the wells and the plates washed five times.

Substrate solution (100 µl) was dispensed into each well and the plate was left at room temperature protected from direct sunlight for 30 minutes. The reaction was stopped by adding stop solution and absorbance was read at 450 nm. Azidothymidine (AZT) (20 µg/ml) and dimethyl sulphoxide were used as the positive and negative controls, respectively. The percentage inhibition of each extract was calculated using formula 4:

% inhibition = [(A450 control – A450 sample) /A450 control] x 100

Formula 4

3.3.5.4 Assay for HIV-1c induced cytopathic effect

The protective effect of the selected mushroom extracts against HIV-1c induced cytopathogenicity was measured using the MTT viability assay described in Section 3.3.5.2.

3.3.5.5 Analysis of the anti-HIV mushroom crude extracts by non-targeted LC – MS

Three of the mushroom crude extracts that had the highest percentage inhibition against HIV-

1 RT (89.9 - 92.6 %) and two extracts that had the highest percentage inhibiton against HIV-1 replication from the HIV-1 p24 ELISA assay, were further analysed using an Agilent HPLC

1260 System coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF)

mass spectrometer. The same procedure described in section 3.3.4.6 was carried out.

3.4 STATISTICAL ANALYSIS

Experimental values are given as means ± standard deviation (SD). Graph-Pad Prism was used to analyse the data. Statistical significance was determined by one way variance analysis

(ANOVA) (Version 5.03, GraphPad Software Inc.). Differences at P < 0.05 were considered to be significant.

CHAPTER FOUR:

RESULTS

4.0 RESULTS

4.1 COLLECTION, IDENTIFICATION AND PREPARATION OF MUSHROOMS

A total of ten different mushrooms, both edible and non-edible, were collected from the woodlands of Zimbabwe (Table 4.1). After drying for seven days (Figure 4.1), the mushrooms were ground to powder using an electrical grinder (Siebtechnik Steel Pulverizer 2,

GmbH, Germany).

Table 4.1: Different types of mushrooms collected from the woodlands of Zimbabwe

Latin name Local Shona Name Edibility

Amanita zambiana Nhedzi Edible

Amanita sp. - Non-edible

Boletus edulis Dindindi Edible

Cantharellus miombooensis Chihombiro Edible

Cantharellus symoensii Jongwe/Firifiti Edible

Cantharellus heinemannianus Tsvuketsvuke Edible

Coprinus sp. - Non-edible

Ganoderma lucidum Howa danda Non-edible

Lactarius kabansus Nzeveyambuya Edible

Trametes strumosa Howa danda Non-edible

Figure 4.1: Sliced pieces of Cantharellus miomboensis during the drying process.

4.2 CHARACTERIZATION OF MUSHROOMS

4.2.1 Quantitative Determination of the Total Protein Content

The percentage total protein content of the different mushrooms are shown in Figure 4.2 . The percentage values ranged from 9.3 % ± 1.0 to 30.8 % ± 1.3. Boletus edulis, Coprinus sp.,

Lactarius kabansus and Amanita zambiana had very high protein content (30.8 % ± 1.3, 30.7

% ± 4.4, 30.1 % ± 0.3 and 28.6 ± 2.4, respectively), followed by Cantharellus symoensii,

Cantharellus heinemannianus and Cantharellus miomboensis with percentage values of 22.9

% ± 0.6, 21.1 % ± 2.4 and 16.8 ± 0.3, respectively. Ganoderma lucidum and T. strumosa had the lowest protein content values (9.3 % ± 1.0 and 10.0 % ± 1.1, respectively).

% total% protein content 0

B. edulis G. lucidum A. zambiana C. symoensiiCoprinus sp L. kabansusT. strumosa C. miomboensisMushroom type C. heinemannianus

Figure 4.2: Percentage total protein content of nine different mushrooms. Results are expressed as ± standard deviation of two measurements. N = 2. Error bars represent standard error of the mean. Boletus edulis had the highest total protein content of 30.8 % ± 1.3 while Ganoderma lucidum had the lowest protein content value of 9.3 % ± 1.0.

4.2.2 Qualitative Determination of Carbohydrates

The results from both the Molisch’s and the Benedict’s tests (Figure 4.3) showed that most of the mushrooms contained carbohydrates as shown in Table 4.2. The mushrooms showed varying levels of carbohydrates, as shown by the different intensities of the positive results, in the following sequence; A. zambiana and L. kabansus > Amanita sp., C. symoensii, and T. strumosa > B. edulis, C. heinemannianus and C. miomboensis > G. lucidum. Mushrooms A. zambiana, C. heinemannianus and C. miomboensis showed high levels of reducing sugars.

A B

Purple colour Red for precipitate positive for positive results results

1 2 3 4 1 2 3 4 5 6 7

Figure 4.3: Analysis of carbohydrates showing positive tests for (A) Benedict’s test for reducing sugars and (B) Molisch’s test for carbohydrates. A1: C. miomboensis, A2: T. strumosa, A3: A. zambiana, A4: Glucose (positive control), B1: Sucrose (positive control), B2: C. symoensii, B3: C. heinemannianus, B4: B. edulis, B5: G. lucidum, B6: Coprinus sp., B7: Starch (5%) (positive control)

Table 4.2: Qualitative analysis of carbohydrate content in mushrooms using Molisch’s and Benedict’s tests

Mushroom Molisch’s test Benedict’s test (reducing

(carbohydrates) sugars)

A. zambiana ++++ +++

Amanita sp. +++ +

B. edulis ++ ++

C. miomboensis ++ +++

C. heinemannianus ++ +++

C. symoensii +++ ++

Coprinus sp. ++ ++

G. lucidum + ++

L. kabansus ++++ +

T. strumosa +++ ++

Glucose (positive control) +++

Starch (positive control) ++++

(+) Extracts showed positive tests. All the mushrooms tested positive for both the carbohydrates and reducing sugars. The increasing number of positive signs corresponds to the increasing intensity of the colour or precipitate.

4.2.3 Determination of Lectins

The results of the haemagglutination assays of the ten different mushrooms in this study using two types of red blood cells, namely, sheep and goat, are shown in Figure 4.4 and Table 4.3.

The results showed that three of the ten mushrooms, namely Amanita sp., B. edulis and L. kabansus were able to agglutinate both the sheep and goat red blood cells.

A Negative control B Amanita sp. showing C haemagglutination

D activity

E T. strumosa F

G A. zambiana H

Figure 4.4: Haemagglutination assay of three of the ten mushroom species with sheep erythrocytes showing representative results. The negative control in the first two rows (A-B) had 0.9% saline azide. Rows C-D, E-F and G-H contain Amanita species, T. strumosa and A. zambiana, respectively. Amanita species showed haemagglutination activity.

Table 4.3: Haemagglutination assay of the ten mushroom species with sheep and goat erythrocytes

Mushroom Sheep RBCs Goat RBCs

A. zambiana - -

Amanita sp. + +

B. edulis + +

C. miomboensis - -

C. heinemannianus - -

C. symoensii - -

Coprinus sp. - -

G. lucidum - -

L. kabansus + +

T. strumosa - -

(+) denotes haemagglutination activity, (-) no haemagglutination activity observed. Three mushrooms, namely, Amanita sp., Boletus edulis and Lactarius kabansus, showed haemagglutination activity.

The specific haemagglutination activity of the three mushrooms varied, with B. edulis having the highest specific activity of 617 HAU/ mg mushroom, and L. kabansus having the lowest activity of 5 HAU/ mg mushroom as shown in Table 4.4. However, although the three mushroom lectins could agglutinate both sheep and goat erythrocytes, Amanita sp. and L. kabansus showed a little higher activity with sheep erythrocytes, while B. edulis had much higher activity with goat erythrocytes.

Table 4.4: Specificity activity of crude extracts of mushrooms showing haemaggutination activity

Mushroom Specific activity (HAU/ mg mushroom)

Sheep RBCs Goat RBCs

Amanita sp. 39 10

B. edulis 154 617

L. kabansus 77 5

The hemagglutination titer, defined as the reciprocal of the highest dilution exhibiting agglutination, represented one hemagglutination unit (HAU). Specific activity was determined as the number of hemagglutination units/mg mushroom. Boletus edulis had the highest specific activity, showing high lectin content.

4.3 CHARACTERIZATION OF MUSHROOM EXTRACTS

4.3.1 Determination of Protein Content of the Crude Extracts

Results from the protein analysis in Figure 4.5 showed significantly high yields from the cold water extracts. However, a few boiled extracts gave higher yields, while some of the protein yields, although lower than the cold water extracts, were not significantly different. Protein yields from methanol extracts followed the water extracts while ethanol and acetone gave similar low yields. A zambiana, C symoensii, C. heinemannianus, G. lucidum, L. kabansus had similar trends: cold water > hot water > methanol > ethanol > acetone, whilst Amanita sp, B. edulis, Coprinus sp., and T. strumosa had a similar trend, apart from hot water having greater absolute values than cold water. C. miomboensis had a high acetone protein content value.

3 Methanol

Ethanol

Acetone 2 Cold Water

Hot Water

Protein content 1

(mg BSA DW) /100 g

B. edulis Amanita sp G. lucidum A. zambiana C. symoensii Coprinus sp L. kabansusT. strumosa C. miomboensis C. heinemannianus

Figure 4.5: Protein content of mushrooms extracted by methanol, ethanol, acetone, cold water and hot water. Data expressed as mean ± SD; N = 3. Hot and cold water solvents extracted more proteins than the organic solvents, as shown by high protein content of the aqueous extracts.

From the ten different mushroom types studied, B. edulis and Amanita sp. gave high yields of protein in the cold and boiled water extracts. Boletus edulis also gave the highest yield in all the methanol extracts. Trametes mushroom had the lowest protein yield.

4.3.2 Determination of Total Phenolic Content

The results of the total phenolic composition of the different mushrooms from the crude extracts are shown in Figure 4.6. With a few exceptions, extracts from cold and boiled water gave the highest levels of total phenolics (17.71 ± 1.1 to 503.70 ± 20.6 mg GAE/100 g dry weight mushroom), followed by methanolic extracts (6.60 ± 0.8 to 341.47 ± 16.3 mg

GAE/100 g dry mushroom), while acetone extracts overly gave the lowest values (4.78 ± 0.2 to 99.88 ± 2.1 mg GAE/100 g dry mushroom). However, most of the yields from the acetone

and ethanol extracts were not significantly different. Cantharellus symoensii, C. heinemannianus, Coprinus sp., and L. kabansus had similar trends: cold water > hot water > methanol > ethanol > acetone, whilst Amanita sp. had a similar trend, apart from hot water having a greater absolute value than cold water. Amanita zambiana and G. lucidum had similar above trends as well, with the exception of methanol having a greater absolute value than hot water. Boletus edulis had high hot water and methanol phenol content whilst C. miomboensis had a high acetone phenol content value. Statistical analysis by two way

ANOVA showed that there was significant difference in the effect of solvents in extracting total phenols (4 df, F = 7.815, P-value = 0.000122) and that the total phenolic composition was also dependant on the mushroom type (9 df, F = 4.984, P value = 0.000224). From the ten different mushroom types studied, Boletus was observed to have the highest total phenolic compounds followed by Amanita sp., while T. strumosa had the lowest phenolic content.

600 Methanol

Ethanol

Acetone 400 Cold Water

Hot Water

200

Totalphenolic content (mg /100 GAE g DW)

B. edulis Amanita sp G. lucidum A. zambiana C. symoensii Coprinus sp L. kabansusT. strumosa C. miomboensis C. heinemannianus

Figure 4.6: Total phenolic content of mushrooms extracted by methanol, ethanol, acetone, cold water and hot water. Data expressed as mean ± SD; N = 3. Hot and cold water solvents extracted more phenolic compounds than the organic solvents, as shown by high total phenolic content of the aqueous extracts, with the exception of B. edulis.

4.3.3 Determination of the Antibacterial Activity of Mushroom Crude Extracts

The different mushroom extracts exhibited various degrees of inhibition of bacterial growth

(6.3 – 14 mm diameter). The highest in vitro antibacterial activity (14.0 ± 1.0 mm) was shown by the cold water extract of C. miomboensis against S. typhi. This was followed by C. symoensii (11.5 ± 1.0 mm, acetone extract), C. miomboensis (11.0 ± 2.0 mm, methanol extract), G. lucidum (10.7 ± 1.2 mm, ethanol extract) and C. symoensii (10.0 ± 0 mm, ethanol extract). The antibacterial activities of the extracts from the ten different mushrooms in this study with respect to the solvent used for extraction (methanol, ethanol, acetone, cold and hot water) are shown in Tables 4.5 to 4.9. The results showed that all the mushrooms exhibited inhibitory activities against at least one of the bacteria tested, as shown by the clear zone of inhibition measurements around the tested mushroom extracts.

Table 4.5: Antibacterial activities of methanol extracts of mushrooms on test organisms

Zone of inhibition diameter (mm)

Gram positive bacteria Gram negative bacteria

S. aureus S. pneumoniae E. coli S. typhi

A. zambiana 7.0 ± 0 9.0 ± 0.20 7.0 ± 0.0 8.5 ± 0.0

B. edulis - 7.5 ± 0.87 8.0 ± 0.0 7.33 ± 0.29

C. miomboensis 6.5 ± 0 6.84 ± 0.29 - 11.0 ± 2.0

C. symoensii 7.23 ± 0.25 8.14 ± 0.90 7.6 ± 1.15 9.5 ± 0.5

heinemannianus - 8.67 ± 0.76 8.5 ± 1.73 8.0 ± 0.0

Coprinus sp. - 8.0 ± 0.0 - 7.33 ± 0.58

G. lucidum - 8.33 ± 1.16 - 8.0 ± 0.0

L. kabansus - 9.33 ± 1.16 - 7.5 ± 0.5

T. strumosa - 7.43 ± 0.0 - 8.33 ± 0.29

DMSO - - - -

Vancomycin Nt Nt 12.4 ± 0.7 15.3 ± 0.3

Kanamycin 15.4 ± 0.7 16.5 ± 1.1 14 ± 0.3 18.5 ± 0.9

The paper discs (6 mm) impregnanted with methanol extracts of mushrooms or antibiotic were placed on agar inoculated with E. coli, S. typhi, S. aureus or S. pneumoniae. After 2 hours incubation at 4 ℃, inoculated plates were incubated at 37 ℃ for 18 hours in an incubator and at the end of the incubation period, the zones of inhibition around the discs were measured. (-): no inhibition. Nt: not tested. DMSO: dimethyl sulfoxide (negative control). Each value is expressed as mean ± SD (n = 3). Statistical significance was determined by one way variance analysis (ANOVA), p < 0.05.

Table 4.6: Antibacterial activities of ethanol extracts of mushrooms on test organisms

Zone of inhibition diameter (mm)

Gram positive bacteria Gram negative bacteria

S. aureus S. pneumoniae E. coli S. typhi

A. zambiana 7.33 ± 0.58 7.83 ± 0.76 7.2 ± 0.0 8.67 ± 0.76

Amanita sp. 8.23 ± 1.25 7.07 ± 0.12 8.0 ± 0.0 9.0 ± 1.0

B. edulis 7.5 ± 0.5 6.6 ± 0.0 8.67 ± 0.58 7.5 ± 0.0

C. miomboensis 8.16 ± 0.29 7.67 ± 0.76 7.77 ± 0.25 9.17 ± 0.29

C. symoensii 8.2 ± 0.76 8.94 ± 0.31 7.4 ± 0.17 10.0 ± 0.0

heinemannianus 8.83 ± 0.29 8.4 ± 0.53 - 8.18 ± 0.58

Coprinus sp. - 8.0 ± 0.0 - 8.0 ± 0.0

G. lucidum - 8.0 ± 0.0 7.67 ± 1.16 10.67 ± 1.16

L. kabansus 7.83 ± 1.04 8.5 ± 0.87 - 8.0 ± 0.5

T. strumosa - 7.33 ± 0.76 - 6.5 ± 0.0

DMSO - - - -

Vancomycin Nt Nt 12.4 ± 0.7 15.3 ± 0.3

Kanamycin 15.4 ± 0.7 16.5 ± 1.1 14 ± 0.3 18.5 ± 0.9

The paper discs (6 mm) impregnanted with ethanol extracts of mushrooms or antibiotic were placed on agar inoculated with E. coli, S. typhi, S. aureus or S. pneumoniae. After 2 hours incubation at 4 ℃, inoculated plates were incubated at 37 ℃ for 18 hours in an incubator and at the end of the incubation period, the zones of inhibition around the discs were measured. (-): no inhibition. Nt: not tested. DMSO: dimethyl sulfoxide (negative control). Each value is expressed as mean ± SD (n = 3). Statistical significance was determined by one way variance analysis (ANOVA), p < 0.05.

