Identification of Traditional Medicinal Plant Extracts with Novel Anti-Influenza Activity

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IDENTIFICATION OF TRADITIONAL MEDICINAL PLANT EXTRACTS WITH NOVEL ANTI-INFLUENZA ACTIVITY

A thesis submitted for the degree of Doctor of Philosophy

Dhivya Rajasekaran

ENVIRONMENT AND BIOTECHNOLOGY CENTRE SWINBURNE UNIVERSITY OF TECHNOLOGY AUSTRALIA 2014

ABSTRACT

The emergence of drug resistant variants of the influenza virus has led to a need to identify novel antiviral agents. As an alternative to synthetic drugs, the consolidation of empirical knowledge with ethnopharmacological evidence of medicinal plants offers a novel platform for the development of antiviral drugs. The aim of this study was to identify plant extracts with proven activity against the influenza virus. Extracts of fifty medicinal plants, originating from the tropical rainforests of Borneo, used as herbal medicines by traditional healers to treat various infections including flu-like symptoms, were initially tested against Mem-Bel (H3N1) and PR8 (H1N1) viruses.

In the preliminary phase, in vitro micro-inhibition assays along with cytotoxicity screenings were performed on MDCK cells. Most plant extracts were found to be minimally cytotoxic, indicating that the compounds linked to an ethnomedical framework were relatively innocuous, and eleven crude extracts exhibited viral inhibition against both strains. All extracts inhibited the enzymatic activity of viral neuraminidase (NA) and four extracts were also shown to act through the hemagglutination inhibition (HI) pathway. Furthermore, the samples that acted through both HI and NA inhibition evidenced more than 90% reduction in virus adsorption and penetration, thereby indicating potent actions in the early stages of viral replication. Concurrent studies involving Receptor Destroying Enzyme (RDE) treatments of HI extracts indicated the presence of sialic acid-like component(s) that may be responsible for HI.

Extracts that exhibited both HI and NA inhibition were investigated for antiviral activities against Oseltamivir resistant former seasonal and pandemic Type A (H1N1) (H275Y) viruses; NA inhibitor resistant Type B (D197E) virus along with the corresponding wild type viruses. All extracts actively inhibited the viruses including the NA inhibitor resistant strains, in fact NA inhibition (NAI) was demonstrated against all viruses, whereas HI activity was demonstrated by only three extracts against pandemic A and Type B viruses. RDE treatment of extracts further indicated the presence of HI active sialic acid (SA) mimics. Additionally, RDE-treated extracts were studied in a NA inhibition assay; the results indicated the presence of non-sialic acid mimics that work synergistically with sialic acid-like components to inhibit the virus. The active

Abstract

components in the extract may affect viral proteins apart from HA and NA or the different stages of viral replication. The manifestation of both modes of viral inhibition in a single extract suggests that there may be a synergistic effect implicating more than one active component.

One of the extracts was fractionated using solid phase extraction (SPE) columns in order to correlate chemical composition with anti-viral activities. Two fractions that were active against influenza viruses were obtained and several in vitro analyses were performed. Gas Chromatograph Mass spectrometer (GC-MS) analyses showed the distribution of several components in more than one fraction, suggesting that the antiviral activity may have been reduced by fractionation; in vitro experiments also indicated that crude extract had a more potent antiviral activity than the isolated active fractions. Cyclic polyols, fatty acids, and amides, which are known to be influenza inhibitory, were predominant in these plant samples. This study serves as a starting place to further chemical investigations that could lead to the identification and isolation of active compounds.

Overall, these results provide substantial support for the use of Borneo traditional plants as promising sources of novel anti-influenza drug candidates. Furthermore, the different pathways of viral inhibition could be a solution to the global occurrence of viral strains resistant to the current NAI drugs.

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ACKNOWLEDGEMENTS

This research would not have been possible without the guidance and warm support of my supervisors, Dr. Lara Grollo, Dr. Enzo Palombo and Dr. Francois Malherbe. Lara’s attention to detail always led the project in the right direction and I genuinely appreciate the many times that she went out of her way to help solve a problem. Enzo was always willing to assist in any way that he could and his contacts at the Sarawak Biodiversity Centre (SBC) played a vital role in the progress of this research work. I am extremely grateful to Francois, for having supported me right from the days when I was a Masters student in India and also provided an opportunity to be a part of the Swinburne research community.

I would like to acknowledge our collaborators, Chu Lee Tu, Tiong Chia Yeo and Diana Lim Siok Ley, Kuching, Malaysia for supporting us with medicinal extracts as and when required. I would also like to express my appreciation for the advice and directions provided by researchers at The University of Melbourne, Dr. Lorena Brown, Dr. Brad Gilbertson and Dr. Patrick Reading. I am grateful for Dr. Simon Crawford’s assistance with Transmission electron microscopy (TEM) studies at the same university. Many thanks to Ms. Cheryl Colson, Asia Pacific Centre for Animal Health, Veterinary School, The University of Melbourne for having offered chicken blood, several times for the mode of action assays. I would like to acknowledge Dr. Lorena Brown, The University of Melbourne and Dr. Aeron Hurt, Victorian Infectious Diseases Reference Laboratory, Melbourne for providing several strains of influenza viruses. I would like to appreciate Ms. Savitri Galappathie and Dr. Peter Mahon’s help with the chemical analyses at Swinburne.

I am grateful to the Faculty of Science, Engineering and Technology (FSET), Swinburne University and my supervisors for the financial support especially during conference presentations and, Environment and Biotechnology Centre (EBC) for providing the lab facilities. I also appreciate the research staff at Swinburne University and all my colleagues who made the lab environment, most enjoyable to work with.