Table 4.7: Antibacterial activities of acetone extracts of mushrooms on test organisms

Zone of inhibition diameter (mm)

Gram positive bacteria Gram negative bacteria

S. aureus S. pneumoniae E. coli S. typhi

A. zambiana - 7.5 ± 0.0 - 9.0 ± 0.0

Amanita sp. 6.67 ± 0.29 7.67 ± 0.76 6.3 ± 0.0 9.0 ± 1.0

B. edulis 7.0 ± 0.0 7.33 ± 0.29 7.83 ± 0.58 8.17 ± 0.76

C. miomboensis 7.67 ± 0.29 6.67 ± 0.29 7.73 ± 0.25 8.67 ± 0.29

C. symoensii 8.67 ± 0.58 8.17 ± 0.29 - 11.5 ± 1.0

heinemannianus 8.07 ± 0.12 7.0 ± 0.0 - 9.17 ± 0.76

Coprinus sp. - 7.0 ± 0.0 - 7.5 ± 0.5

G. lucidum - 8.27 ± 0.64 8.0 ± 0.0 8.33 ± 0.76

L. kabansus - 8.43 ± 0.81 7.5 ± 0.5 9.5 ± 0.5

T. strumosa 9.5 ± 1.8 8.17 ± 0.76 - 7.5 ± 0.5

DMSO - - - -

Vancomycin Nt Nt 12.4 ± 0.7 15.3 ± 0.3

Kanamycin 15.4 ± 0.7 16.5 ± 1.1 14 ± 0.3 18.5 ± 0.9

The paper discs (6 mm) impregnanted with acetone extracts of mushrooms or antibiotic were placed on agar inoculated with E. coli, S. typhi, S. aureus or S. pneumoniae. After 2 hours incubation at 4 ℃, inoculated plates were incubated at 37 ℃ for 18 hours in an incubator and at the end of the incubation period, the zones of inhibition around the discs were measured. (-): no inhibition. Nt: not tested. DMSO: dimethyl sulfoxide (negative control). Each value is expressed as mean ± SD (n = 3). Statistical significance was determined by one way variance analysis (ANOVA), p < 0.05.

Table 4.8: Antibacterial activities of cold water extracts of mushrooms on test organisms

Zone of inhibition diameter (mm)

Gram positive bacteria Gram negative bacteria

S. aureus S. pneumoniae E. coli S. typhi

A. zambiana - - - -

Amanita sp. - - - -

B. edulis - - - 7.33 ± 0.29

C. miomboensis - - - 14.0 ± 1.0

C. symoensii - - - -

heinemannianus - - - -

Coprinus sp. - - - -

G. lucidum - - - -

L. kabansus - - - 7.5 ± 0.0

T. strumosa - - - -

Water - - - -

Vancomycin Nt Nt 12.4 ± 0.7 15.3 ± 0.3

Kanamycin 15.4 ± 0.7 16.5 ± 1.1 14 ± 0.3 18.5 ± 0.9

The paper discs (6 mm) impregnanted with cold water extracts of mushrooms or antibiotic were placed on agar inoculated with E. coli, S. typhi, S. aureus or S. pneumoniae. After 2 hours incubation at 4 ℃, inoculated plates were incubated at 37 ℃ for 18 hours in an incubator and at the end of the incubation period, the zones of inhibition around the discs were measured. (-): no inhibition. Nt: not tested. Water (negative control). Each value is expressed as mean ± SD (n = 3). Statistical significance was determined by one way variance analysis (ANOVA), p < 0.05.

Table 4.9: Antibacterial activities of hot water extracts of mushrooms on test organisms

Zone of inhibition diameter (mm)

Gram positive bacteria Gram negative bacteria

S. aureus S. pneumoniae E. coli S. typhi

A. zambiana - - - -

Amanita sp. - - 7.0 ± 0.0 -

B. edulis - - - -

C. miomboensis - - - -

C. symoensii - - 6.5 ± 0.0 -

heinemannianus - - 6.5 ± 0.0 -

Coprinus sp. - - - -

G. lucidum - - - -

L. kabansus - - - -

T. strumosa - Nt - -

Water - - - -

Vancomycin Nt Nt 12.4 ± 0.7 15.3 ± 0.3

Kanamycin 15.4 ± 0.7 16.5 ± 1.1 14 ± 0.3 18.5 ± 0.9

The paper discs (6 mm) impregnanted with hot water extracts of mushrooms or antibiotic were placed on agar inoculated with E. coli, S. typhi, S. aureus or S. pneumoniae. After 2 hours incubation at 4 ℃, inoculated plates were incubated at 37 ℃ for 18 hours in an incubator and at the end of the incubation period, the zones of inhibition around the discs were measured. (-): no inhibition. Nt: not tested. Water (negative control). Each value is expressed as mean ± SD (n = 3). Statistical significance was determined by one way variance analysis (ANOVA), p < 0.05.

Species C. miomboensis, C. symoensii, Amanita sp. and B. edulis all had the highest number of total crude extracts inhibiting at least one of the bacteria (12 each), closely followed by A. zambiana and C. heinemannianus (10 each), while Coprinus had the least (6) (Tables 4.5 to

4.9). All the mushrooms samples, except Coprinus sp., G. lucidum and T. strumosa had

inhibitory effect on all the four bacteria tested. Species C. heinemannianus and C. symoensii had the highest effect on inhibition of bacteria as indicated by having the most extracts which had high inhibitory properties ranging from 8-14 mm [15.4 % (8) each] followed by G. lucidum [13.5 % (7)], while B. edulis, Coprinus species and T. strumosa had the least [5.8 %

(3 each)].

The organic solvent, ethanol, was the most effective in extracting antibacterial compounds as shown by its highest number of bacterial growth inhibiting crude extracts (33), followed by acetone (31) and methanol (28). In addition, ethanol extracts showed the strongest antibacterial activity (8-14 mm) among the five solvents used, followed by methanol and acetone. Water extracts exhibited the lowest number of antibacterial activity, despite having the extract with the highest inhibitory effect. Ethanol, acetone and methanol extracts were all effective against all the four bacteria, indicating the broad spectrum of antibacterial activity of the extracts. Gram negative bacteria were more susceptible to the extracts than gram positive bacteria (52 and 46 extracts, respectively). Among the four bacteria tested, S. typhi was the most susceptible as indicated by its highest number of inhibitions as well as the highest number of the most potent extracts in the 8-14 mm diameter range.

Based on the results obtained from the antibacterial assays (Section 4.3.3), the sixteen extracts that showed high inhibitory levels against the tested bacteria were selected for further characterization (Table 4.10).

Table 4.10: Extracts selected for further study, exhibiting high antibacterial activity (9 – 14 mm zones of inhibition) at a concentration of 200 µg/ml

Mushroom type Solvent used for Zone of inhibition Bacteria inhibited

extraction (mm)

C. miomboensis Cold water 14.0 ± 1.0 S. typhi

C. symoensii Acetone 11.5 ± 1.0 S. typhi

C. miomboensis Methanol 11.0 ± 2.0 S. typhi

G. lucidum Ethanol 10.7 ± 1.2 S. typhi

C. symoensii Ethanol 10.0 ± 0 S. typhi

C. symoensii Methanol 9.5 ± 0.5 S. typhi

L. kabansus Acetone 9.5 ± 0.5 S. typhi

T. strumosa Acetone 9.5 ± 1.8 S. aureus

Amanita sp. Methanol 9.3 ± 1.2 S. pneumoniae

L. kabansus Methanol 9.3 ± 1.2 S. pneumoniae

C. heinemannianus Acetone 9.2 ± 0.8 S. typhi

C. miomboensis Ethanol 9.2 ± 0.3 S. typhi

A. zambiana Methanol 9.0 ± 0.2 S. pneumoniae

A. zambiana Acetone 9.0 ± 0 S. typhi

Amanita sp. Ethanol 9.0 ± 1.0 S. typhi

Amanita sp. Acetone 9.0 ± 1.0 S. typhi

Total number of extracts 16

Acetone had the highest number of extracts that showed high antibacterial activity while hot water had none. S. typhi was the most susceptible bacteria as it was inhibited by most of the extracts selected (12 out of 16 extracts).

4.4 CHARACTERIZATION OF MUSHROOM EXTRACTS SHOWING

ANTIBACTERIAL ACTIVITY

4.4.1 Analysis of the Extracts using Absorption Spectroscopy and TLC

The UV spectra of mushroom extracts that exhibited antibacterial activity showed absorbance peaks ranging from 227 – 308 nm; 734 – 745 nm and 586 nm as shown in Table 4.11.

Table 4.11: Absorption spectrum peaks obtained from the selected crude extracts of mushrooms that showed high antibacterial activity

Mushroom sample Absorbance Peak (nm)

Methanol Ethanol Acetone Cold water

A manita sp. 266, 638 236, 259, 586 227, 259

G. lucidum 234, 308

T. strumosa 228, 740

C. heinemmanianus 272

C. symoensii 308 279 235, 745

C. miomboensis 276 295 228, 734

L. kabansus 256 231, 738

A. zambiana 257, 735 264

The absorbance peak values obtained varied from one extract to another showing that the secondary metabolite profile of each mushroom extract was different.

The absorbance profile obtained from the different mushrooms using the same extraction solvent type varied with each mushroom species (Figures 4.7 – 4.9).

Figure 4.7: Representative UV spectra obtained from acetone (Ac) and water (H2O) extracts of mushrooms. Am: Amanita sp., Tr: T. strumosa, Az: A. zambiana, Lk: L. kabansus, Cs: C. symoensii, Ch: C. heinemannianus and Cm: C. miomboensis

Figure 4.8: Representative UV spectra obtained from ethanol (Eth) extracts of mushrooms. Am: Amanita sp., Cs: C. symoensii, Cm: C. miomboensis and G: G. lucidum

Figure 4.9: Representative UV spectra obtained from methanol (Meth) extracts of mushrooms. Am: Amanita sp., Az: A. zambiana, Lk: L. kabansus, Cs: C. symoensii and Cm: C. miomboensis

Each mushroom type also exhibited a unique absorption profile from the different solvents as shown in Figure 4.10.

A B

C D

Figure 4.10: Representative UV spectra obtained from different solvent extracts of the same mushroom. (A): methanol (Meth) and acetone (Ac) extracts of L. kabansus (Lk), (B): methanol, acetone and ethanol (Eth) extracts of Amanita sp. (Am), (C) ethanol, acetone and methanol extracts of C. symoensii (Cs), (D): ethanol, cold water (Cw) and methanol extracts of C. miomboensis (Cm), (E): methanol and acetone extracts of A. zambiana (Az)

Analysis of the mushroom crude extracts by TLC and staining with vanillin - sulphuric acid revealed different profiles as shown in Figure 4:11 .

1 2 3 4 5 6 7 8

Figure 4.11: Representative chromatograms of mushrooms extracts developed in Toluene Ethyl acetate Methanol (TEM) solvent and sprayed with vanillin-sulphuric acid. (1): acetone extract of L. kabansus, (2): methanol extract of A. zambiana, (3): ethanol extract of G. lucidum, (4): methanol extract of C. symoensii, (5): methanol extract of C. miomboensis, (6): acetone extract of T. strumosa, (7): acetone extract of L. kabansus and (8): cold water extract of C. miomboensis

4.4.2 Separation of Mushroom Crude Extracts by TLC

The retention factor (Rf) values that were obtained from the mushroom extracts using the six different mobile systems are shown in Table 4.12. The TEM mobile phase gave the highest total number of separated bands, followed by CM1 with a total of 93 and 92 bands, respectively. The EEW mobile phase gave the least number of bands (42). The TEM mobile phase was selected for the PTLC separation of methanol extracts of Amanita species, A. zambiana, L. kabansus, C. miomboensis and C. symoensii; acetone extracts of A. zambiana,

C. heinemannianus, L. kabansus and T. strumosa and the ethanol extracts of C. symoensii and 78

G. lucidum. The CM1 mobile phase was selected for the PTLC separation of the remaining extracts, namely, the water extract of C. miomboensis, the acetone extracts of Amanita species and C. symoensii and the ethanol extracts of Amanita species and C. miomboensis. These two mobile phases were selected for separation of components of the 16 mushroom extracts

(Table 4.10) by PTLC based on the high number of bands obtained and the good resolution of bands.

Table 4.12: The Rf values of fractions separated from selected mushroom extracts using different TLC mobile phases

Rf Values: Mushroom- Toluene.- Chloroform- Chloroform- Toluene- Ethyl Ethyl acetate- Extracting Ethyl acetate- Methanol Methanol Ethyl acetate acetate- Methanol- solvent Methanol 9:1 (v/v) 9.5:0.5 (v/v) 9.5:0.5 (v/v) Ethanol-Water Water 8:1:1 (v/v/v) 8.2.0.2 (v/v/v) 100:13.5:10 (v/v/v)

A. zambiana- 0.04, 0.16 0.11, 0.14 0.03, 0.07 0.05, 0.16 0.14, 0.18 0.07, 0.16 Acetone 0.33, 0.58 0.34, 0.46 0.13, 0.19 0.26, 0.31 0.34, 0.95 0.26, 0.41 0.65, 0.88 0.74, 0.88 0.43, 0.78 0.86, 0.90 0.98 (5) 0.96 (5) 0.92, 0.97 0.95, 0.99 (8) 0.90, 0.97 0.98 (7) 0.99 (9) 0.98 (9)

A. zambiana- 0.19, 0.51 0.82, 0.99 (2) 0.67, 0.97 (2) 0.06, 0.26 (2) 0.97 (1) 0.41, 0.98 (2) Methanol 0.80 (3)

Amanita sp- 0.05, 009 0.08, 0.30 0.05, 0.07 0.12, 0.20 0.10, 0.18 0.08, 0.18 Acetone 0.15, 0.35 0.41, 0.84 0.10, 0.16 0.30, 0.79 0.21, 0.34 0.32, 0.37 0.50, 0.55 0.94, 0.99 0.23, 0.41 0.98 (5) 0.94, 0.96 (6) 0.96 (5) 0.59, 0.85 0.99 (7) 0.61, 0.68 0.96, 0.99 (10) 0.83, 0.96, 0.98 (11)

Amanita sp- 0.55, 0.88 (2) 0.87, 0.99 (2) 0.05, 0.57 0.10, 0.30 (2) 0.94 (1) 0.12, 0.97 (2) Ethanol 0.97 (3)

Amanita sp- 0.05, 0.52 0.81, 0.93 0.06, 0.68 0.10, 0.30 (2) 0.98 (1) 0.11, 0.94 (2) Methanol 0.86, 0.98 (4) 0.99 (3) 0.98 (3)

C. heinemannianus- 0.26, 0.50 0.89, 0.99 (2) 0.40, 0.45 0.04, 0.09 0.97 (1) 0.07, 0.23 Acetone 0.55, 0.64 0.73, 0.93 0.27, 0.86 0.46, 0.98 (4) 0.82, 0.99 (6) 0.96 (5) 0.96 (5)

C. miomboensis- 0.04, 0.16 0.12, 0.79 0.08, 0.15 0.05, 0.08 0.34, 0.97 (2) 0.07, 0.22 Ethanol 0.29, 0.41 0.84, 0.90 0.34, 0.37 0.28 (3) 0.42, 0.97 (4) 0.51, 0.57 0.99 (5) 0.51, 0.60 084 (7) 0.80, 0.95 (8)

C. miomboensis- 0.29, 0.63 0.84, 0.99 (2) 0.59, 0.85 0.03, 0.07 (2) 0.97 (1) 0.99 (1) Methanol 0.88 (3) 0.96 (3)

C. miomboensis- 0.07, 0.11 (2) Water

C. symoensii- 0.04, 0.20 0.13, 0.81 0.05, 0.07 0.05, 0.10 0.08, 0.15 0.09, 0.23 Acetone 0.30, 0.40 0.81, 0.88 0.11, 0.13 0.27, 0.59 0.32, 0.97 (4) 0.44, 0.99 (4) 0.55, 0.63 0.93, 0.99 (6) 0.16, 0.18 0.77, 0.99 (6) 0.70, 0.87 0.28, 0.33 0.96 (9) 0.46, 0.57 0.61, 0.66 0.79, 0.96, 0.98 (15)

C. symoensii- 0.18, 0.27 0.11, 0.82 0.18, 0.61 0.08, 0.27 (2) 0.32, 0.97 (2) 0.07, 0.21 Ethanol 0.38, 0.49 0.87, 0.94 0.71, 0.91 0.43, 0.99 (4) 0.55, 0.63 0.99 (5) 0.96 (5) 0.80, 0.99 (8)

C. symoensii- 0.19, 0.28 0.10, 0.80 0.51, 0.62 0.06 0.97 (1) 0.08, 0.20 Methanol 0.49, 0.55, 0.83 (5) 0.98 (3) 0.82, 0.96 (4) 0.26 (2) 0.41, 0.99 (4)

G. lucidum- 0.24, 0,46 0.08, 0.21 0.13, 0.62 0.10, 0.21 0.42, 0.94 0.40, 0.84 Ethanol 0.55, 0.60 0.27, 0.78 0.74, 0.84 0.30, 0.98 (4) 0.98 (3) 0.98 (3) 0.66, 0.85 0.91, 0.99 (6) 0.90, 0.97 0.92, 0.99 (8) 0.99 (7)

L. kabansus- 0.04, 0.08 0.30, 0.39 0.12, 0.17 0.10, 0.30 0.10, 0.20 0.07, 0.17 Acetone 0.16, 0.55 0.79, 0.97 0.67, 0.90 0.80, 0.98 (4) 0.35, 0.96 0.31, 0.36 0.60, 0.72 0.99 (5) 0.97, 0.99 (6) 0.98 (5) 0.94 (5) 0.85, 0.96, 0.99 (9)

L. kabansus- 0.54, 0.85 0.99 (1) 0.79, 097 (2) 0.10, 0.30 (2) 0.6, 0.93 0.59, 0.75 Methanol 0.99 (3) 0.94 (3) 0.91 (3)

T. strumosa- 0.28, 0.38 0.32, 0.43 0.13, 0.17 0.02, 0.05 0.10, 0.17 0.07, 0.17 Acetone 0.45, 0.5 0.76, 0.86 0.45, 0.71 0.09, 0.30 0.82, 0.88 0.89, 0.87 0.62, 0.87 0.99 (5) 0.95, 0.98 0.65, 0.84 0.97, 0.98 (6) 0.96 (5) 0.97 (7) 0.99 (7) 0.94 (7) The TEM mobile phase gave the highest total number of separated bands (93) while the EEW mobile phase gave the least number of bands (42). The TEM and CM1 mobile phases were selected for separation of compounds from the 16 mushtoom extracts that were further analysed. Rf values highlighted in bold represent the mobile phase that was used to separate each mushroom crude extract during PTLC. Values in parenthesis show the total number of bands separated.

4.4.3 Isolation of Components of Mushroom Crude Extracts by PTLC

After separation of the sixteen crude extracts by PTLC, each of the bands was scrapped off together with silica gel and eluted with ethyl acetate. A total of one hundred and thirty one fractions (CP1 to CP131) were obtained from the 16 extracts as shown in Table 4.13.

Table 4.13: Number of fractions obtained after scrapping bands from each of the separated crude extract on the PTLC plate

Mushroom crude extract Number of fractions obtained C. miomboensis – methanol 7 (CP1 to CP7) C. symoensii – methanol 8 (CP8 to CP15) A. zambiana – methanol 7 (CP16 to CP22) C. heinemannianus – acetone 8 (CP23 to CP30) A. zambiana – acetone 9 (CP31 to CP38) C. symoensii – ethanol 9 (CP39 to CP47) T. strumosa – acetone 8 CP48 to CP55) Amanita sp. – methanol 7 (CP56 to CP62) L. kabansus – acetone 9 (CP63 to CP71) G. lucidum – ethanol 8 (CP72 to CP80) L. kabansus – methanol 10 (CP81 to CP90) C. symoensii – acetone 7 (CP91 to CP97) C. miomboensis – water 6 (CP98 to CP103) C. miomboensis – ethanol 11 (CP104 to CP114) Amanita sp. – acetone 8 (CP115 to CP122) Amanita sp. – ethanol 9 (CP123 to CP131) Total number of fractions 131 The ethanol extract of C. miomboensis had the highest number of compounds separated (11), closely followed by the methanol extract of L. kabansus (10).

4.4.4 Determination of the Antibacterial Activity of the Isolated Fractions

The compounds (CP1 – CP131) isolated from the mushroom crude extracts exhibited varying degrees of inhibition of bacterial growth ranging from 0 to 99.8 % (Appendix 8.3). Thirteen of the isolated compounds exhibited very high inhibitory activity against growth of S. typhi

ranging from 86.6 to 99.8 %, while one of the components (CP50) exhibited high inhibitory activity against growth of S. aureus (87.5 %), as shown in Table 4.14. The highest bacterial inhibitory activities were observed from the compounds isolated from the ethanol extract of

G. lucidum (CP73) and the acetone extract of C. symoensii (CP94), each with 99.8% inhibitory activity against S. typhi, closely followed by the compound from acetone extract of

L. kabansus (CP70) (99.7 %) against S. typhi. However, CP70 was the most potent isolate as it exhibited the lowest IC50 value of 206 µg/ml, followed by CP94 and the compound isolated from the ethanol extract of C. symoensii (CP44) with IC50 values of 223 µg/ml and 245 µg/ml, respectively.

Table 4.14: Percentage inhibition of growth of S. typhi and S. aureus by the fourteen potent compounds isolated from different mushroom crude extracts using PTLC

Isolate Rf % inhibition IC50 Source of Bacteria no. value 200 µg/ml 400 µg/ml 600 g/ml 800 µg/ml (µg/ml) Isolate tested

CP25 0.26 15.4 ± 5.7 41.3 ± 9.9 99.0 ± 0 98.7 ± 0 413 Acetone extract CP26 0.55 7.1 ± 10.0 11.3 ± 16.0 82.1 ± 0 97.4 ± 0 505 of C. S. typhi CP27 0.63 10.8 ± 2.0 62.2 ± 0 88.4 ± 0 95.7 ± 1.8 350 heinemmanianus CP39 0.09 6.2 ± 5.0 38.6 ± 9.1 69.7 ± 3.2 86.6 ± 1.3 462 Ethanolic extract S. typhi CP44 0.50 33.0 ± 0 86.5 ± 5.2 92.7 ± 0 92.9 ± 0.1 245 of C. symoensii CP50 0.20 11.4 ± 0.0 13.5 ± 0.1 57.6 ± 0.1 87.5 ± 0.3 564 Acetone extract S. aureus of T. strumosa CP70 0.76 29.3 ± 29.6 98.7 ± 0.3 99.7 ± 0 99.7 ± 0 206 Acetone extract S. typhi of L. kabansus CP73 0.20 0.0 ± 0 2.3 ± 3.0 78.3 ± 29 99.8 ± 0.2 573 Ethanolic extract S. typhi CP76 0.38 13.9 ± 18.5 10.0 ± 14.1 95.5 ± 5.3 98.3 ± 1.4 467 of G. lucidum CP77 0.43 2.4 ± 2.1 41.1 ± 4.4 97.4 ± 1.4 94.9 ± 3.7 415 CP78 0.56 0.0 ± 0 0.6 ± 0.9 97.5 ± 2.4 97.7 ± 0 506 CP92 0.07 0.0 ± 0 0.0 ± 0 38.0 ± 16 98.4 ± 0.2 619 Acetone extract S. typhi CP94 0.55 16.1 ± 22.7 98.2 ± 0 99.7 ± 0 99.8 ± 0 223 of C. symoensii CP98 0.03 4.3 ± 6.0 93.3 ± 5.6 95.7 ± 2.2 96.9 ± 0 291 Cold water S. typhi extract of C. miomboensis Ampicillin 96.9 ± 0.4 S. typhi (50 µg/ml) 91.3 ± 0.5 S. aureus The negative control was used to calculate the percentage inhibition of bacterial growth by each extract. The highest bacterial inhibitory activities were observed from the compounds isolated from the ethanol extract of G. lucidum (CP73) and the acetone extract of C. symoensii (CP94), each with 99.8% inhibitory activity against S. typhi, closely followed by the compound from acetone extract of L. kabansus (CP70) (99.7 %) against S. typhi. However, CP70 was the most potent isolate as it exhibited the lowest IC50 value of 206 µg/ml.

4.4.5 Identification of the Most Potent Antibacterial Compounds of Mushroom Extracts by LC-MS

Analysis of seven of the most potent isolated mushroom components (Section 4.4.4) using an

Agilent HPLC 1260 System coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-

Flight (Q-TOF) mass spectrometer revealed the presence of the following compounds: lucidenic acid M, C16 sphinganine, stearamide, palmitic amide, cavipetin D, notoginsenoside

R2, sorbitan oleate, boviquinone 4, 11-amino-undecanoic acid, 26-methyl nigranoate, goshonoside and Z-13-oxo-9-octadecenoic acid as shown in Table 4.15.

Table 4.15: Compounds identified from seven of the most potent components isolated from the antibacterial crude extracts of mushrooms

Isolate no. Compound name Formula

CP25 Sorbitan oleate C24H44O6

Boviquinone 4 C26H36O4

CP27 Boviquinone 4 C26H36O4

CP44 Palmitic amide C16H33NO

Stearamide C18H37NO

CP50 Lucidenic acid M C27H42O6

11-amino-undecanoic acid C11H23NO2

26-methyl nigranoate C31H48O4

CP70 Notoginsenoside R2 C41H70O13

Lucidenic acid M C27H42O6

Cavipetin D C25H38O5

Goshonoside C26H44O8

CP77 Z-13-oxo-9-octadecenoic acid C18H32O3

11-amino-undecanoic acid C11H23NO2

CP94 Palmitic amide C16H33NO

C16 sphinganine C16H35NO2

Lucidenic acid M C27H42O6 Lucidenic acid M was the most common compound that was identified in three different extracts, the acetone extracts of C. symoensii, L. kabansus and T. strumosa. Compounds Boviquinone 4 and palmitic amide were each identified in two different extracts. Most of the compounds were unique to each mushroom extract.

4.5 DETERMINATION OF THE ANTI-HIV ACTIVITY OF MUSHROOM CRUDE

EXTRACTS

4.5.1 Determination of Anti-HIV-1 Reverse Transcriptase Activity

The HIV-1 RT inhibition ratio of mushroom extracts from the ten wild edible and non-edible mushrooms used in this study ranged from 0.7 % to 92.6 % as shown in Figures 4.12 to 4.13 .

The cold water extracts from all the mushrooms gave the highest levels of RT inhibitory activity, which was all above 50%, followed by the boiled water extracts, while acetone extracts gave the lowest values. The cold water extract of the mushroom L. kabansus demonstrated the highest inhibition ratio of 92.6 %, closely followed by the cold water extracts of Amanita sp., B. edulis, T. strumosa, A. zambiana, C. heinemannianus and C. miomboensis with inhibition ratios of 91.3 %, 89.9 %, 89.6 %, 88 %, 86.2 % and 81.2 %, respectively. From the ten different mushroom types studied, Amanita sp. and C. miomboensis had the most number of extracts exhibiting high anti-RT activity.

1 2 3 4 5 6 7 8

Figure 4.12: Representative assay for anti-HIV RT activity of mushroom crude extracts. A1- B1: negative control, C1-D1: positive control (0.2 ng/µl), columns 2-8: different mushroom crude extracts (100 µg/ml) in duplicate wells for each extract. The resulting signal intensity, which was measured spectrophotometrically, was directly proportional to the actual RT activity. The more intense green colour represented extracts that were less effective in inhibiting HIV RT than the lighter coloured wells.

100 Methanol

80 Ethanol Acetone 60 Cold Water Hot Water

% Inhibition % 40

B. edulis Amanita sp G. lucidum A. zambiana C. symoensiiCoprinus sp L. kabansusT. strumosa C. miomboensis C. heinemannianus

Figure 4.13: HIV-1 Reverse transcriptase inhibitory activity of crude extracts (100 µg/ml) from edible and non-edible mushrooms. The absorbance values for the negative control were used to calculate the percentage inhibition of each extract. The cold water extracts from all the mushrooms gave the highest levels of RT inhibitory activity, followed by the boiled water extracts, while acetone extracts gave the lowest values. From the ten different mushroom types studied, Amanita sp. and C. miomboensis had the most number of extracts exhibiting high anti-RT activity. Data expressed as mean ± SD; N = 2

4.5.2 Cytotoxicity Assay of Mushroom Extracts

Eight out of the fifty mushroom extracts (CME1 to CME50) had CC50 values below 5 µg/ml, while thirty six of the extracts exhibited CC50 values that were above 20 µg/ml (Figure 4.14 and Table 4.16). Three out of the ten acetone extracts showed high toxic effects to the

PBMCs, with values ranging from 0.0004 – 0.2 µg/ml. The ethanol extracts had the highest non toxic effects to the PBMCs with the values ranging from 59.86 µg/ml to over 10 000

µg/ml, followed by the hot water and cold water extracts. Species A. zambiana and T. strumosa had all the five extracts exhibiting low or no toxic effect to the cells, while C.

miomboensis, C. symoensii, G. lucidum and L. kabansus had at least four of the five extracts showing very low or no toxic effects to the PBMCs.

Figure 4.14: Representative curve used to derive the CC50 values of the different mushroom extracts. The graph represents the curve for the ethanol extract from Coprinus species. Results are shown as mean ± SD (N = 3).

Table 4.16: The CC50 values of mushroom extracts showing varying levels of inhibitory effects to the growth of PBMCs

CC50 (µg/ml) of mushroom extracts

Mushroom Acetone Ethanol Methanol Cold water Hot water

Amanita sp. 0.0004 123.4 0.03 19.7 80.82

A. zambiana 111.6 >10 000 >1 000 890.4 784.7

B. edulis 6.48 718.5 >10 000 >10 000 16.8

C. heinemannianus 0.006 NC 2 591 126.1 >10 000

C. miomboensis >1 000 149.8 46.84 4.07 208.6

C. symoensii 142.8 59.86 5.81 547.1 494.7

Coprinus sp. 7.1 1 184 0.11 0.27 >10 000

G. lucidum 0.2 >10 000 138.5 >10 000 1 740

L. kabansus 331.2 224.2 2 612 167.3 0.3

T. strumosa 153.1 3 444 1 009 57.14 2 425

The ethanol extracts had the highest non toxic effects to the PBMCs with the values ranging from 59.86 µg/ml to over 10 000 µg/ml, followed by the hot water and cold water extracts. Species A. zambiana and T. strumosa had all the five extracts exhibiting low or no toxic effect to the cells. NC – data did not converge.

4.5.3 Determination of the in vitro Anti-HIV-1 Activity using HIV-1 p24 Antigen

Expression Assay

Twenty extracts of the CME1 to CME50 set, with CC50 values ranging from 80.82 to over 10

000 µg/ml were selected for the anti-HIV-1 activity on PBMCs. The percentage inhibition of

HIV-1c infection by the selected mushroom crude extracts ranged from 16.8 % to 97.8 % as shown in Table 4.17. Two of the twenty extracts, namely, the boiled water extracts from

Coprinus species and C. heinemannianus, exhibited high inhibition of HIV-1c infection (92.8

± 1.7% and 97.8 % ± 0, respectively), with IC50 values of 24.3 µg/ml and 33.8 µg/ml, respectively and no cellular cytotoxicity to the cells (Table 4.18).

Table 4.17: Inhibitory effects of different mushroom extracts on HIV-1 replication Mushroom Extracting solvent HIV-1 p24 inhibition (%) 50 µg/ml 100 µg/ml 150 µg/ml C. miomboensis Ethanol 42.8 ± 2.4 41.1 ± 0.0 52.3 ± 0.9 B. edulis Ethanol 38.5 ± 3.7 38.9 ± 1.5 32.3 ± 10.9 T. strumosa Ethanol 24.7 ± 2.4 45.8 ± 8.3 46.8 ± 1.7 C. heinemannianus Methanol 16.8 ± 6.4 29.2 ± 5.2 32.5 ± 6.1 L. kabansus Methanol 49.3 ± 5.2 54.5 ± 22.0 40.3 ± 9.2 T. strumosa Methanol 17.1 ± 0.5 33.9 ± 2.8 40.4 ± 10.7 G. lucidum Methanol 71.7 ± 0 67.0 ± 0 73.3 ± 0 B. edulis Methanol 52.4 ± 6.3 48.0 ± 3.4 59.0 ± 1.0 L. kabansus Cold water 39.5 ± 20.9 59.8 ± 16.8 59.1 ± 6.9 C. symoensii Cold water 45.6 ± 0 65.5 ± 0 59.0 ± 2.9 C. heinemannianus Cold water 44.0 ± 5.6 48.8 ± 7.7 39.6 ± 5.3 B. edulis Cold water 48.7 ± 6.0 51.8 ± 7.2 34.8 ± 1.4 G. lucidum Cold water 68.5 ± 0 60.1 ± 18.8 54.9 ± 2.8 Amanita species Hot water 43.4 ± 3.2 - 39.5 ± 12.0 T. strumosa Hot water 49.6 ± 22.4 49.9 ± 19.9 38.7 ± 9.7 A. zambiana Hot water 67.9 ± 7.7 37.6 ± 14.6 68.7 ± 24.1 C. symoensii Hot water 52.2 ± 12.6 50.5 ± 5.3 59.5 ± 19.6 C. heinemannianus Hot water* 65.6 ± 0.2 54.3 ± 1.9 97.8 ± 0 G. lucidum Hot water 54.2 ± 0 60.1 ± 28.4 73.3 ± 0 Coprinus species Hot water* 75.8 ± 16.7 92.6 ± 1.4 92.8 ± 1.7 Azidothymidine (20 µg/ml) 85.4 ± 2.6 (-) results not converging. Results are shown as mean ± SD (n = 2). The absorbance values obtained from the negative control were used to calculated the percentage inhibition for each extract. Rows highlighted in bold indicated extracts that exhibited high inhibitory effects of above 90 %. Samples marked with asterisk* were selected for analysis of compound profile by LC – MS.