I would like to express my gratitude to family and friends, especially my parents, husband, Tom and cousin, Preethi for their prayers, love and support.

DECLARATION

I, Dhivya Rajasekaran declare, to the best of my knowledge, that the material contained in this thesis entitled, “Identification of traditional medicinal plant extracts with novel anti-influenza activity”, has not been accepted for the award of any other degree, and has not been previously published or written by another person, except where due reference is made in the text. Furthermore, where the work is based on joint research or publications, the thesis discloses the relative contributions of the respective collaborators and/ or authors.

Dhivya Rajasekaran

April 2014

COMMUNICATIONS

Publication

- Rajasekaran, D., Palombo, E.A., Yeo, T.C., Ley, D.L.S., Tu, C.L., Malherbe, F., Grollo, L., 2013. Identification of traditional medicinal plant extracts with novel anti-influenza activity, In: PLOS One, Volume 8 (11), e79293, 27 November, 2013

Conference presentations

- Rajasekaran, D.*, Palombo, E.A., Yeo, T.C., Ley, D.L.S., Tu, C.L., Malherbe, F., Grollo, L., 2012. Identification of traditional medicinal plant extracts with novel anti-influenza activity, Oral presentation, In: Influenza 2012: One Influenza, One World, September 2012, St Hilda’s College, Oxford, United Kingdom

- Rajasekaran, D., Palombo, E.A., Yeo, T.C., Ley, D.L.S., Tu, C.L., Malherbe, F., Grollo, L.*, 2012. Identification of traditional medicinal plant extracts with novel anti-influenza activity, Oral presentation, In: Australian Society of Microbiology (ASM) Conference, July, 2012, Brisbane, Queensland, Australia

- Rajasekaran, D., Palombo, E.A., Yeo, T.C., Ley, D.L.S., Tu, C.L., Malherbe, F., Grollo, L., 2012. Identification of traditional medicinal plant extracts with anti-influenza activity, Poster presentation, In: ASM Conference, July, 2011, Hobart, Tasmania, Australia

* Speaker

Table of Contents

TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

DECLARATION ...... v

COMMUNICATIONS ...... vi

TABLE OF CONTENTS ...... vii

LIST OF TABLES ...... xiii

LIST OF FIGURES ...... xv

ABBREVIATIONS ...... xvii

CHAPTER 1 LITERATURE REVIEW ...... 1

1.1. Influenza virus infection ...... 1

1.2. Influenza pandemics ...... 1

1.3. Taxonomic classification of influenza viruses ...... 3

1.4. Virion structure and organization ...... 3

1.5. Structure of the viral genome ...... 5

1.6. Structure and functions of transmembrane proteins ...... 6

1.6.1. Hemagglutinin (HA) glycoprotein ...... 7

1.6.2. M2 proton channel ...... 9

1.6.3. Neuraminidase (NA) ...... 9

1.7. Other Proteins of influenza virus ...... 11

1.8. Influenza virus replication ...... 11

1.8.1. Viral entry into the host cell ...... 12

1.8.2. Release of vRNPs into the host cell nucleus ...... 14

1.8.3. Transcription and replication of the viral genome ...... 14

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Table of Contents

1.8.4. Export of vRNPs from the nucleus of the host cell ...... 15

1.8.5. Viral assembly and budding ...... 15

1.9. Clinical Manifestations of Influenza Viruses ...... 15

1.10. Prevention and treatment of influenza virus infection ...... 16

1.10.1. Vaccines ...... 16

1.10.2. Anti-influenza drugs ...... 18

1.10.2.1. M2 inhibitors...... 18

1.10.2.2. Neuraminidase inhibitors (NAIs)...... 19

1.10.2.3. Other investigational agents for influenza...... 20

1.11. Mutations that lead to antigenic variation of influenza virus ...... 23

1.12. The need for third generation anti-influenza drugs ...... 25

1.13. Plants- a potential source for antimicrobial compounds ...... 26

1.14. Historical evidences of medicinal plants’ usage from simple blends to compound drugs...... 27

1.15. Natural products in drug discovery ...... 28

1.16. Strategies for the discovery of antimicrobial compounds from plant sources ...... 30

1.17. Antiviral agents from plant sources ...... 30

1.18. Antiviral activity of plants against influenza viruses ...... 35

1.19. Chemical classes of plant metabolites showing anti-influenza activity ...... 39

1.19.1. Phenolic compounds ...... 39

1.19.2. Saponins ...... 40

1.19.3. Alkaloids ...... 40

1.19.4. Other compounds ...... 40

1.20. Aims of this study ...... 41

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Table of Contents

CHAPTER 2 MATERIALS AND METHODS ...... 42

2.1. Cells ...... 42

2.2. Viruses ...... 42

2.3. Plant Extracts ...... 44

2.4. Cytotoxicity studies of extracts ...... 45

2.5. In vitro micro-inhibition assay ...... 45

2.6. Time-of-addition assay ...... 46

2.7. Virus binding (attachment) assay...... 46

2.8. Penetration assay ...... 46

2.9. Neuraminidase (NA) inhibition assay ...... 47

2.10. Hemagglutination inhibition (HI) test ...... 47

2.11. RDE Treatment ...... 48

2.12. Trypsin treatment ...... 48

2.13. Analysis of crude extract and fractions with influenza virus using transmission electron microscope (TEM) ...... 48

2.14. Plaque neutralisation assay ...... 49

2.15. Solid phase extraction (SPE) ...... 50

2.16.GC-MS analysis of extract and fractions ...... 50

2.17. Statistical analysis ...... 51

CHAPTER 3 CYTOTOXICITY AND ANTIVIRAL SCREENING OF PLANT EXTRACTS ...... 52

3.1. Introduction ...... 52

3.2. Results ...... 54

3.2.1. Cytotoxicity studies of all fifty plant extracts ...... 54

3.2.2. Inhibitory effects of plant extracts on influenza virus ...... 57

3.3. Discussion ...... 63

Table of Contents

CHAPTER 4 MODE OF ACTION STUDIES ON ANTI-INFLUENZA MEDICINAL PLANT EXTRACTS ...... 65

4.1. Introduction ...... 65

4.2. Results ...... 67

4.2.1. Anti-influenza extracts studied with a time-of-addition assay ...... 67

4.2.2. Inhibitory effects of extracts on the attachment of H3N1 and H1N1 ...... 72

4.2.3. Inhibitory effects of extracts on the penetration of influenza virus ...... 75

4.2.4. NAI effects of plant extracts ...... 78

4.2.5. Inhibitory effects of plant extracts on influenza virus-induced hemagglutination...... 79

4.2.6. Effect of RDE treatment on the antiviral activity of extracts ...... 82

4.2.7. Effect of Trypsin treatment on the antiviral activity of extracts ...... 85

4.2.8. TEM study of Extract 8...... 87

4.3. Discussion ...... 90

CHAPTER 5 ANTIVIRAL ACTIVITY OF SELECTED MEDICINAL PLANT EXTRACTS AGAINST TYPE A AND B INFLUENZA VIRUSES INCLUDING NAI RESISTANT STRAINS ...... 93

5.1. Introduction ...... 93

5.2. Results ...... 95

5.2.1. Inhibitory effects of plant extracts against seasonal Type A H1N1 virus and Oseltamivir resistant seasonal Type A H1N1 virus with H275Y mutation ...... 95

5.2.2. Inhibitory effects of plant extracts against pandemic Type A H1N1 virus and Oseltamivir resistant pandemic Type A H1N1 virus with H275Y mutation ...... 98

5.2.3. Inhibitory effects of plant extracts against Type B virus and Type B virus with D197E mutation ...... 100

5.2.4. NAI effects of plant extracts ...... 102

5.2.5. Inhibitory effects of plant extracts on viral HA ...... 104

5.2.6. Effect of RDE treatment on the antiviral activity of plant extracts ...... 106

5.2.7. Effect of RDE treatment on the HI activity of plant extracts ...... 108

Table of Contents

5.2.8. Effects of RDE treatment on the NAI activity of plant extracts ...... 110

5.3. Discussion ...... 112

CHAPTER 6 STUDIES TO DETERMINE THE CHEMICAL PROPERTIES OF MEDICINAL EXTRACT 8 ...... 117

6.1. Introduction ...... 117

6.2. Results ...... 119

6.2.1. Bioactivity-guided fractionation of Extract 8 ...... 119

6.2.2. Inhibitory activities of Extract 8 and its fractions ...... 121

6.2.3. Inhibitory effects of Extract 8 fractions against six influenza viruses ...... 123

6.2.4. Effects of plant extract against influenza virus-induced hemagglutination ...... 125

6.2.5. NA inhibitory effects of plant extract fractions ...... 127

6.2.6. Effects of RDE treatment on the antiviral activity of Extract 8 and its fractions...... 129

6.2.7. GC-MS analyses of crude extract and fractions ...... 131

6.3. Discussion ...... 135

CHAPTER 7 SUMMARY AND CONCLUSIONS ...... 139

7.1. Cytotoxicity and antiviral screening ...... 139

7.2. Mode of action studies ...... 140

7.3. Antiviral activities of selected medicinal plant extracts against Type A and B viruses, including NAI resistant strains ...... 141

7.4. Chemical fractionation and antiviral assessment of Extract 8 ...... 143

BIBLIOGRAPHY ...... 145

APPENDICES ...... 161

APPENDIX I ...... 161

AI.1. Tropical rainforests of Sarawak ...... 161

AI.2. Mussaenda elmeri (Rubiaceae) ...... 161

AI.3. Calophyllum lanigerum (Clusiaceae)...... 162

Table of Contents

AI.4. Albizia corniculata (Fabaceae) ...... 162

AI.5. Trigonopleura malayana (Euphorbiaceae) ...... 162

AI.6. Santiria apiculata (Burseraceae) ...... 163

AI.7. Anisophyllea disticha (Anisophylleaceae) ...... 163

AI.8. Trivalvaria macrophylla (Annonaceae) ...... 163

AI.9. Baccaurea angulata (Euphorbiaceae) ...... 164

AI.10. Tetracera macrophylla (Dilleniaceae) ...... 164

APPENDIX II ...... 165

APPENDIX III ...... 166

APPENDIX IV ...... 167

APPENDIX V ...... 168

APPENDIX VI ...... 177

APPENDIX VII ...... 178

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LIST OF TABLES

Table 1.1. Influenza A pandemics………………………………………………………2

Table 1.2. Antiviral agents for influenza virus under development……………………21

Table 1.3. Some plants with reported antiviral activities against herpes virus………...32

Table 1.4. Antiviral activities of plants against HIV…………………………………..33

Table 1.5. Antiviral activities of plants against other viruses………………………….34

Table 1.6. Antiviral activity of plants against influenza viruses……………………….36

Table 1.7. Herbal drugs for the treatment of influenza……………………………...... 38

Table 2.1. List of influenza viruses that were employed to perform antiviral assays….43