Table 4.18: Cytotoxicity and anti-HIV-1 activity of the hot water extracts of Coprinus species and C. heinemannianus using HIV-1c (MJ4) in PBMC cells

Mushroom Extracting solvent *CC50 (µg/ml) *IC50 (µg/ml) *TI Coprinus species Hot water >10 000 24.3 ± 6.6 >400 C. heinemannianus Hot water >10 000 33.8 ± 0.7 >290 *CC50: cytotoxic concentration of the mushroom extracts that caused the reduction of viable cells by 50%. Data presented as means ± SD, each time in triplicate. *IC50: concentration of the mushroom extracts that resulted in 50% inhibition in HIV-1 infection. Data presented as means ± SD, each time in duplicate. *TI: therapeutic index = CC50/IC50.

The cold and boiled water extracts had the highest number of extracts exhibiting moderate (50

– 70%) to high inhibitory activity on HIV-1 infection. Both the hot water extracts of Coprinus species and C. heinemannianus exhibited a dose-dependent inhibitory activity (Figure 4.15).

150 Ch - HW Co - HW

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% inhibition%

0 0 50 100 150 200 Concentration (µg/ml)

Figure 4.15: Anti-HIV activity of the hot water extracts of Coprinus sp. and C. heinemannianus, showing a dose-dependent inhibitory activity. Results are shown as mean ± SD (n = 2). Ch-HW = hot water extract of C. heinemannianus, Co-HW = hot water extract of Coprinus species

4.5.4 Analysis of the Anti-HIV Mushroom Crude Extracts by Non-targeted LC – MS

Three of the mushroom crude extracts that exhibited high percentage inhibition against HIV-1

RT (89.9 - 92.6 %) and two extracts that had high percentage inhibition against HIV-1 replication from the HIV-1 p24 ELISA assay, were further analysed using an Agilent HPLC

1260 System coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF)

Mass Spectrometer. Analysis of the crude extracts revealed the presence of a variety of secondary metabolites which included alkaloids, penicillins, carboxylic acid esters, fatty acids and fatty acid derivatives, diterpenes, diterpenoid alkaloids, amino acid derivatives, sesqueterpenoids, terpene derivatives, and phospholidids.

CHAPTER FIVE:

DISCUSSION

5.1 CHARACTERIZATION OF MUSHROOM FRUITING BODIES

5.1.1 Quantitative Determination of the Total Protein Content

Nutritionally, edible mushrooms are highly valued due to high protein content and other nutrients than most of the plants. Some investigators have equated the amino acid composition of mushrooms to that of animal proteins (Boda et al., 2012; Reis et al., 2011).

The results obtained confirm that the edible mushrooms used in this study are good sources of protein. Overall, the edible mushrooms such as B. edulis, L. kabansus and A. zambiana had high levels of protein content reaching 30.8 % ± 1.3 when compared to non-edible mushrooms such as G. lucidum (9.3 % ± 1.0) and T. strumosa (10.0 % ± 1.1) as shown in

Figure 4.2. Proteins are the workhorses of the cell that carry out all the key functions such as digesting food, building tissue, transporting oxygen in the blood to the body tissues, dividing cells and powering muscles. A good protein source can prevent the occurrence of protein deficiency diseases such as kwashiorkor and marasmus. Malnutrition, with its constituents of protein and micronutrient deficiencies, is a major health burden in developing countries. It is the most important risk factor for illness and death globally, affecting hundreds of millions of pregnant women and young children (Henchion et al., 2017; Luchuo et al., 2013; Schonfeldt et al., 2013). In Zimbabwe, around 650,000 children under 5 years (27 %) suffer from chronic malnutrition (stunting) (USAID, 2018). Thus, local edible mushrooms such as A. zambiana,

B. edulis, Cantharellus species and L. kabansus can be used in the development of mushroom based protein products that are able to minimize protein-energy malnutitrion, particularly stunting in children. These mushrooms can be used to enrich human diets, especially in developing countries where animal protein may not be readily available and out of the reach of most household budgets (Teklit, 2015).

Mushrooms have been reported to contain protein levels ranging from 15 % to 35 % dry weight (Masamba and Kazombo-Mwale, 2010). The values obtained from this study ranged from 9.3 % ± 1.0 to 30.8 % ± 1.3 dry weight, which compare favourably well with the general protein content of mushroom species reported in previous studies (Masamba and Kazombo-

Mwale, 2010). In addition, the three Cantharellus species (C. heinemannianus, C. miomboensis and C. symoensii), had different levels of protein content (21.1 % ± 2.4, 16.8 %

± 0.3 and 30.7% ± 4.8, respectively). Similar studies where mushrooms of the same genus gave different protein content values on a dry weight basis have been reported (Masamba and

Kazombo-Mwale, 2010).

5.1.2 Qualitative Determination of Carbohydrates

Carbohydrates are a major constituent of mushroom dry matter (usually about 50 - 60 %) comprising of various compounds; monosaccharides and their derivatives, oligosaccharides

(commonly called sugars) and polysaccharides (Kalac, 2012). The results from both the

Molisch’s and the Benedict’s tests showed that most of the mushrooms contained varying levels of carbohydrates, based on the different intensities of the positive reactions (Table 4.2).

Species Amanita, C. symoensii, L. kabansus and T. strumosa showed high levels of carbohydrates while, C. heinemannianus and C. miomboensis showed high levels of reducing sugars. High levels of both carbohydrates and reducing sugars were exhibited by A. zambiana when compared to the other mushrooms studied. To a certain extent, the aforementioned mushroom species can in addition to providing protein nutrition (section 5.1.1), augment the energy providing portions of household diets to result in a greater chance of balanced diets in

Zimbabwean communities and lower tier developing countries. Several reports have also highlighted the variation in the carbohydrate content in different species of mushrooms (Boda et al., 2012; Cheung, 2008).

5.1.3 Determination of Lectins

Mushrooms produce many kinds of proteins with important biological activities, including lectins and ribonucleases. In this study, the lectin activity of the ten different wild mushrooms, using two types of red blood cells, namely sheep and goat, were studied. The results showed that three of the ten mushrooms, namely Amanita sp., B. edulis and L. kabansus contained varying levels of lectin content with B. edulis having the highest specific activity of 617 HAU/ mg mushroom, and L. kabansus having the lowest activity of 5 HAU/ mg mushroom (Table 4.4). In other related studies, high content of lectins in mushrooms has also been detected in B. edulis, Lactarius and Amanita sp. (Dhamodharan and Mirunalini,

2011; Singh and Bhari, 2014). Lectins obtained from mushrooms are reportedly able to bind to abnormal and cancer cells and label these cells for destruction by the human body’s immune system (Hassan et al., 2015). Thus, the mushrooms under study, particularly B. edulis and L. kabansus, could be an excellent source of cancer-fighting macromolecules and can be used in the human diet to promote health. Extracts from the mushrooms can be obtained and commercialized as dietary supplements for their ability to enhance immune function and antitumor activity.

5.2 CHARACTERIZATION OF MUSHROOM CRUDE EXTRACTS

5.2.1 Determination of Protein Content of Crude Extracts

Results from the protein analysis in Figure 4.5 showed high yields of protein from the cold and boiled water extracts. The high protein yields observed in the boiled water extracts could be due to increase in protein availability during boiling as a result of hydrolysis of insoluble protein to soluble protein. Protein yields from methanol extracts followed the aqueous extracts while ethanol and acetone gave similar low yields. The mushroom species Amanita gave significantly high yields of protein in the cold and boiled water extracts, while B. edulis

had high yields in the cold and boiled water extracts, as well as in all the methanol extracts.

Thus, both cold and boiled water would be the solvents of choice in the development of mushroom protein extracts that could be used as healthy dietary supplements. The results also indicate that boiling mushrooms is the method of choice for cooking the edible mushrooms under study, due to the enhanced availability of the protein content. The yields of protein obtained correlated with the results of the total protein content by the Kjeldhal method

(section 4.2.1), particluary for the B. edulis mushroom which gave the highest yield and T. strumosa which gave the lowest yield in both the assays. It has been reported that the amount of protein varies from species to species in the same genus as observed in the two different species of the genus Cantharellus (C. cibarius, 1.057 mg/ml of protein and C. subcibarius,

1.567 mg/ml of protein) (Boda et al., 2012). Similarly, the three different species of the genus

Cantharellus gave different protein yields (Figure 4.5).

5.2.2 Determination of Total Phenolic Content of Crude Extracts

Phenolic compounds are crucial due to their free radical scavenging activity, hence, they protect the human body from cell damage by reactive oxygen species (Gan et al., 2013). Total phenolic compounds have been reported as the major naturally occurring antioxidant compounds in the wild edible mushrooms (Wang and Xu, 2014). In this study, crude mushroom extracts were prepared from the mushrooms using cold water, boiling water, methanol, ethanol and acetone, and total phenolic compounds were determined. High levels of total phenolics were observed in both the cold and boiled water extracts (Figure 4.6), when compred to the organic extracts. Similar trends, where cold water extracts had high total phenolic yields followed by hot water extracts, while acetone extracts gave the least yields, were observed by Wang and Xu (2014). The high values in water extracts could be explained by the high polarity of water compared to the other organic solvents, hence, more polar

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compounds dissolving in water. From the ten different mushroom types studied, B. edulis had the highest total phenolic compounds followed by Amanita species. A diet rich in antioxidants protects cells from free radicals, helping the body cope with the normal oxidative stress that damages healthy cells. The ability of phenolic compounds to protect against several degenerative disorders, including brain dysfunction, cancer and cardiovascular diseases, is related to their capacity to act as antioxidants (Zhang et al., 2015). Thus, local edible mushrooms such as B. edulis can be used as healthy foods for the prevention of a range of illnesses including cancer, diabetes and arteriosclerosis. Powder formulations of these mushrooms could be prepared and used in diets as antioxidants.

5.2.3 Determination of Antibacterial Activity of Crude Extracts

The results of the antibacterial activities of methanol, ethanol, acetone, cold and hot water extracts from ten different mushrooms used in this study highlighted that all the mushrooms possess inhibitory activities against at least one of the bacteria tested. This was shown on clear zones of inhibition around the tested mushroom extracts. It has been reported that mushroom species possess different constituents and in different concentrations which account for their differential antimicrobial activity (Akyuz et al., 2010; Padmavathy et al.,

2014). In this study, the highest in vitro antibacterial activity was shown by the cold water extract of C. miomboensis against S. typhi (14 mm zone of inhibition). This was followed by the acetone extract of C. symoensii, the methanol extract from C. miomboensis and the ethanol extracts of G. lucidum and C. symoensii (section 4.3.3). Extracts of these species, using the identified optimal solvents for extraction, can potentially be used for antibacterial medicinal purposes similar to antibiotics. In a separate study, the edible mushroom species C. cibarius showed antimicrobial activity against some Gram-positive and Gram-negative bacteria (Rahi and Malik, 2016).

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Species C. miomboensis, Amanita sp. and B. edulis all had the highest number of total extracts inhibiting at least one of the bacteria tested (section 4.3.3). Species C. heinemannianus and C. symoensii had the highest effect on inhibition of bacteria as indicated by having the most extracts which had high inhibitory properties ranging from 8-14 mm [15.4 % (8) each], followed by G. lucidum [13.5% (7)]. This shows that C. heinemannianus, C. symoensii and G. lucidum extracts contain compounds that have more inhibitory potential against the bacteria studied than the rest of the mushroom extracts used in this study. In similar studies carried out by Quereshi et al. (2010), methanol, ethanol, acetone and cold water extracts of G. lucidum from India showed antimicrobial activity against the S. aureus, S. typhi and E. coli bacterial culture collections. From this study, the methanol extracts showed no inhibition to S. aureus and E. coli, while the ethanol and acetone extracts inhibited growth of both E. coli and S. typhi but did not inhibit growth of S. aureus. Ethanol extracts of G. lucidum from Turkey inhibited growth of E. coli while the methanol extract showed no inhibition (Celik et al.,

2014). In another study, acetone and ethanol extracts of C. cibarius collected in Turkey, exhibited antibacterial activity against E. coli and S. aureus but showed no inhibition against

S. typhi (Dulger et al., 2004). Results of another study in Nigeria showed that methanol and ethanol extracts of C. cibarius inhibited E. coli and S. typhi growth but showed no inhibition against S. aureus and S. pneumoniae (Aina et al., 2012). Similarly, the research findings in this study showed that methanol, ethanol and acetone extracts of the three Cantharellus species studied exhibited various degrees of inhibition against the four bacteria tested. This shows that different species of mushrooms exhibit different antimicrobial activity due to a number of factors such as the presence of different antimicrobial components, type of the extracting medium, geographical location of the mushroom and the type of organism being tested.

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Extracts obtained from ethanol gave the highest number of bacterial growth inhibition (33), followed by acetone (31) and methanol (28). In addition, ethanolic extracts showed the strongest antibacterial activity (8 - 14 mm) among the five extracts against the bacterial strains, followed by methanol and acetone. Water extracts exhibited the lowest number of antibacterial activity, despite having the extract with the highest inhibitory effect. This indicates that the active compounds from the mushrooms studied, which inhibit the growth of susceptible bacteria, may dissolve better in the organic solvents than in aqueous solvents.

These results are consistent with already reported literature that extracts from organic solvents give more consistent antimicrobial activity than water extracts (Kamra and Bhatt, 2012;

Tiwari et al., 2011).

It is interesting to note that, although cold and hot water extractions gave highest values of total phenolic compounds (Figure 4.6), these had the least effect on most bacteria. This shows that the antibacterial activity in the mushroom extracts depends not only on the presence of phenolic compounds, but also on the presence of various other secondary metabolites.

Ethanol, acetone and methanol extracts were all effective against all the four bacteria tested, indicating the broad spectrum of antibacterial activity of the crude extracts. However, Gram- negative bacteria were slightly more susceptible to the extracts than Gram-positive bacteria

(52 and 46 extracts, respectively). Several different classes of antibiotics block steps in the synthesis of peptidoglycan, resulting in cells being more susceptible to osmotic lysis.

Although all bacteria have an inner cell wall, Gram-negative bacteria have a unique outer membrane which prevents certain drugs and antibiotics from penetrating the cell. Thus, antibiotics that affect the cell wall will impair Gram-positive bacteria and not Gram-negative bacteria. The results obtained in this study suggest that the antibacterial extracts obtained may

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act by affecting not just the cell wall, but other cell growth mechanisms like protein synthesis, bacterial DNA replication and transcription.

Among the four bacteria tested, S. typhi was the most susceptible bacteria as shown by its highest number of inhibitions as well as the highest number of most potent extracts in the 8 -

14 mm diameter range. A decline in the number of multidrug resistant clinical isolates (S. typhi) has been reported (Madhulika et al., 2004). Thus, this study shows that the S. typhi isolate studied, may be a phage type that is susceptible to most antibiotics. The antibacterial activity of the ethanolic, methanolic and acetone extracts against E. coli, S. typhi, S. aureus and S. pneumoniae is of great importance in the human healthcare system. Streptococcus pneumoniae is the most common cause of community acquired pneumoniae (CAP) in children while E. coli accounts for more than 70 % of the infections of the urinary tract worldwide (Blossom et al., 2006; Sangeeth et al., 2014). Species S. typhi is the cause of typhoid fever, which has been an epidemic in Zimbabwe. The S. aureus is the most common cause of bacterial infections and abscesses of skin, joints and bones (Stanely et al., 2013).

Resistance to antibiotics has been reported in S. aureus, S. pneumoniae, S. typhi and E. coli

(Blossom et al., 2006; Okonko et al., 2009; Rowe et al., 1997; Sangeeth et al., 2014; Stanely et al., 2013). The bacterial strains used were clinical isolates. Species E. coli and S. aureus are mostly encountered in urinary tract infections while isolated cases of S. typhi are common.

Thus, the antibacterial activity found in the mushroom extracts can be further investigated for future use in the development of therapeutic agents to treat infections caused by these bacteria. The species with the highest antibacterial activity have potential application in clinical situations where patients are not responding to orthodox antibiotic regimes.

Application of a cocktail of the identified antibacterial extracts may potentially succeed in minimising occurrence of multi-drug resistant strains of bacterial pathogens [such as the

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extended-spectrum β-lactamase-producing and gentamicin resistant Enterobacteriaceae

(Magwenzi et al., 2017) and the methicillin-resistant Staphylococcus aureus (Nikaido, 2009)] that arise through selective pressures of the continual antibiotic environment. Thus, bacteria that would normally develop antibiotic resistance mutations in the single antibiotic environment, would face lethality from another mushroom extract component in the mushroom extract cocktail.

5.2.4 Characterization of Mushroom Extracts Showing High Antibacterial Activity

During mushroom growth, a variety of primary and secondary metabolites can be accumulated as intracellular and extracellular products, including phenolics, polyketides, terpenoids, steroids, antibacterial or antifungal proteins, and fatty acids (Shen et al., 2017).

Different mushroom species usually have characteristic metabolite profiles, although they may show similar antimicrobial activities. Many phenolic compounds, especially the low- molecular weight phenolic compounds, have been identified in various mushrooms, and their antimicrobial properties have been demonstrated (Shen et al., 2017). Analysis of the mushroom crude extracts using absorption spectroscopy confirmed that each extract had a unique secondary metabolite profile.

5.2.4.1 Analysis of the extracts using absorption spectroscopy and TLC

The UV spectra of mushroom extracts showed absorbance peaks ranging from 227 – 308 nm;

734 – 745 nm and 586 nm (Table 4.11). The absorbance peaks from the same extraction solvent type varied with each mushroom type. Although the spectrum of the mushrooms extracted with the same solvent showed some similarities, changes could be seen in the peak heights at similar wavelengths for the different extracts. This may be a result of different compounds being present in each of the extracts. Each mushroom type also exhibited different

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absorption peaks from the different solvents. Some chemical components appeared to be very similar among the extracts. For example, the ethanol and acetone extracts of Amanita species were characterised by strong absorption at the wavelength of 259, while the acetone extract of

T. strumosa and the cold water extract of C. miomboensis had a strong absorption at a wavelength of 228 nm. However, each crude extract had unique characteristic absorption peaks to reveal its fingerprint feature. The results indicate that the crude extracts obtained contain a variety of compounds depending on the type of extracting solvent and the type of mushroom. Absorption spectrum results also indicate that there may be high levels of phenolic compounds in most of the extracts as these compounds can exhibit maximum absorption at wavelengths of 220 - 310 nm (Carvalho et al., 2015). Analysis of the extracts by TLC and staining with vanillin sulphuric acid further confirmed the unique profile of each extract. These generated profiles can also aid in the identification of unknown mushroom tissues in conjunction with other morphological and genetic techniques to assist in forensic cases. This can further be used in distinguishing between almost identical poisonous and non- poisonous mushroom species resulting from environmental phenotypic plasticity and determining the effect of the environment on the genome.

5.2.4.2 Separation of mushroom compounds by TLC and Isolation of components of mushroom extracts by PTLC

The different retention factors (Rf) indicated the presence of a variety of compounds in each extract ranging from 6 – 11 compounds that could be visualized, depending on the type of extracting medium and the mushroom (section 4.4.2). The acetone extracts had the highest number of compounds separated, with an average of 9 bands per sample for the TEM mobile phase, indicating that acetone extracted more compounds when compared to the other solvents. It was noted that PTLC yielded more bands than the ones obtained during analytical

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TLC using the same mobile phases. This difference may be due to the quantity of the samples spotted on the plate, being too low to be detected during the analytical TLC while these became more visible when larger amounts of samples were applied.

5.2.4.3 Determination of Antibacterial Activity of the Isolated Compounds

The isolated components (CP1 – CP131) of the mushroom crude extracts exhibited varying degrees of inhibition of bacterial growth ranging from 0 to 99.8 % (section 4.4.4). This suggests that the bioactive products which are contained in mushrooms are in different concentrations which exhibit varying degrees of antimicrobial activity. This result is in agreement with the findings of Chu (2013) where fractions obtained from different mushrooms were reported to show varying inhibitory effects on growth of the microorganism.

Thirteen of the isolated components exhibited very high percentage inhibition activity against growth of S. typhi ranging from 86.6 to 99.8 %, while one of the components (CP50) exhibited high inhibitory activity against growth of S. aureus (87.5 %), as shown in Table

4.14. The compound CP70 was the most potent isolate as it exhibited the lowest IC50 value of

206 µg/ml, followed by the compounds CP94 and CP44, with IC50 values of 223 µg/ml and

245 µg/ml, respectively.

Some of the mushroom crude extracts that had shown high potency against the bacteria under study did not yield any components that had high antibacterial activity when tested against the same bacteria. A number of reasons for this lack of potency may be possible, one of which may be that the compounds responsible for the actibacterial activity may have been acting in synergy, hence separating the compounds reduced their potency. Karmegam et al. (2012) reported that medicinal plant extracts used in combination exhibited higher antibacterial activity against E. coli and Bacillus cereus than the individual extracts. The other reason may

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be that the concentrations of the compounds of interest were too low to exert antibacterial activity.

5.2.4.4 Analysis of Antibacterial Components of Mushroom Extracts by LC-MS

Analysis of seven of the most potent isolated mushroom components using LC-MS revealed the presence of a number of terpenoid derivatives namely; lucidenic acid M, cavipetin D, notoginsenoside R2, boviquinone 4, 26-methyl nigranoate and goshonoside (Table 4.15). The compound lucidenic acid M (Figure 5.1) was identified in 3 of the isolates obtained from the acetone extracts of C. symoensii, L. kabansus and T. strumosa. The results indicate that the acetone organic solvent extracted the same compound in the three different mushrooms, which was not present in the ethanol extract of the same mushroom, C. symoensii (CP44).

Thus, the results are in agreement with the reported findings that the bioactive compounds isolated vary depending on the type of solvent used for extraction and the mushroom type

(Shen et al., 2017). Lucidenic acid M, which is also known as lucidenate M, is a member of a class of organic compounds known as triterpenoids (Chen et al., 2017). Mushroom terpenoids are a large group of secondary metabolites belonging to terpenes with different functional groups, which have been identified in various mushroom genera/species, including sesquiterpenoids from Lentinus conatus, Lactarius sp. and Flammulina velutipes, and triterpenoids from Ganoderma sinense and Ganoderma pfeifferi (Shen et al., 2017). The

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Figure 5.1: Structure of Lucidenic acid M

diverse biological activity of lucidenic acid, including its antibacterial activity has been reported (Basnet et al., 2017; Chen et al., 2017; Hsu and Yen, 2014).

Notoginsenoside R2 and 26-methyl nigranoate are also both members of the triterpenoid class of compounds (Lee et al., 2010; Sun et al., 1996), which were found in the isolates from the acetone extracts of L. kabansus and T. strumosa, respectively. Cavipetin D (C25H38O5) and boviquinone 4 are both members of compounds known as diterpenoids. Cavipetin D (Figure

5.2) is a constituent of edible mushrooms and has been reported to exhibit antibacterial activity (Shen et al., 2009). In this study, cavipetin D was identified in the component isolated from the acetone crude extract of L. kabansus, an edible mushroom, confirming reports that the compound is found in edible mushrooms. Boviquinone 4 has also been reported in mushrooms (Velisek and Cejpek, 2011). Goshonoside, identified in the acetone extract of L. kabansus, is a diterpenoid glycoside.

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Figure 5.2: Structure of Cavipetin D

Another class of compounds that were identified in the isolates were the fatty acid derivatives namely, palmitic amide, stearamide, sorbitan oleate, 11-amino-undecanoic acid (a fatty acid amine) and Z-13-oxo-9-octadecenoic acid. Palmitic amide was common in two of the isolates:

CP44 and CP94, while stearamide was found present in CP44. Palmitic amide (Figure 5.3) and stearamide are both primary fatty acid amides coming from palmitic acid and stearic acid, respectively. Primary fatty acid amides (R-CO-NH2) are a class of compounds that have only recently been isolated and characterized from biological sources. Stearamide is often used in the synthesis of organic chemicals and surfactants and is reported to have antibacterial activity

(Ahmed et al., 2017). Sorbitan oleate belongs to the class of organic compounds known as fatty acid esters and was isolated from the acetone extract of C. heinemannianus. The compound is an emulsifier and clarification agent in food preparations (Grant et al., 2006).

The compound Z-13-oxo-9-octadecenoic acid was isolated from the ethanol extract of G. lucidum and its antimicrobial activity has been reported (Idan et al., 2015; Prost et al., 2005).

Figure 5.3: Structure of Palmitic amide

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The compound C16 sphinganine, which was identified in the acetone extract of C. symoensii, is a sphingolipid metabolite derived from the same group of compounds as phytosphingosine.

Phytosphingosine (Figure 5.4) is a phospholipid and sphingolipid metabolites such as sphingosine and ceramide are highly bioactive compounds that are involved in diverse cell processes, such as cell-cell interaction, cell proliferation, differentiation, and apoptosis. The compound is one of the most widely distributed natural sphingoid bases, which is abundant in fungi and plants and is known to inhibit the growth of both Gram-positive and Gram-negative bacterial strains (Fischer et al., 2012).

Figure 5.4: Structure of Phytosphingosine

Analysis of the isolates further indicated that the majority of the components isolated from the mushroom crude extracts by preparative TLC contained more than one compound (Table

4.15). This may mean that the compounds present in the fractions have similar polarities, hence the mobile phase used and the time for the PTLC run may not have been enough to separate the different compounds. However, each mushroom component exhibited a unique profile of compounds, although some similar compounds were found in more than one fraction. The results thus show that a variety of secondary metabolites, that is, tepernoids, phospholipids and fatty acids isolated from the selected mushrooms are part of the key compounds responsible for the antibacterial activity observed. These compounds may be acting in synergy or as individual compounds. 111

5.2.5 Determination of the Anti-HIV Activity of Mushroom Crude Extracts

5.2.5.1 Determination of anti-HIV-1 Reverse Transcriptase Activity

Although anti-HIV-1 treatment has achieved a lot of success by suppressing viral replication to undetectable levels and has improved the quality of life of HIV-1 infected individuals, complete, long-term suppression of HIV-1 replication is still a major challenge due to the rapid emergence of drug resistance. Hence, continuous search for new anti-HIV-1 agents and novel targets is still an urgent priority as part of a global strategy to combat the spread of

HIV-1 infection. The HIV-1 encodes three enzymatic proteins, reverse transcriptase (RT), integrase, and protease, which are critical for its replication. The RT is critical during the early steps of the viral replication cycle since the enzyme is necessary for reverse transcription of the viral genome (Dahake et al., 2013; Jadaun et al., 2016; Leteane et al.,

2012; Orozco et al., 2016; Tietjen et al., 2016; Zhiming et al., 2016;). In this study, fifty aqueous and organic crude extracts from the ten mushrooms selected were screened for inhibitory properties against HIV-1 reverse transcriptase (RT). The cold water extracts of the mushrooms L. kabansus, Amanita species, B. edulis, T. strumosa, A. zambiana and C. heinemannianus, demonstrated high inhibition ratios of 92.6%, 91.3 %, 89,9 %, 89.6 %, 88 % and 86.2, respectively (Figure 4.13). The high levels of RT inhibition by the cold water extracts, indicate that the active compounds from the mushrooms studied, that inhibit the

HIV-1 RT activity, dissolve better in the aqueous solvents than in organic solvents. From the ten different mushroom types studied, Amanita species and C. miomboensis had the most number of extracts exhibiting high anti-RT activity.

Of note is the observation that cold water extracts gave the highest values of total phenolic compounds in the total phenol assay (section 4.3.2). Thus, the high levels of RT inhibitory activity observed in this study may indicate a correlation between total phenolic compounds

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and anti-HIV-1 RT activity. Hot water extracts of A. zambiana, B. edulis, C. miomboensis and

Coprinus sp. had inhibitory values that were above 50 %. Similar trends were reported elsewhere, where hot water extracts of different edible and medicinal mushroom species screened for HIV-1 RT inhibitory activity exhibited over 50 % inhibition (Wang et al., 2007).

The results obtained from this assay showed that most of the mushrooms selected for this study are potential candidates for development of anti-HIV therapeutic agents. The advantages of developing HIV-1 RT inhibitors based on mushrooms are the availablility in large quantities of the basidiomes, and the amenability of the majority of the species to large- scale fermentation to yield mycelia. Additionally, natural products extracted from mushrooms, especially edible medicinal fungi exhibit lower toxicity and fewer side effects than chemical drugs (Orozco et al., 2016).

5.2.5.2 Cytotoxicity Assay of Mushroom Extracts

The results obtained from the general cytotoxicity assay of the fifty mushroom extracts showed that the mushrooms under study generally had low toxic effects or were not toxic to the cells. However, the type of solvent may have an effect on the level of toxicity of some of the extracts. In this study, the ethanol extracts had the highest non toxic effects to the PBMCs with CC50 values ranging from 59.86 µg/ml to over 10 000 µg/ml, followed by the hot and cold water extracts.

5.2.5.3 Determination of the in vitro Anti-HIV-1 Activity using p24 Antigen Expression

Assay

Twenty mushroom crude extracts with CC50 values ranging from 80.82 µg/ml to over 10 000

µg/ml were evaluated for their effect on HIV-1 replication by measuring the levels of p24 antigen. The results obtained from the assay showed that some of the mushrooms under study

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contain bioactive compounds that have the potential to prevent entry of HIV-1 viral particles into the human cells. Four of the twenty extracts exhibited percentage inhibition of HIV-1c infection ranging from above 70%, while two of the extracts, namely, the boiled water extracts from Coprinus species and C. heinemannianus, exhibited percentage inhibition of

HIV-1c infection of over 90 % (Table 4.17) and section 4.5.3. Therapeutic indices (TI) of the hot water extracts of Coprinus species and C. heinemannianus were calculated as a ratio of

CC50 to IC50. A TI of >200 by both extracts strongly suggest that these mushroom types and their extracts are good candidates for further anti-HIV studies. The cold and boiled water extracts had the highest number of extracts exhibiting moderate to high inhibitory activity on

HIV-1 infection. The results tally with the observation made from the anti-HIV-1 RT assay

(section 4.5.1), that the active compounds from the mushrooms studied, which inhibit the

HIV-1 infection, dissolve better in the aqueous solvents than in organic solvents.

The extracts of Ganoderma mushroom expressed moderate levels of inhibition of HIV-1 infection (up to 73.3 %). From the studies conducted by Haoses-Gorases and Goraseb (2013) in Zambia, Tanzania and Namibia, results showed that the use of Ganoderma mushroom significantly improved the health status of HIV/AIDS patients. The findings show that the mushrooms studied, in particular, C. heinemannianus, Coprinus species and G. lucidum, contain compounds that may inhibit the early stages of HIV infection. These identified mushroom extracts may assist as alternative therapeutic interventions, as part of a holistic integrated therapeutic management of HIV, which includes other therapeutic interventions currently being employed.

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5.2.5.4 Analysis of the Anti-HIV Mushroom Crude Extracts by LC-MS

Three of the mushroom crude extracts that exhibited high percentage inhibiton against HIV-1

RT (section 4.5.1) and two extracts that had high percentage against HIV-1 replication from the HIV-1 p24 ELISA assay (Table 4.17), were further analysed using an Agilent HPLC 1260

System coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer. Analysis of the crude extracts revealed the presence of a variety of secondary metabolites which included alkaloids, penicillins, carboxylic acid esters, fatty acids and fatty acid derivatives, amino acid derivatives, diterpenes, diterpenoid alkaloids, sesqueterpenoids and other terpene derivatives, and phospholidids. The results obtained are in agreement with several reports of the anti-HIV activities of a variety of these secondary metabolites obtained from mushrooms (Adotey et al., 2011; De Silva et al., 2013; El Dine et al., 2008; Finimundy et al., 2014; Narayan and Rai, 2016; Rahi and Malik, 2016; Wang et al., 2007). For example, phytosphingosine and C16 sphinganine were both identified in the cold water extracts of B. edulis and the hot water extract of Coprinus species. Phytosphingosine was further identified in the cold water extract of L. kabansus. Both compounds whose anti-HIV activities have been reported, belong to the class of compounds known as phospholipids. The sphinganine- and phytosphingosine-based compounds have been reported to possess anti-HIV-1 activity by disrupting the early and late HIV-1 membrane fusion mechanisms (Ashkenazi et al., 2012;

Klug et al., 2014). Sesquiterpenoids and triterpenes, which were common in most of the crude extracts analysed, have reportedly exhibited anti-HIV-1 activity (El Dine et al., 2008; Wang et al., 2014).