Table 2.2. Extracts obtained from plant materials……………………………………..44

Table 3.1. Cellular toxicity concentrations of all extracts tested………………………55

Table 3.2. Medicinal plant extracts from Sarawak demonstrating antiviral activity against H3N1 and H1N1 strains………………………………………………………..60

Table 3.3. Inhibitory concentrations of extracts against H3N1 and H1N1 strains……..62

Table 4.1. Inhibitory effects of plant extracts against H3N1 at various time points of viral inoculation………………………………………………………………………...68

Table 4.2. Inhibitory effects of plant extracts against H1N1 at various time points of viral inoculation……………………………………………………………………...... 71

Table 4.3. Inhibitory effects of anti-influenza extracts on the binding of H3N1 and H1N1 strains…………………………………………………………………………....74

Table 4.4. Inhibitory effects of anti-influenza extracts on the penetration of H3N1 and H1N1 strains at 30 and 120 min………………………………………………………..76

Table 4.5. NAI activity of extracts……………………………………………………..78

Table 4.6. NAI assay using commercial drugs…………………………………………79

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List of Tables

Table 5.1. Inhibitory concentration of anti-influenza extracts…………………………96

Table 5.2. NAI activity of anti-influenza extracts…………………………………….103

Table 6.1. Inhibitory activities of crude extract and fractions in reducing Mem-Bel and PR8 viral plaques……………………………………………………………………...121

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LIST OF FIGURES

Figure 1.1. Structure of influenza A virus...... 3

Figure 1.2. Genomic organisation of influenza A virus...... 5

Figure 1.3. Reproductive cycle of influenza virus...... 13

Figure 1.4. Antigenic variations of influenza virus...... 25

Figure 3.1. Cytotoxicity effects of plant extracts...... 56

Figure 3.2. Inhibitory effects of plant extracts on H3N1 virus...... 58

Figure 3.3. Inhibitory effects of plant extracts on H1N1 influenza virus...... 59

Figure 4.1. Inhibitory effects of plant extracts against H3N1 at various time points of viral inoculation...... 69

Figure 4.2. Inhibitory effects of plant extracts against H1N1 at various time points of viral inoculation...... 70

Figure 4.3. Inhibitory effects of plant extracts on the binding of H3N1 and H1N1 virus...... 73

Figure 4.4. Inhibitory effects of anti-influenza extracts on the penetration of H3N1 and H1N1 strains at 60 min...... 77

Figure 4.5. Inhibitory effects of plant extracts on influenza virus-induced hemagglutination...... 80

Figure 4.6. Effect of RDE treatment on plant extracts’ activity...... 82

Figure 4.7. Effect of RDE treatment on the antiviral activity of plant extracts against H3N1...... 83

Figure 4.8. Effect of RDE treatment on the antiviral activity of plant extracts against H1N1...... 84

Figure 4.9. Effect of trypsin treatment on the antiviral activity of plant extracts...... 86

List of Figures

Figure 4.10. TEM image of PR8 virus...... 88

Figure 4.11. TEM image of PR8 virus treated with crude Extract 8...... 89

Figure 5.1. Inhibitory effects of plant extracts against seasonal H1N1 viruses...... 97

Figure 5.2. Inhibitory effects of plant extracts against pandemic H1N1 viruses...... 99

Figure 5.3 Inhibitory effects of plant extracts against Type B influenza virus...... 101

Figure 5.4. Inhibitory effects of plant extracts on influenza virus-induced hemagglutination...... 105

Figure 5.5 Effects of RDE treatment on the antiviral activity of plant extracts...... 107

Figure 5.6. Effect of RDE treatment on the HI activity of plant extracts...... 109

Figure 5.7 Effect of RDE treatment on the NAI activity of plant extracts...... 111

Figure 6.1. Inhibitory activities of Extract 8 fractions...... 120

Figure 6.2.Inhibition of viral plaques by Extract 8 and its fractions...... 122

Figure 6.3. Inhibitory activities of Extract 8 fractions against influenza viruses...... 124

Figure 6.4. Effects of fractions against influenza virus-induced hemagglutination...... 126

Figure 6.5. NAI effects of the 40% ACN fraction and Extract 8...... 128

Figure 6.6. Effect of RDE treatment on the inhibitory activity of plant fractions...... 130

Figure 6.7. Components of Extract 8...... 132

Figure 6.8. Components of 40% ACN fraction of Extract 8...... 133

Figure 6.9. Components of 100% ACN fraction of Extract 8...... 134

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ABBREVIATIONS

ºC Degree Celsius

µg Microgram

µL Micro litre

µm Micrometer

ACN Acetonitrile

ANOVA Analysis of Variance

BM2 Type B influenza virus matrix protein 2

CC50 50% Cytotoxic Concentration

CDC Centres for Disease Control and Prevention

CL Chemiluminescence

CM2 Type C influenza virus matrix protein 2

CO2 Carbon dioxide

CPSF Cleavage and Polyadenylation Specificity Factor

CPE Cytopathic Effect

CRBC Chicken Red Blood Cell

CRM1 Chromosome Region Maintenance 1

CTD Carboxy Terminal Domain

xvii

Abbreviations

DMSO Dimethyl Sulfoxide

FBS Foetal Bovine Serum

FL Fluorescence

FP Fusion Peptide

GC Gas Chromatography

GC-MS Gas Chromatograph Mass spectrometer h Hour

HA Hemagglutinin

HEF Hemagglutinin esterase fusion protein

HI Hemagglutination Inhibition (OR) Hemagglutination Inhibitory

HIV Human Immunodeficiency Virus

HPLC High Performance Liquid Chromatography

IC50 50% Inhibitory Concentration

Ig Immunoglobulin

MAb Monoclonal Antibody

MDCK Madin Darby Canine Kidney min Minute mL Millilitre

xviii

Abbreviations

mRNA Messenger Ribonucleic Acid

MPLC Medium-Pressure Liquid Chromatography

MS Mass Spectral

MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

MU 4-methylumbelliferone

MUNANA 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid

M1 Matrix Protein 1

M2 Matrix Protein 2

N (or) NA Neuraminidase

NAI Neuraminidase Inhibitor(y)