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CHAPTER SIX:

CONCLUSION

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6.0 CONCLUSION

In this study, the nutritional and phytochemical composition of ten wild edible and non-edible mushroom species found in Zimbabwe, were analysed. The antibacterial and anti-HIV potential of the bioactive constituents of the selected mushrooms were evaluated. Although there are many studies on edible and wild mushroom species globally, the reports are based on species that are country - specific or are popular for consumption in certain geographical locations. Very little or no work, to the author’s best knowledge, has been carried out in

Zimbabwe to characterize and compare the nutritive value and bioactive composition among local wild edible and non-edible mushrooms. Only few disparate information related to the ten mushroom species used in this study can be found. In addition, the role of a large variety of mushrooms in human health is still a largely unexplored area of research. Characterization of mushrooms provides information on the biological properties of mushrooms occurring in

Zimbabwe and potentially provides a stepping stone towards the development of new natural infection - fighting strategies to control viral and microbial infections.

The mushrooms used in this study, especially edible ones, contained considerable amounts of proteins and carbohydrates which are vital in supplementing nutrition to humans. The nutritive contents with respect to protein and carbohydrates varied amomg the ten mushroom species. The mushrooms studied exhibited high levels of total phenolic compounds, which play a key role in the antioxidant activity of the mushrooms, potentially contributing to cancer prevention strategies. Some of the mushrooms studied also contained lectins with varying levels of specific activity. Thus, local wild mushrooms are a valuable source of lectins for further research into drug development. Furthermore, the mushrooms can be developed into functional foods or medicines for prevention and treatment of chronic diseases. From this study, it can be concluded that B. edulis is the mushroom of choice for augumenting a healthy

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and balanced diet due to the high levels of proteins, lectins and total phenolic content as well as moderate levels of carbohydrates exhibited by the mushroom.

Extracts of natural materials are a good source for the characterization and development of compounds with various biological activities. By screening extracts of natural sources such as mushrooms, it is possible to discover a multitude of structurally diverse drugs and enzyme inhibitors. Based on the results obtained from the present study, it can be concluded that wild edible and non-edible mushrooms found in Zimbabwe are rich in resources that possess a multitude of biological activities. The mushrooms possess compounds than can be used as antibacterial agents against some common bacterial infections. The tepernoids, phospholipids and fatty acids were identified as some of the compounds responsible for the antibacterial activity of mushrooms. From the study, C. symoensii, C. miomboensis and L. kabansus are recommended as the mushrooms of choice for the development of antibacterial agents against

S. typhi due to the high number and effectiveness of the most potent crude extracts and isolated compounds that the mushrooms exhibited. Similarly, L kabansus and Amanita species are the mushrooms of choice for screening for antibacterial agents against

Streptococcus pneumoniae while T. strumosa can be further investigated for the antibacterial activity against S. aureus.

The replicative cycle of HIV comprises a number of steps that could be considered as targets for therapeutic intervention, hence any effective treatment of HIV- 1 infection should target as many aspects of viral life cycle as possible. From this study, it can be concluded that the local mushrooms have compounds that are able to inhibit the entry as well as the reverse transcription stages of the HIV replication cycle. Thus, these mushroom extracts may have potential for either prophylactic or therapeutic intervention in HIV infection. The anti-HIV

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activity of the mushroom extracts may be attributed to the secondary metabolites present in the mushrooms. From the anti-HIV-1 assays, it can be concluded that mushroom species L. kabansus, Amanita species and B. edulis are the mushrooms of choice for the development of anti-HIV agents that inhibit reverse transcriptase activity due to their high percentage inhibition. Similarly, C. heinemannianus and Coprinus species as well as G. lucidum, are the best candidates for development of anti-HIV agents that can block the entry stage of HIV.

This study confirmed the reports that the chemical profile of the extracts as well as the antibacterial and anti-HIV effects of mushrooms vary depending on the type of mushroom and the extracting solvent medium used. Of the five solvents tested, water was determined to be the solvent of choice for isolation of anti-HIV-1 compounds from the mushrooms studied, while ethanol and acetone were determined to be the solvents of choice for isolation of antibacterial compounds.

6.1 RECOMMENDATIONS

This study represents a novel starting point for future studies in which extracts of the selected mushrooms and more can be used in different fields, such as medicine and pharmaceuticals. It is, therefore, recommended that further purification, identification and determination of the mechanism of action of the antibacterial and anti-HIV compounds be carried out to assist in the optimization of the compounds activity. Isolation and identification of more bioactive compounds from the local mushrooms is necessary as this will contribute towards the development of new therapeutic agents against bacterial and HIV infections. It is also recommended that the compounds identified in this study namely, lucidenic acid M, cavipetin

D, notoginsenoside R2, boviquinone 4, 26-methyl nigranoate, goshonoside, C16 sphinganine, palmitic amide, stearamide, sorbitan oleate, 11-amino-undecanoic acid and Z-13-oxo-9-

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octadecenoic acid, be further purified and each compound’s antibacterial effect be tested, either individually or in combination with other known therapeutic agents to evaluate their potency as well as assess whether there is enhanced treatment and prevention of drug resistance. In addition the possibility of compounds acting in synergy needs to be explored.

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8.0 APPENDICES

8.1 PUBLICATONS ARISING FROM THE WORK PRESENTED IN THIS THESIS

8.1.1 Reid T, Kashangura C, Chidewe C, Benhura MA, Mduluza T. (2016). Antibacterial properties of wild edible and non-edible mushrooms found in Zimbabwe. African Journal of Microbiology Research, 10(26): 977-984.

8.1.2 Reid T, Munyanyi M, Mduluza T. (2017). Effect of cooking and preservation on nutritional and phytochemical composition of the mushroom Amanita zambiana. Food Science and Nutrition, 5(3): 538–544.

8.1.3 Reid T, Kashangura C, Chidewe C, Benhura MA, Stray-Pedersen B, Mduluza T. Characterization of anti-Salmonella typhi compounds from mushroom extracts in Zimbabwe. International Journal of Medicinal Mushrooms, Manuscript accepted for publication.

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8.2 REAGENTS

0.9% Saline azide

8.766 g NaCl, 2 g NaN3, 0.9895 g MnCl2 and 0.735 CaCl2

Fill to 1 L with distilled water.

Alsever solution

0.05 g glucose, 0.80 g Sodium citrate and 0.42 g NaCl

Fill to 100 ml with distilled water.

Vanillin-sulphuric reagent

1 g vanillin 100 ml ethanol 1.5 ml concentrated sulphuric acid .

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8.3 PERCENTAGE INHIBITION OF BACTERIAL GROWTH BY COMPONENTS ISOLATED FROM DIFFERENT MUSHROOM EXTRACTS USING PTLC

Table 8.3.1: Percentage inhibition of bacterial growth by components isolated from different mushroom extracts using PTLC Isolate Rf % inhibition Source of isolate Bacteria no. value 200 µg/ml 400 µg/ml 600 µg/ml 800 µg/ml tested CP1 0.10 1.0 ± 1.4 1.1 ± 1.6 34.7 ± 0 70.0 ± 0 CP2 0.17 0.0 ± 0 5.6 ± 0 64.9 ± 0 62.8 ± 0 CP3 0.21 6.0 ± 2.3 3.9 ± 1.4 2.4 ± 2.3 4.2 ± 4.8 CP4 0.31 3.0 ± 3.3 2.7 ± 3.4 0.1 ± 0.1 0.3 ± 0.4 Methanolic S. typhi CP5 0.35 0.2 ± 0.3 0.4 ± 0.5 0.0 ± 0 0.0 ± 0 extract of C. CP6 0.41 0.4 ± 0.5 0.0 ± 0 0.0 ± 0 0.0 ± 0 miomboensis CP7 0.70 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.6 ± 0.9 CP8 0.02 0.1 ± 0.2 0.5 ± 0.8 0.3 ± 0.2 45.1 ± 21.5 CP9 0.06 0.8 ± 1.2 0.0 ± 0 0.0 ± 0 5.0 ± 7.0 CP10 0.13 1.3 ± 1.8 1.0 ± 1.5 2.3 ± 1.9 0.3 ± 0 CP11 0.22 27.9 ± 15.4 41.5 ± 3.5 - - Methanolic S. typhi CP12 0.27 47.0 ± 6.9 35.6 ± 3.1 43.4 ± 10.1 - extract of C. CP13 0.47 3.9 ± 5.5 0.0 ± 0 2.7 ± 1.4 0.0 ± 0 symoensii CP14 0.79 0.0 ± 0 0.0 ± 0 0.0 ± 0 4.1 ± 5.7 CP15 0.95 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.00 ± 0 CP16 0.01 0.0 ± 0 6.9 ± 2.0 0.0 ± 0 2.4 ± 0.6 CP17 0.19 0.0 ± 0 8.9 ± 3.8 3.9 ± 0.04 17.5 ± 0.6 CP18 0.23 0.0 ± 0 11.6 ± 0.1 7.9 ± 0.03 25.0 ± 7.4 Methanolic S. CP19 0.49 7.5 ± 10.6 7.9 ± 11.2 11.6 ± 7.2 20.6 ± 0.7 extract of A. pneumoniae CP20 0.67 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 zambiana CP21 0.78 0.0 ± 0 2.1 ± 2.9 1.3 ± 1.9 13.4 ± 1.3 CP22 0.97 2.1 ± 2.9 0.0 ± 0 0.9 ± 1.2 4.4 ± 6.2 CP23 0.02 0.7 ± 1.0 0.0 ± 0 0.0 ± 0 0.0 ± 0 Acetone extract S. typhi CP24 0.14 0.70 ± 1.0 2.25 ± 3.2 0.00 ± 0 0.00 ± 0 of C. CP25 0.26 15.42 ± 5.7 41.30 ± 9.9 99.0 ± 0 98.7 ± 0 heinemmanianus CP26 0.55 7.1 ± 10.03 11.3 ± 16.0 82.1 ± 0 97.4 ± 0 CP27 0.63 10.8 ± 2.0 62.2 ± 0 88.4 ± 0 95.7 ± 1.8 CP28 0.71 8.8 ± 1.7 13.7 ± 8.7 33.7 ± 40.5 62.5 ± 8.4 CP29 0.74 5.2 ± 2.7 7.5 ± 0 0.0 ± 0 0.0 ± 0 CP30 0.98 10.3 ± 2.6 7.1 ± 2.3 0.00 ± 0 0.00 ± 0 CP31 0.01 6.7 ± 1.1 5.5 ± 6.3 29.6 ± 3.6 77.9 ± 0 Acetone extract S. typhi CP32 0.14 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 of A. zambiana CP33 0.19 4.1 ± 0.1 5.5 ± 4.5 5.51 ± 0.7 6.2 ± 2.7 CP34 0.23 13.0 ± 0.8 1 66 ± 2.3 - - CP35 0.25 5.07 ± 7.2 2.91 ± 4.1 - - CP36 0.47 5.6 ± 0.7 32.2 ± 7.9 - - CP37 0.59 36.0 ± 3.5 34.9 ± 1.1 - - CP38 0.74 4.1 ± 5.8 5.19 ± 5.1 - - CP39 0.09 6.2 ± 5.0 38.6 ± 9.1 69.7 ± 3.2 86.6 ± 1.3 Ethanolic extract S. typhi CP40 0.22 0.0 ± 0 0.0 ± 0 7.9 ± 3.7 25.9 ± 1.4 of C. symoensii CP41 0.29 0.0 ± 0 15.8 ± 20.5 0.0 ± 0 0.9 ± 0 CP42 0.33 0.0 ± 0 0.0 ± 0 38.0 ± 0 74.9 ± 0 CP43 0.37 8.2 ± 2.0 9.6 ± 1.7 5.6 ± 0.3 6.51 ± 0.3 CP44 0.50 33.0 ± 0 86.5 ± 5.2 92.7 ± 0 92.9 ± 0.1 CP45 0.56 5.9 ± 2.0 6.3 ± 1.6 7.3 ± 0.8 5.9 ± 1.8 139

CP46 0.78 17.8 ± 0.9 2.1 ± 0.7 0.00 ± 0 0.0 ± 0 CP47 0.97 14.6 ± 9.1 6.3 ± 8.9 63.9 ± 47.7 67.7 ± 38.0 CP48 0.11 21.2 ± 1.4 21.7 ± 0.8 22.9 ± 0.3 24.3 ± 0.7 Acetone extract S. aureus CP49 0.14 10.9 ± 0.1 17.3 ± 0.4 22.4 ± 0.3 24.8 ± 0.01 of T. strumosa CP50 0.20 11.4 ± 0.0 13.5 ± 0.1 57.6 ± 0.1 87.5 ± 0.3 CP51 0.24 15.7 ± 0.2 22.4 ± 0.5 22.6 ± 0.3 24.2 ± 0.1 CP52 0.27 0.0 ± 0 1.8 ± 0.1 7.5 ± 0.2 6.2 ± 6.9 CP53 0.33 1.4 ± 0.01 9.1 ± 0.4 10.3 ± 0.1 10.6 ± 0.3 CP54 0.46 0.0 ± 0 0.0 ± 0 3.6 ± 0.02 6.8 ± 0.2 CP55 0.73 0.0 ± 0 4.6 ± 0.1 5.6 ± 0.2 17.7 ± 0.3 CP56 0.01 0.00 ± 0 0.00 ± 0 0.00 ± 0 0.00 ± 0 Methanolic S. CP57 0.09 0.0 ± 0 4.1 ± 0.01 3.0 ± 0.04 0.00 ± 0 extract of pneumoniae CP58 0.22 0.0 ± 0 4.0 ± 0.04 4.6 ± 0.1 4.0 ± 0 Amanita sp CP59 0.25 0.0 ± 0 0.0 ± 0 0.00 ± 0 0.00 ± 0 CP60 0.44 0.0 ± 0 0.0 ± 0 0.00 ± 0 6.8 ± 0.01 CP61 0.73 0.0 ± 0 0.0 ± 0 10.7 ± 0.4 7.6 ± 0 CP62 0.98 0.0 ± 0 0.0 ± 0 16.9 ± 0.04 36.4 ± 0.2 CP63 0.01 9.7 ± 0.8 1.6 ± 2.2 16.5 ± 6.0 6.4 ± 4.6 Acetone extract S. typhi CP64 0.10 27.0 ± 3.7 24.1 ± 3.4 23.3 ± 14.6 25.2 ± 16.2 of L. kabansus CP65 0.24 39.4 ± 4.1 52.5 ± 3.3 - 82.0 ± 2.5 CP66 0.28 25.1 ± 7.1 27.8 ± 4.6 14.8 ± 2.6 15.7 ± 2.7 CP67 0.31 0.0 ± 0 39.1 ± 10.2 - - CP68 0.49 0.0 ± 0 2.0 ± 2.9 - - CP69 0.56 21.2 ± 1.2 26.1 ± 0.2 29.2 ± 7.7 28.3 ± 8.4 CP70 0.76 29.3 ± 29.6 98.7 ± 0.3 99.7 ± 0.01 99.7 ± 0 CP71 0.98 2.4 ± 3.3 18.0 ± 25.4 50.1 ± 24.8 79.7 ± 1.6 CP72 0.03 1.1 ± 0.3 10.1 ± 14.3 1.2 ± 1.7 5.0 ± 7.1 Ethanolic extract S. typhi CP73 0.20 0.0 ± 0 2.3 ± 3.0 78.3 ± 28.7 99.8 ± 0.2 of G. lucidum CP74 0.26 0.0 ± 0 0.0 ± 0 0.0 ± 0 68.4 ± 15.3 CP75 0.29 57.1 ± 2.8 46.9 ± 4.5 - - CP76 0.38 13.9 ± 18.5 10.0 ± 14.1 95.5 ± 5.3 98.3 ± 1.4 CP77 0.43 2.4 ± 2.1 41.1 ± 4.4 97.4 ± 1.4 94.9 ± 3.7 CP78 0.56 0.0 ± 0 0.6 ± 0.9 97.5 ± 2.4 97.7 ± 0 CP79 0.79 4.3 ± 1.2 1.51 ± 2.1 0.00 ± 0 9.3 ± 13.2 CP80 0.99 38.1 ± 6.6 54.7 ± 14.5 75.7 ± 1.3 - CP81 0.01 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 Methanolic S. CP82 0.15 0.0 ± 0 0.0 ± 0 29.6 ± 0.1 45.0 ± 0.1 extract of L. pneumoniae CP83 0.25 0.0 ± 0 0.0 ± 0 0.00 ± 0 0.0 ± 0 kabansus CP84 0.29 0.0 ± 0 37.7 ± 0.03 18.1 ± 0.1 27.8 ± 0.1 CP85 0.33 0.0 ± 0 39.9 ± 1.1 35.6 ± 0.02 39.6 ± 0.7

CP86 0.46 0.0 ± 0 0.0 ± 0 0.00 ± 0 0.0 ± 0 CP87 0.53 0.0 ± 0 0.0 ± 0 0.00 ± 0 17.5 ± 0.03 CP88 0.73 0.0 ± 0 0.0 ± 0 11.6 ± 0.03 28.3 ± 0.4 CP89 0.90 0.0 ± 0 0.0 ± 0 0.00 ± 0 0.00 ± 0 CP90 0.99 0.0 ± 0 0.0 ± 0 1.3 ± 1.8 19.5 ± 1.0 CP91 0.04 0.0 ± 0 0.0 ± 0 0.00 ± 0 9.3 ± 13.2 Acetone extract S. typhi CP92 0.07 0.0 ± 0 0.0 ± 0 38.0 ± 15.9 98.4 ± 0.2 of C. symoensii CP93 0.28 0.0 ± 0 4.9 ± 6.9 7.2 ± 10.2 8.3 ± 11.7 CP94 0.55 16.1 ± 22.7 98.2 ± 0 99.7 ± 0 99.8 ± 0.01 CP95 0.59 0.8 ± 1.1 0.0 ± 0 0.0 ± 0 0.0 ± 0 CP96 0.88 0.6 ± 0.9 7.4 ± 10.5 10.5 ± 14.8 16.5 ± 19.7

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CP97 0.96 7.2 ± 10.2 6.7 ± 9.4 4.1 ± 5.8 5.0 ± 4.0 CP98 0.03 4.3 ± 6.0 93.3 ± 5.6 95.7 ± 2.2 96.9 ± 0 Cold water S. typhi CP99 0.08 0.0 ± 0 2.2 ± 3.2 6.6 ± 9.3 7.7 ± 11.0 extract of C. CP100 0.13 7.8 ± 0.6 0.0 ± 0 0.0 ± 0 0.0 ± 0 miomboensis CP101 0.20 0.0 ± 0 15.5 ± 4.5 64.9 ± 0.4 - CP102 0.31 4.4 ± 4.5 0.0 ± 0 0.0 ± 0 0.6 ± 0.9 CP103 0.98 15.9 ± 7.4 35.7 ± 6.4 48.2 ± 13.9 79.0 ± 0 CP104 0.02 0.0 ± 0 0.0 ± 0 0.0 ± 0 44.6 ± 63.1 Ethanolic extract S. typhi CP105 0.15 8.7 ± 4.2 7.2 ± 1.2 0.0 ± 0 0.0 ± 0 of C. CP106 0.20 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 miomboensis CP107 0.26 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 CP108 0.47 0.0 ± 0 0.0 ± 0 0.0 ± 0 8.3 ± 11.7 CP109 0.54 0.0 ± 0 0.0 ± 0 0.0 ± 0 1.4 ± 2.0 CP110 0.60 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.8 ± 1.1 CP111 0.67 53.2 ± 4.1 76.7 ± 1.0 76.8 ± 2.2 - CP112 0.75 0.0 ± 0 0.0 ± 0 3.5 ± 3.4 11.9 ± 13.6 CP113 0.86 0.0 ± 0 1.2 ± 1.7 18.9 ± 2.5 15.0 ± 6.8 CP114 0.96 0.0 ± 0 9.2 ± 10.8 11.0 ± 0.5 8.4 ± 11.9 CP115 0.02 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 Acetone extract CP116 0.09 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 of Amanita sp S. typhi CP117 0.15 0.0 ± 0 0.0 ± 0 26.3 ± 9.0 26.7 ± 13.4 CP118 0.35 1.1 ± 1.6 0.0 ± 0 17.7 ± 3.7 15.0 ± 6.6 CP119 0.49 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.0 ± 0 CP120 0.62 0.0 ± 0 2.9 ± 4.03 7.5 ± 5.06 12.9 ± 2.2 CP121 0.82 23.3 ± 32.9 48.5 ± 68.6 48.8 ± 69.0 49.0 ± 69.3 CP122 0.96 0.0 ± 0 0.0 ± 0 0.00 ± 0 1.3 ± 1.9 CP123 0.02 0.0 ± 0 0.0 ± 0 1.64 ± 2.3 5.4 ± 7.6 Ethanolic extract S. typhi CP124 0.08 1.7 ± 2.4 12.2 ± 0.9 - - of Amanita sp CP125 0.17 0.0 ± 0 0.0 ± 0 8.9 ± 12.5 8.3 ± 11.7 CP126 0.36 1.4 ± 2.0 0.0 ± 0 0.0 ± 0 0.0 ± 0 CP127 0.55 0.0 ± 0 0.7 ± 1.1 2.6 ± 3.6 10.3 ± 14.6 CP128 0.60 0.0 ± 0 5.2 ± 7.3 3.0 ± 4.3 0.0 ± 0 CP129 0.72 0.0 ± 0 8.0 ± 5.1 33.8 ± 11.2 61.2 ± 3.5 CP130 0.86 0.0 ± 0 4.2 ± 3.6 0.5 ± 0.7 12.7 ± 0.5 CP131 0.98 0.0 ± 0 3.6 ± 5.1 8.1 ± 11.5 29.0 ± 12.7 Ampicillin 96.9 ± 0.4 S. typhi (50 µg/ml) 91.3 ± 0.5 S. aureus 92.4 ± 0.7 S. pneumoniae

The negative control values were used to calculate the percentage inhibition of bacterial growth by each extract.

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8.4 LC-MS CHROMATOGRAMS OUTPUT OF ISOLATED COMPONENTS OF MUSHROOM SAMPLES

Figure 8.4.1: LC-MS output of chromatograms of isolated components of mushroom samples that exhibited high anti-bacterial activity analyzed in positive mode with a column.

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8.5 LC-MS PROFILES OF MUSHROOM CRUDE EXTRACTS

Table 8.5.1: Showing compounds present in the hot water crude extract of Coprinus sp. After analysis by LC-MS in positive mode with a column. The crude extracts exhibited high inhibition of HIV-1 replication during the HIV-1 p24 ELISA assay

Cpd Label Name Formula 1 Cpd 1: 0.793 2 Cpd 2: 0.793 3 Cpd 3: 0.794 4 Cpd 4: 0.795 5 Cpd 5: Brunfelsamidine Brunfelsamidine C5 H7 N3 6 Cpd 6: 3-(Pyrazol-1-yl)-L-alanine 3-(Pyrazol-1-yl)-L-alanine C6 H9 N3 O2 7 Cpd 7: Pterolactam Pterolactam C5 H9 N O2 8 Cpd 8: 0.820 9 Cpd 9: Propamocarb Propamocarb C9 H20 N2 O2 10 Cpd 10: 0.868 11 Cpd 11: 0.940 12 Cpd 12: Amino acid(Arg-) Amino acid(Arg-) C6 H14 N4 O2 13 Cpd 13: 0.962 14 Cpd 14: Cytosine Cytosine C4 H5 N3 O 15 Cpd 15: L-Cyclo(alanylglycyl) L-Cyclo(alanylglycyl) C5 H8 N2 O2 16 Cpd 16: 4-Methylaminobutyrate 4-Methylaminobutyrate C5 H11 N O2 17 Cpd 17: 4- 4- C7 H7 N2 (Hydroxymethyl)benzenediazonium(1+) (Hydroxymethyl)benzenediazonium( O 1+) 18 Cpd 18: Vidarabine Vidarabine C10 H13 N5 O4 19 Cpd 19: Benzyl glycinate Benzyl glycinate C9 H11 N O2 20 Cpd 20: Cordycepin Cordycepin C10 H13 N5 O3 21 Cpd 21: 2-Aminobut-2-enoate 2-Aminobut-2-enoate C4 H7 N O2 22 Cpd 22: N(alpha)-t-Butoxycarbonyl-L- N(alpha)-t-Butoxycarbonyl-L-leucine C11 H21 N leucine O4 23 Cpd 23: N-Acetylserine N-Acetylserine C5 H9 N O4 24 Cpd 24: Pterolactam Pterolactam C5 H9 N O2 25 Cpd 25: Pyrroline hydroxycarboxylic Pyrroline hydroxycarboxylic acid C5 H7 N acid O3 26 Cpd 26: 3-Oxo-3-phenylpropanoate 3-Oxo-3-phenylpropanoate C9 H8 O3 27 Cpd 27: 3-Dehydro-L-threonate 3-Dehydro-L-threonate C4 H6 O5 28 Cpd 28: 1-nitrohexane 1-nitrohexane C6 H13 N

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O2 29 Cpd 29: Isonicotinic acid Isonicotinic acid C6 H5 N O2 30 Cpd 30: Pirbuterol Pirbuterol C12 H20 N2 O3 31 Cpd 31: 1,8-Diazacyclotetradecane-2,9- 1,8-Diazacyclotetradecane-2,9-dione C12 H22 dione N2 O2 32 Cpd 32: 3'-O-Methyladenosine 3'-O-Methyladenosine C11 H15 N5 O4 33 Cpd 33: 1.031 34 Cpd 34: 1.043 35 Cpd 35: Lysyl-Tyrosine Lysyl-Tyrosine C15 H23 N3 O4 36 Cpd 36: Arginyl-Phenylalanine Arginyl-Phenylalanine C15 H23 N5 O3 37 Cpd 37: Pyruvophenone Pyruvophenone C9 H8 O2 38 Cpd 38: 1.115 39 Cpd 39: Alanyl-Isoleucine Alanyl-Isoleucine C9 H18 N2 O3 40 Cpd 40: Benzaldehyde Benzaldehyde C7 H6 O 41 Cpd 41: 1.117 42 Cpd 42: Pantothenic Acid Pantothenic Acid C9 H17 N O5 43 Cpd 43: Quinacetol Quinacetol C11 H9 N O2 44 Cpd 44: 5-Phenyl-1,3-oxazinane-2,4- 5-Phenyl-1,3-oxazinane-2,4-dione C10 H9 N dione O3 45 Cpd 45: N(alpha)-t-Butoxycarbonyl-L- N(alpha)-t-Butoxycarbonyl-L-leucine C11 H21 N leucine O4 46 Cpd 46: 1.118 47 Cpd 47: 1.118 48 Cpd 48: Bethanidine Bethanidine C10 H15 N3 49 Cpd 49: L-isoleucyl-L-proline L-isoleucyl-L-proline C11 H20 N2 O3 50 Cpd 50: Pterolactam Pterolactam C5 H9 N O2 51 Cpd 51: 2'-Aminoacetophenone 2'-Aminoacetophenone C8 H9 N O 52 Cpd 52: Benzenepropanenitrile Benzenepropanenitrile C9 H9 N 53 Cpd 53: 2(1H)-Quinolinone 2(1H)-Quinolinone C9 H7 N O 54 Cpd 54: quinaldine quinaldine C10 H9 N 55 Cpd 55: 1.161 56 Cpd 56: 1.166 57 Cpd 57: 1.172 58 Cpd 58: Pinacidil Pinacidil C13 H19 N5 59 Cpd 59: L-1,2,3,4-Tetrahydro-beta- L-1,2,3,4-Tetrahydro-beta-carboline- C12 H12 carboline-3-carboxylic acid 3-carboxylic acid N2 O2 60 Cpd 60: Anisomycin Anisomycin C14 H19 N O4 61 Cpd 61: 1.182 62 Cpd 62: 1.182 63 Cpd 63: Marimastat Marimastat C15 H29 144

N3 O5 64 Cpd 64: 1.211 65 Cpd 65: 1.219 66 Cpd 66: D-Lombricine D-Lombricine C6 H15 N4 O6 P 67 Cpd 67: 1.225 68 Cpd 68: 1.225 69 Cpd 69: 1.226 70 Cpd 70: 1.239 71 Cpd 71: 1,3,8-Trihydroxy-4-methyl-2,7- 1,3,8-Trihydroxy-4-methyl-2,7- C24 H26 diprenylxanthone diprenylxanthone O5 72 Cpd 72: 1.267 73 Cpd 73: 1.286 74 Cpd 74: 1.289 75 Cpd 75: 1.313 76 Cpd 76: 1.319 77 Cpd 77: 1.319 78 Cpd 78: 1.330 79 Cpd 79: Coutaric acid Coutaric acid C18 H27 N3 O4 80 Cpd 80: Ximelagatran Ximelagatran C24 H35 N5 O5 81 Cpd 81: 1.433 82 Cpd 82: 1.511 83 Cpd 83: Leucyl-leucyl-norleucine Leucyl-leucyl-norleucine C18 H35 N3 O4 84 Cpd 84: 1.654 85 Cpd 85: 1.655 86 Cpd 86: 1.791 87 Cpd 87: Tebuconazole Tebuconazole C16 H22 Cl N3 O 88 Cpd 88: 2.064 89 Cpd 89: 2.088 90 Cpd 90: 2.104 91 Cpd 91: 2.108 92 Cpd 92: Kanzonol V Kanzonol V C24 H24 O4 93 Cpd 93: Phenylacetonitrile Phenylacetonitrile C8 H7 N 94 Cpd 94: 2(1H)-Quinolinone 2(1H)-Quinolinone C9 H7 N O 95 Cpd 95: Lupinate Lupinate C13 H18 N6 O3 96 Cpd 96: 5(S),14(R)-Lipoxin B4 5(S),14(R)-Lipoxin B4 C20 H32 O5 97 Cpd 97: Isocolumbin Isocolumbin C20 H22 O6 98 Cpd 98: 5(S),14(R)-Lipoxin B4 5(S),14(R)-Lipoxin B4 C20 H32 O5 99 Cpd 99: 5(S),14(R)-Lipoxin B4 5(S),14(R)-Lipoxin B4 C20 H32 O5 100 Cpd 100: 8.445 101 Cpd 101: 5(S),14(R)-Lipoxin B4 5(S),14(R)-Lipoxin B4 C20 H32 O5 102 Cpd 102: 8.747 145

103 Cpd 103: Ketopelenolide a Ketopelenolide a C15 H22 O3 104 Cpd 104: 1-(3-Furanyl)-6,7-dihydroxy- 1-(3-Furanyl)-6,7-dihydroxy-4,8- C15 H24 4,8-dimethyl-1-nonanone dimethyl-1-nonanone O4 105 Cpd 105: C16 Sphinganine C16 Sphinganine C16 H35 N O2 106 Cpd 106: 12.676 107 Cpd 107: 13.132 108 Cpd 108: 2,3-Dinor-11b-PGF2a 2,3-Dinor-11b-PGF2a C18 H30 O5 109 Cpd 109: N6-Galacturonyl-L-lysine N6-Galacturonyl-L-lysine C12 H22 N2 O8 110 Cpd 110: Macrophorin A Macrophorin A C22 H32 O4 111 Cpd 111: (ent- (ent-2alpha,3beta,15beta,16beta)- C20 H32 2alpha,3beta,15beta,16beta)-15,16- 15,16-Epoxy-2,3-kauranediol O3 Epoxy-2,3-kauranediol 112 Cpd 112: Avenic acid A Avenic acid A C12 H22 N2 O8 113 Cpd 113: 17abeta-Hydroxy-D- 17abeta-Hydroxy-D-homoandrost-4- C20 H30 homoandrost-4-en-3-one en-3-one O2 114 Cpd 114: Macrophorin A Macrophorin A C22 H32 O4 115 Cpd 115: (ent- (ent-2alpha,3beta,15beta,16beta)- C20 H32 2alpha,3beta,15beta,16beta)-15,16- 15,16-Epoxy-2,3-kauranediol O3 Epoxy-2,3-kauranediol 116 Cpd 116: 9,10-Epoxy-18-hydroxystearate 9,10-Epoxy-18-hydroxystearate C18 H34 O4 117 Cpd 117: Phytosphingosine Phytosphingosine C18 H39 N O3 118 Cpd 118: 5,6-Ep-15S-HETE 5,6-Ep-15S-HETE C20 H30 O4 119 Cpd 119: 5,6-Ep-15S-HETE 5,6-Ep-15S-HETE C20 H30 O4 120 Cpd 120: 5,6-Ep-15S-HETE 5,6-Ep-15S-HETE C20 H30 O4 121 Cpd 121: 17.264 122 Cpd 122: Prosolanapyrone II Prosolanapyrone II C18 H24 O4 123 Cpd 123: α-ESA α-ESA C18 H30 O2 124 Cpd 124: Ginsenoyne K Ginsenoyne K C17 H24 O3 125 Cpd 125: Ethyl 2-benzylacetoacetate Ethyl 2-benzylacetoacetate C13 H16 O3 126 Cpd 126: (S)-alpha-Terpinyl glucoside (S)-alpha-Terpinyl glucoside C16 H28 O6 127 Cpd 127: 18.668 128 Cpd 128: 18.679 129 Cpd 129: Polysorbate 60 Polysorbate 60 C22 H42 O8 130 Cpd 130: Sterebin D Sterebin D C18 H30 O3