NAD(P)H Nicotinamide Adenine Dinucleotide Phosphate, reduced form

NB Type B influenza virus matrix protein

NEP Nuclear Export Protein

NIST National Institute of Standards and Technology

NLS Nuclear Localization Signal nm Nanometre nM Nanomolar

NP Nucleoprotein

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Abbreviations

NPs Natural Products

NS1 Non-Structural protein

NS2 Non-Structural protein 2

OD Optical Density

PA Polymeric Acid protein

PABP(II) Poly (A) Binding Protein II

PBS Phosphate Buffered Saline

PB1 Polymerase Basic protein 1

PB2 Polymerase Basic protein 2

PC6 Proprotein Convertase 6 pfu Plaque Forming Unit

Pol (A) Polyadenylation

Pol (II) RNA Polymerase II

RBC Red Blood Cell

RDE Receptor Destroying Enzyme

RdRp Ribonucleic Acid -dependant Ribonucleic Acid polymerase

RNP Ribonucleoprotein

RNA Ribonucleic Acid

Abbreviations

rpm Revolutions Per Minute

RPMI Rosewell Park Memorial Institute

SA Sialic Acid

SBC Sarawak Biodiversity Centre

SD Standard Deviation

SEM Standard Error of the Mean

SnRNA Small Nuclear RNA

SPE Solid Phase Extraction

TCHM Traditional Chinese Herbal Medicine

TCID50 50% Tissue Culture Infectious Dose

TEM Transmission Electron Microscope(y)

VLC Vacuum Liquid Chromatography vRNA Viral Ribonucleic Acid

WHO World Health Organisation

Single letter abbreviations for amino acids and nucleobases

A Adenine

D Aspartic acid

E Glutamic acid

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Abbreviations

H Histidine

I Isoleucine

K Lysine

N Asparagine

R Arginine

S Serine

U Uracil

Y Tyrosine

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Chapter 1

CHAPTER 1 LITERATURE REVIEW

1.1. INFLUENZA VIRUS INFECTION

Influenza viruses cause acute respiratory infections that are generally referred to as “flu”, which, in more severe cases, may lead to secondary infections like pneumonia. Being a major causative agent for recurrent epidemics and pandemics, the virus affects 5% to 10% of the workforce annually, leading to significant impacts on the economy (Rothberg and Rose, 2005). Worsening this situation, about 10% of the world population is annually infected by the virus, contributing to around 250, 000 deaths (McCaughey, 2010). Thus, substantial annual fatality and the occasional pandemic caused by influenza virus affects human and animal health, which in turn have devastating consequences over the global economy (Hudson, 2009). Also, considerable mortality caused within the high risk groups comprising children, the elderly and victims of cardio pulmonary diseases, indicates the infectious nature of influenza viruses (Pleschka et al., 2009). Prior to the advent of airplanes and globalization, about 20-100 million people died due to the 1918 H1N1 Spanish flu (McCaughey, 2010). At present, without proper containment, a flu epidemic like the Spanish flu may affect hundreds of millions of people globally in a few days. Concerns regarding a major pandemic became apparent with the outbreak of A/H5N1, the avian influenza. Though person-to-person transmission of A/H5N1 is limited, the worst case scenario of viruses developing potential to cause efficient human-to-human transmission may cause a death toll higher than the destructive Spanish flu (Grienke et al., 2012).

1.2. INFLUENZA PANDEMICS

Influenza virus causes pandemics on a regular basis. The rapid emergence of a new virus which instigates an epidemic and affects the global population is referred to as a ‘pandemic’. The world has seen at least three influenza pandemics in the last century. The lack of pre-existing immune protection within the human population is a major factor for the spread of a new viral strain during pandemics. Typically, an influenza pandemic occurs once in every 10 to 40 years. The 1918 (H1N1) pandemic is considered as one of the biggest natural disasters that have affected mankind. Table 1.1. details influenza pandemics that resulted in significant mortality (McCaughey, 2010).

Chapter 1

Table 1.1. Influenza A pandemics, adapted from McCaughey. (2010)

Year of Virus subtype Comments Estimated deaths emergence 1889 H2N2 Pandemic 1 million Antigenic type analysed by retrospective serological testing of stored sera.

1900 H3N2 Pandemic uncertain, <1 million Antigenic type analysed by retrospective serological testing of stored sera.

1918 H1N1 ‘Spanish’ Pandemic 20 to 100 million

1957 H2N2 ‘Asian’ Pandemic 1 to 1.5 million

1968 H3N2 Pandemic 0.75 to 1 million ‘Hong Kong’

1977 H1N1 ‘Russian’ Not considered to be a <100 000 pandemic in the sense that severity and attack rate were lower than with previous antigenic shift events. Widely speculated to be a virus of laboratory origin.