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131 Cpd 131: 18.980 132 Cpd 132: 19.340 133 Cpd 133: 19.349 134 Cpd 134: 19.732 135 Cpd 135: 19.763 136 Cpd 136: 19.793 137 Cpd 137: 19.820 138 Cpd 138: 19.847 139 Cpd 139: 19.954 140 Cpd 140: 19.972 141 Cpd 141: 20.005 142 Cpd 142: 20.035 143 Cpd 143: 20.073 144 Cpd 144: 20.093 145 Cpd 145: 20.093 146 Cpd 146: 20.118 147 Cpd 147: 20.137 148 Cpd 148: 20.142 149 Cpd 149: 20.292 150 Cpd 150: 20.536 151 Cpd 151: 3,6-Epoxy-5,5',6,6'-tetrahydro- 3,6-Epoxy-5,5',6,6'-tetrahydro-b,b- C40 H58 b,b-carotene-3',5,5',6'-tetrol carotene-3',5,5',6'-tetrol O5 152 Cpd 152: 20.701 153 Cpd 153: 20.768 154 Cpd 154: (all-E)-6'-Apo-y-caroten-6'-al (all-E)-6'-Apo-y-caroten-6'-al C32 H42 O 155 Cpd 155: Palmitic amide Palmitic amide C16 H33 N O 156 Cpd 156: Oleamide Oleamide C18 H35 N O 157 Cpd 157: 21.281 158 Cpd 158: 23.776 159 Cpd 159: 23.780 160 Cpd 160: PI(18:1(9Z)/20:3(8Z,11Z,14Z)) PI(18:1(9Z)/20:3(8Z,11Z,14Z)) C47 H83 O13 P 161 Cpd 161: Pheophytin a Pheophytin a C55 H74 N4 O5 162 Cpd 162: 7-Hydroxychlorophyll a 7-Hydroxychlorophyll a C55 H72 Mg N4 O6 163 Cpd 163: PE(15:0/24:1(15Z)) PE(15:0/24:1(15Z)) C44 H86 N O8 P

147

Table 8.5.2: Showing compounds present in the cold water crude extract of L. kabansus after analysis by LC-MS in positive mode with a column. The crude extracts exhibited high inhibition of HIV-1 RT during the HIV-1 RT assay

Cpd Label Name Formula 1 Cpd 1: 0.792 2 Cpd 2: 0.792 3 Cpd 3: 0.893 4 Cpd 4: N-(2- N-(2-Methylpropyl)acetamide C6 H13 N O Methylpropyl)acetamide 5 Cpd 5: 0.939 6 Cpd 6: Betazole Betazole C5 H9 N3 7 Cpd 7: 0.942 8 Cpd 8: delta-Guanidinovaleric acid delta-Guanidinovaleric acid C6 H13 N3 O2 9 Cpd 9: 11-amino-undecanoic acid 11-amino-undecanoic acid C11 H23 N O2 10 Cpd 10: Pirbuterol Pirbuterol C12 H20 N2 O3 11 Cpd 11: 0.954 12 Cpd 12: Alanyl-Aspartate Alanyl-Aspartate C7 H12 N2 O5 13 Cpd 13: 0.976 14 Cpd 14: Vinylacetylglycine Vinylacetylglycine C6 H9 N O3 15 Cpd 15: Aminohydroquinone Aminohydroquinone C6 H7 N O2 16 Cpd 16: L-isoleucyl-L-proline L-isoleucyl-L-proline C11 H20 N2 O3 17 Cpd 17: Arecaidine Arecaidine C7 H11 N O2 18 Cpd 18: Isoguvacine Isoguvacine C6 H9 N O2 19 Cpd 19: 0.981 20 Cpd 20: Asparaginyl- Asparaginyl-Hydroxyproline C9 H15 N3 O5 Hydroxyproline 21 Cpd 21: Pantothenic Acid Pantothenic Acid C9 H17 N O5 22 Cpd 22: 0.992 23 Cpd 23: Phlorin Phlorin C12 H16 O8 24 Cpd 24: 1.006 25 Cpd 25: 1.010 26 Cpd 26: Tos-Arg-CH2Cl Tos-Arg-CH2Cl C13 H21 Cl N4 O2 S 27 Cpd 27: 1-Deoxynojirimycin 1-Deoxynojirimycin C6 H13 N O4 28 Cpd 28: (S)-3- (S)-3- C11 H10 N2 O3 [(Cyanophenylmethyl)amino]-3- [(Cyanophenylmethyl)amino]- oxopropanoic acid 3-oxopropanoic acid 29 Cpd 29: N,N-Dihydroxy-L- N,N-Dihydroxy-L-tryptophan C11 H12 N2 O4 tryptophan 30 Cpd 30: 1.041 31 Cpd 31: 2(1H)-Quinolinone 2(1H)-Quinolinone C9 H7 N O 32 Cpd 32: Alanyl-Proline Alanyl-Proline C8 H14 N2 O3 33 Cpd 33: 4-Methylaminobutyrate 4-Methylaminobutyrate C5 H11 N O2 34 Cpd 34: (p-Aminobenzyl)penicillin (p-Aminobenzyl)penicillin C16 H19 N3 O4 S 35 Cpd 35: 1-Nitronaphthalene 1-Nitronaphthalene C10 H7 N O2 36 Cpd 36: 2,5-Xylidine 2,5-Xylidine C8 H11 N 37 Cpd 37: Oxadixyl Oxadixyl C14 H18 N2 O4 38 Cpd 38: Pantothenic Acid Pantothenic Acid C9 H17 N O5 39 Cpd 39: 1.111 40 Cpd 40: Mexacarbate Mexacarbate C12 H18 N2 O2 41 Cpd 41: 1- 1-Pyrrolidinecarboxaldehyde C5 H9 N O Pyrrolidinecarboxaldehyde 148

42 Cpd 42: Histidinyl-Tyrosine Histidinyl-Tyrosine C15 H18 N4 O4 43 Cpd 43: 1.115 44 Cpd 44: 1.116 45 Cpd 45: Licocoumarin A Licocoumarin A C25 H26 O5 46 Cpd 46: (Z)-1-(Methylthio)-5- (Z)-1-(Methylthio)-5-phenyl-1- C12 H12 S phenyl-1-penten-3-yne penten-3-yne 47 Cpd 47: Garcimangosone D Garcimangosone D C19 H20 O9 48 Cpd 48: 2(1H)-Quinolinone 2(1H)-Quinolinone C9 H7 N O 49 Cpd 49: (1xi,2xi)-1-(4- (1xi,2xi)-1-(4- C15 H22 O9 Hydroxyphenyl)-1,2,3-propanetriol Hydroxyphenyl)-1,2,3- 2-O-beta-D-glucopyranoside propanetriol 2-O-beta-D- glucopyranoside 50 Cpd 50: Morusin Morusin C25 H24 O6 51 Cpd 51: sofalcone sofalcone C27 H30 O6 52 Cpd 52: Arbutin Arbutin C12 H16 O7 53 Cpd 53: L-1,2,3,4-Tetrahydro-beta- L-1,2,3,4-Tetrahydro-beta- C12 H12 N2 O2 carboline-3-carboxylic acid carboline-3-carboxylic acid 54 Cpd 54: quinaldine quinaldine C10 H9 N 55 Cpd 55: 1.178 56 Cpd 56: Homoarecoline Homoarecoline C9 H15 N O2 57 Cpd 57: Vanilloloside Vanilloloside C14 H20 O8 58 Cpd 58: 1.183 59 Cpd 59: Microlenin Microlenin C29 H34 O7 60 Cpd 60: Eremopetasitenin B2 Eremopetasitenin B2 C24 H32 O7 S 61 Cpd 61: Salicylanilide Salicylanilide C13 H11 N O2 62 Cpd 62: 1-(3,4-Dimethoxyphenyl)- 1-(3,4-Dimethoxyphenyl)-1,2- C16 H24 O9 1,2-ethanediol 2-O-b-D-glucoside ethanediol 2-O-b-D-glucoside 63 Cpd 63: 9-Azabicyclo[3.3.1]nonan- 9-Azabicyclo[3.3.1]nonan-3- C8 H13 N O 3-one one 64 Cpd 64: 1.221 65 Cpd 65: 6-O-Oleuropeoylsucrose 6-O-Oleuropeoylsucrose C22 H36 O13 66 Cpd 66: Atrazine Atrazine C8 H14 Cl N5 67 Cpd 67: Pummeline Pummeline C15 H13 N O4 68 Cpd 68: 1-Piperidinecarboxaldehyde 1-Piperidinecarboxaldehyde C6 H11 N O 69 Cpd 69: 1.238 70 Cpd 70: 1.241 71 Cpd 71: 1.254 72 Cpd 72: Cyanazine Cyanazine C9 H13 Cl N6 73 Cpd 73: Osajin Osajin C25 H24 O5 74 Cpd 74: 1.278 75 Cpd 75: Lonchocarpenin Lonchocarpenin C27 H28 O6 76 Cpd 76: 3-Hydroxylidocaine 3-Hydroxylidocaine C14 H22 N2 O2 77 Cpd 77: 1.342 78 Cpd 78: 1.378 79 Cpd 79: 3,4-Dihydro-5-propanoyl- 3,4-Dihydro-5-propanoyl-2H- C7 H11 N O 2H-pyrrole pyrrole 80 Cpd 80: trans-O-Methylgrandmarin trans-O-Methylgrandmarin C16 H18 O6 81 Cpd 81: Junosine Junosine C19 H19 N O4 82 Cpd 82: zeleplon zeleplon C17 H15 N5 O 83 Cpd 83: Furmecyclox Furmecyclox C14 H21 N O3 84 Cpd 84: 1.693 85 Cpd 85: 1.720

149

86 Cpd 86: 1.911 87 Cpd 87: 1.952 88 Cpd 88: 1.980 89 Cpd 89: 2.010 90 Cpd 90: 2.060 91 Cpd 91: Lumichrome Lumichrome C12 H10 N4 O2 92 Cpd 92: N4-Phosphoagmatine N4-Phosphoagmatine C5 H15 N4 O3 P 93 Cpd 93: Ptelatoside A Ptelatoside A C19 H26 O10 94 Cpd 94: Lymecycline Lymecycline C29 H38 N4 O10 95 Cpd 95: Ononin Ononin C22 H22 O9 96 Cpd 96: 3-Methylene-indolenine 3-Methylene-indolenine C9 H7 N 97 Cpd 97: 3.523 98 Cpd 98: 3.523 99 Cpd 99: Hexyl glucoside Hexyl glucoside C12 H24 O6 100 Cpd 100: 3.812 101 Cpd 101: 4.459 102 Cpd 102: hexamethylene hexamethylene bisacetamide C10 H20 N2 O2 bisacetamide 103 Cpd 103: 5.514 104 Cpd 104: Heptopargil Heptopargil C13 H19 N O 105 Cpd 105: 8.079 106 Cpd 106: Isocolumbin Isocolumbin C20 H22 O6 107 Cpd 107: 8.742 108 Cpd 108: Ferimzone Ferimzone C15 H18 N4 109 Cpd 109: Isogingerenone B Isogingerenone B C22 H26 O6 110 Cpd 110: 2-Methylbenzaldehyde 2-Methylbenzaldehyde C8 H8 O 111 Cpd 111: Styrene Styrene C8 H8 112 Cpd 112: Isogingerenone B Isogingerenone B C22 H26 O6 113 Cpd 113: Sapidolide A Sapidolide A C14 H18 O5 114 Cpd 114: 13.016 115 Cpd 115: Ginsenoyne K Ginsenoyne K C17 H24 O3 116 Cpd 116: N6-Galacturonyl-L-lysine N6-Galacturonyl-L-lysine C12 H22 N2 O8 117 Cpd 117: 17.266 118 Cpd 118: 17.897 119 Cpd 119: Tributyl phosphate Tributyl phosphate C12 H27 O4 P 120 Cpd 120: Prosolanapyrone II Prosolanapyrone II C18 H24 O4 121 Cpd 121: (S)-alpha-Terpinyl (S)-alpha-Terpinyl glucoside C16 H28 O6 glucoside 122 Cpd 122: 18.672 123 Cpd 123: Polysorbate 60 Polysorbate 60 C22 H42 O8 124 Cpd 124: 18.985 125 Cpd 125: 19.032 126 Cpd 126: 19.046 127 Cpd 127: 19.047 128 Cpd 128: 19.058 129 Cpd 129: Goshonoside F2 Goshonoside F2 C26 H44 O8 130 Cpd 130: 19.344 131 Cpd 131: 19.352 132 Cpd 132: 19.707 133 Cpd 133: 13-Demethylspirolide C 13-Demethylspirolide C C42 H61 N O7 134 Cpd 134: 19.738 135 Cpd 135: 19.751 150

136 Cpd 136: 19.768 137 Cpd 137: 19.772 138 Cpd 138: 19.791 139 Cpd 139: 19.799 140 Cpd 140: 19.828 141 Cpd 141: Iriomoteolide 1a Iriomoteolide 1a C29 H46 O7 142 Cpd 142: 19.852 143 Cpd 143: 19.959 144 Cpd 144: 19.975 145 Cpd 145: 20.009 146 Cpd 146: 20.039 147 Cpd 147: 20.071 148 Cpd 148: 20.096 149 Cpd 149: 20.098 150 Cpd 150: Methyl (7Z,9Z,9'Z)-6'-apo- Methyl (7Z,9Z,9'Z)-6'-apo-y- C33 H44 O2 y-caroten-6'-oate caroten-6'-oate 151 Cpd 151: 20.119 152 Cpd 152: 20.134 153 Cpd 153: 20.142 154 Cpd 154: 20.253 155 Cpd 155: Pipericine Pipericine C22 H41 N O 156 Cpd 156: Docosatetraenoyl Docosatetraenoyl C24 H41 N O2 Ethanolamide Ethanolamide 157 Cpd 157: 20.537 158 Cpd 158: 20.539 159 Cpd 159: Docosatrienoic Acid Docosatrienoic Acid C22 H38 O2 160 Cpd 160: 20.542 161 Cpd 161: PEP-16:0/18:1(11Z)) PEP-16:0/18:1(11Z)) C39 H76 N O7 P 162 Cpd 162: 20.646 163 Cpd 163: 20.666 164 Cpd 164: 20.722 165 Cpd 165: 20.729 166 Cpd 166: 20.751 167 Cpd 167: 20.770 168 Cpd 168: 20.774 169 Cpd 169: 20.783 170 Cpd 170: 20.798 171 Cpd 171: Palmitic amide Palmitic amide C16 H33 N O 172 Cpd 172: Oleamide Oleamide C18 H35 N O 173 Cpd 173: Caffeoylcycloartenol Caffeoylcycloartenol C39 H56 O4 174 Cpd 174: 21.285 175 Cpd 175: 21.298 176 Cpd 176: 23.764 177 Cpd 177: 23.776 178 Cpd 178: PI(18:1(9Z)/20:3(8Z,11Z,14Z)) C47 H83 O13 P PI(18:1(9Z)/20:3(8Z,11Z,14Z)) 179 Cpd 179: 7-Hydroxychlorophyll a 7-Hydroxychlorophyll a C55 H72 Mg N4 O6 180 Cpd 180: Pheophytin a Pheophytin a C55 H74 N4 O5 181 Cpd 181: PE(15:0/24:1(15Z)) PE(15:0/24:1(15Z)) C44 H86 N O8 P

151