2009 H1N1 2009 Pandemic <100 000 ‘Mexican swine’

Chapter 1

1.3. TAXONOMIC CLASSIFICATION OF INFLUENZA VIRUSES

Influenza virus belongs to the family Orthomyxoviridae, a family of RNA viruses that consists of six genera: Influenza virus A, Influenza virus B, Influenza virus C, Isavirus, Thogotovirus and a newly discovered genus which is still unnamed (Presti et al., 2009). Influenza viruses A, B and C affect vertebrates, including birds, humans and other mammals. Isavirus infects salmon whereas thogotovirus infects both vertebrates and invertebrates (Jones and Nuttall, 1989, Raynard et al., 2001).

1.4. VIRION STRUCTURE AND ORGANIZATION

Electron microscopy studies reveal that influenza viruses possess spherical or filamentous organisation. The spherical and filamentous forms of types A and B are on the order of 100 nm and 300 nm, respectively. Influenza viruses are classified into three types based on structural differences (Rossman and Lamb, 2011). Figure 1.1 illustrates the structure of the influenza A virus (Flint, 2009).

Figure 1.1. Structure of influenza A virus

Influenza A virus composed of an eight segmented negative sense single stranded RNA genome covered by a matrix protein and a lipid envelope is shown along with the viral proteins, adapted from Flint, 2009.

Chapter 1

The genomes of types A and B comprise eight segments of negative-sense single- stranded RNA, whereas the one of Type C is composed of seven segments. Eleven proteins are encoded by the eight segmented RNA genome, and include hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), matrix protein 2 (M2), nucleoprotein (NP), non-structural protein (NS1), non-structural protein 2 (NS2 or nuclear export protein, NEP), polymeric acid protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase basic protein 1–F2 (PB1-F2). Of these, eight proteins are packaged into the virion. As shown in Figure 1.1, HA and NA glycoproteins, which serve as the main antigenic determinants of influenza virus, are arranged as spikes on the surface of the virus. The matrix ion channels (M2), the third integral membrane protein that spans the viral envelope, are limited in number (Rossman and Lamb, 2011, Samji, 2009).

The ratio of HA: NA is approximately 4:1 while M2: HA is 1:10-100. About 80% of the virus envelope protein is made up of HA, whereas NA accounts for 17% of the total transmembrane proteins. Also, 16 to 20 molecules of M2 are present in a virion, thus constituting a very minor component of the viral envelope (Samji, 2009). The virion core is surrounded by a matrix (M1) protein, which, in turn, is surrounded by the lipid envelope. Interior to the matrix protein are the NEP and the ribonucleoprotein (RNP) complex, which is made up of viral segments coated with NP and RNA polymerase. Influenza B viruses possess four envelope proteins in their structure with similar organization for HA and NA but NB (Influenza B virus protein), an ion channel protein encoded by RNA segment 6 which also encodes NA and BM2 (Type B influenza virus matrix protein 2), is present instead of M2. The Type C viruses which form cord-like structures in the order of 500 µm has only one glycoprotein, the hemagglutinin-esterase- fusion (HEF) protein which performs the role of both HA and NA, along with a minor envelope protein, CM2 (Type C influenza virus Matrix protein 2) (Bouvier and Palese, 2008, McCaughey, 2010, Hatta and Kawaoka, 2003).

Type A and B viruses are the predominant causes of human influenza infections (Nguyen et al., 2011), with Type A being the major causative agent for epidemics. Definite antigenic properties are conferred onto the virus by virion surface glycoproteins, HA and NA. Based on the antigenicity of the two surface glycoproteins, HA and NA, there are currently 17 HA (H1-H16) and 9 NA (N1-N9) subtypes

Chapter 1 recognized in Type A virus, with most subtypes present in waterfowl and shorebirds (Krauss et al., 2007, Tong et al., 2012).

1.5. STRUCTURE OF THE VIRAL GENOME

The types A and B viruses possess eight negative-sense; single stranded viral RNA segments whereas Type C virus has a seven-segment genome. The segments are numbered with respect to their length starting from the most elongated (Bouvier and Palese, 2008). Figure 1.2 shows the genomic organisation of the Type A virus (Flint, 2009).

Figure 1.2. Genomic organisation of influenza A virus

The eight-segmented RNA genome of influenza A virus that encodes different viral proteins is shown. Five segments (1, 3, 4, 5, 6) code for one protein (PB2, PA, HA, NP, NA) and three segments (2, 7 and 8) code for two proteins each (PB1& PB1-F2, M1& M2, NS1& NEP), adapted from Flint, 2009.

As shown in Figure 1.2, the RNA segments 1, 3, 4 and 5 of types A and B viruses code for PB2, PA, HA and NP proteins, respectively. The polymerase protein, PB1, is encoded on segment 2 in all influenza viruses; some strains of Type A virus also code for an accessory protein PB1-F2 which is pro-apoptotic. In Type A virus, segment 6 encodes only NA protein, but in Type B, an additional NB matrix protein which functions like that of M2 in Type A virus, is also encoded. The M1 matrix protein is encoded by segment 7 in both Type A and B viruses. Segment 8 of types A and B

Chapter 1 viruses express the interferon-antagonist NS1 protein and the NEP/NS2 protein, involved in viral RNP export from the host cell nucleus. Since the HEF protein of influenza C replaces both HA and NA proteins, the virus genome of Type C has one segment less than that of influenza viruses A and B (Bouvier and Palese, 2008).

The helical hairpin structure formed by the ends of each viral RNA (vRNA ) segment is bound by the RNA polymerase complex. The rest of the segment is coated with arginine-rich NP. The net positive charge of NP binds the negatively charged phosphate backbone of the vRNA. Non-coding regions of different lengths are present at both 3′ and 5′ ends of each vRNA segment. These regions also consist of the mRNA polyadenylation signal and a segment of the packaging signals required for viral assembly. All virus segments have highly conserved ends that function as the promoter for vRNA replication and transcription processes by the viral polymerase complex (Bouvier and Palese, 2008).

1.6. STRUCTURE AND FUNCTIONS OF TRANSMEMBRANE PROTEINS

The influenza A virus membrane comprises three proteins: HA, NA and M2. Influenza NA, a glycoside hydrolase, and HA identify carbohydrate structures and attach to terminal sialic acid (SA) components on the surface of the host cell (Grienke et al., 2012). The primary role of the HA protein occurs at the initial stages of infection and include SA-receptor binding; fusion of virus and cell membranes after virus uptake into endosomes. The M2 protein functions to transfer protons into the infecting virus in endosomes and dissociate the matrix protein M1 from the genome–transcriptase complex at acidic pH, so that the uncoated complex is transported to the nucleus after membrane fusion. At the end of infection, SA from the virus and cell glycoconjugates is cleaved by NA in order to release newly-made viruses from infected cells. Thus HA facilitates entry, NA regulates release and M2 aids in viral uncoating in target cells (Russell et al., 2008). Since HA and NA play opposing roles, a stable coaction is vital for effective entry and release of influenza virus (Grienke et al., 2012).

Chapter 1

1.6.1. Hemagglutinin (HA) glycoprotein

The HA gene segment of the influenza virus codes for HA, a glycoprotein that is expressed on the viral capsid. To date, 17 HA (H1-H17) subtypes have been identified, with most subtypes being present in waterfowl and shorebirds. The subtypes of HA are further grouped into two phylogenetic groups: Group 1, which includes sub-types H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and Group 2, which includes sub-types H3, H4, H7, H10, H14 and H15. H17 is closely related to Group 1 HAs but its definitive taxonomic classification is still pending (Tong et al., 2012, Velkov et al., 2013).

The principal function of HA is to initiate internalization of the virus into the host cell by binding to the cell’s SA receptors. SAs are monosaccharides belonging to a multifarious family of sugars containing a nine carbon backbone that exists in branched and unbranched organizations, and associated with different chemical groups. Generally, SAs are present at the distal end of glycan chains which is a characteristic of all cell types. As a result, the capacity to bind to the different types of SAs is a vital factor for the infectivity of human influenza strains (Wilks et al., 2012).

The binding specificity of HA partly defines the ability of influenza viruses to infect birds or humans. Generally, HAs of human strains of influenza preferentially bind to SAs attached through a α2-6 linkage to the terminal galactose (SAα2-6) of the oligosaccharides on the cell surface. These types of linkages are abundant in human respiratory epithelia, whereas, the HA of avian strains bind preferentially to 2,3-linked SAs (SAα2-3), which are frequent in the avian intestinal tract (Ramos et al., 2011). The preferred binding of human influenza virus to α2-6 linked SAs is related to a structural topography. Cone-like and umbrella-like structural conformations of SAs exist in the glycan of host cells. SAs of adequate length acquire an umbrella-like form due to the angle constraint imposed by the α2-6 linkage. Shorter SAs and SAs with a α2-3 linkage take up the cone-like arrangement. Viruses which possess the capacity to bind to the umbrella-like conformation acquire potential for human transmission. Therefore, mutations that confer binding to the umbrella conformation of SA are selected by the virus to infect humans. Strains possessing the ability to bind α2-6 linkages but exhibiting a low level of transmission may have the potential to bind only the shorter SAs. Hence, binding of the HA to SA receptor represents a primary step in viral replication (Wilks et al., 2012). 7

Chapter 1

Each chain of an individual HA molecule is synthesized as a precursor polypeptide, HA0, which is cleaved into two subunits, a globular surface unit HA1 and stalk-like membrane unit HA2, by the host’s proteases. Type A viruses cannot infect the host cell unless HA undergoes proteolytic cleavage, thereby exposing the N-terminal hydrophobic regions. Virulent and avirulent avian influenza viruses have different sequences at the HA cleavage sites. The virulent strains possess a series of basic amino acids at the HA cleavage sites, which are not present in the avirulent strains. These multiple basic residues are recognized by the host cell’s proteases furin and proprotein convertase 6 (PC6) that cleave the HAs of virulent viruses, leading to systemic infection throughout the body. In contrast, the HAs of avirulent viruses are not cleaved by the proteases as they lack these basic residues. They are nonetheless vulnerable to proteases, which are most likely contained within the respiratory and/or intestinal tract, thus leading to localized viral infection. Thus, HA is an influential factor for the severity of infections in avian strains (Goto et al., 2001).

The subunits HA1 and HA2 play different roles in the process of viral entry; HA1 promotes receptor binding onto the host cell surface, whereas HA2 facilitates membrane fusion. Despite glycosylation, folding and trimerisation, HA is unable to conciliate membrane fusion and the host cell proteases cleave it at a linker sequence connecting HA1 and HA2 subunits. Cleavage at the carboxyl terminus of an arginine present between HA1 and HA2 results in the formation of N-terminus of mature HA2, while the C-terminus of mature HA1 is formed by the removal of arginine via the action of a carboxypeptidase. These cellular enzymes are prerequisites for viral infection (Bertram et al., 2010).

Once viral entry is facilitated, multiple endocytic pathways result. Membrane fusion is facilitated by a low pH environment in the endosomal vesicle that confers conformational changes upon HA2. Successful membrane fusion requires the combined action of definite elements in HA2, including an N-terminal fusion peptide (FP), transmembrane domain and a central coiled coil. The pH change causes a loop-to-helix transition of a sequence present between FP and the coiled coil. As a result of this transformation, the hydrophobic FP is pushed towards the target cell and implants into the cellular membrane. Stable post-fusion structures, where FP and the membrane fusion anchor of HA are in close contact, result when the C-terminal extracellular

Chapter 1 portion of HA2 folds onto the central coiled coil in an antiparallel manner. Consequently, the viral and host cell membrane are dragged in close proximity and the fusion process occurs (Bertram et al., 2010, Du et al., 2012b).

1.6.2. M2 proton channel

The M2 protein of the virus, a small integral membrane protein that functions as an acid-activated ion channel, is essential for the organized release of the nucleocapsid after viral fusion with the endosomal membrane. The M2 proton channel is stimulated by the low endosomal pH; the basic structure is a tetramer composed of monomers whose transmembrane domains form a channel that acts as a proton-selective ion channel. Each monomer consists of 97 residues with the N-terminal residues positioned at three parts of the viral structure: peripheral to the viral membrane are 25 residues, the next 21 residues form a single transmembrane helix and the hydrophobic-hydrophilic interface has the 16-residue amphipathic helix at the viral membrane. The C-terminal 35 residues are positioned within the virus. The amphipathic helix is correlated with virus budding whereas M1 binding is a function of the C-terminal portion. The M2 proton channel is lined by the H37-W41 cluster, which guards proton transport through the channel. Proton selectivity and activation by low pH are functions of H37, while W41 acts like a gate that precludes the proton current from flowing outward. Acidification within the endosome, due to the entry of protons into the interior of the virus through the M2 proton channel, promotes viral uncoating during the replication of influenza virus (Du et al., 2012a) .

1.6.3. Neuraminidase (NA)

Influenza neuraminidase, a glycoside hydrolase, is an antigenic glycoprotein on the surface of influenza. The major roles played by neuraminidase glycoprotein are efficient release of viral progeny particles from viral exit sites by cleaving the terminal SA receptors on cell membranes from the infected cell and mobilization of virus in the respiratory tract. The sialidase activity of NA cleaves the α2-3 and α2-6 glycosidic terminal SAs, thereby, decreasing the amount of HA receptor binding sites on the host cell. This process results in the detachment of mature virus from the host cell, thereby preventing the self-aggregation caused by the HA. Also, NA cleaves the glycan structure of mucus, thereby helping influenza virus to mobilize within the respiratory

Chapter 1 tract. Thus, NA plays a crucial role in spreading the virus within infected tissues and facilitates virus transmission during infection.

NA consists of four identical subunits forming a mushroom shaped homotetramer. The head of this tetramer is made up of four catalytic domains and the stalk region is formed by the extended N-terminal sequences of the four subunits. Within each subunit, conserved residues form the catalytic active site. Variations in length and glycosylation patterns of the stalk amino sequence may be present in the different subtypes, although a membrane anchoring helix is conserved for each monomer (Grienke et al., 2012).

The nine subtypes of NA are classified on the basis of antigenicity and are further grouped phylogenetically: Group 1 consists of N1, N4, N5 and N8 subtypes and Group 2 consists of N2, N3, N6, N7 and N9 subtypes. N1 and N2 are the only subtypes currently found in viral strains that cause human infections, while all subtypes are found in avian hosts. NAs possess well-conserved residues around the active sites. Group 1 NAs are structurally different from Group 2. A pocket formation at the 150-loop where it opens a cavity adjacent to the active site is a characteristic feature of Group 1 NAs, but the 2009 H1N1 virus lacks this cavity (Du et al., 2012a).

Apart from facilitating the release of viral progeny from infected cells and helping in the spread of virus within the respiratory tract, NA is also important for viral entry (Hsieh et al., 2012b). The role of NA in removing SA residues from HA could improve fusion and infectivity of influenza virus following three mechanisms:

I. Glycoconjugates carried by various lipids and proteins expressed at the surface of the host cell could interfere with the receptor binding site of HA spikes. By masking these glycoconjugates, NA enables the binding of HA to SA receptors present at the host cell surface II. Desialylation of HA by NA could also remove acid sialic residues of HA protein, and this partial unmasking could facilitate further binding of HA to the target cell surface receptors, thereby augmenting HA-mediated fusion III. Desialylation of HA may help in the proteolytic cleavage of the HA0 precursor into its functional subunits HA1 and HA2 where the removal of N-glycosylation sites near HA0 cleavage site could modify its approach to cellular proteases (Su et al., 2009b).

Chapter 1

Recent reports also indicate an additional function of NA in viral assembly and budding from host cells (Ernst et al., 2013).

1.7. OTHER PROTEINS OF INFLUENZA VIRUS

Apart from the transmembrane proteins, the influenza virus has other functional proteins required for efficient replication and spread. Influenza RNA polymerase is composed of two polymerase complex proteins (PB1 and PB2) and one polymerase acidic subunit (PA), and its main functions are to initiate transcription and replication of viral RNA using the host cell machinery. Nucleoprotein (NP) plays a major role in the encapsidation of segmented RNA and binding with the three polymerase subunits, PA, PB1 and PB2 to create ribonucleoprotein particles (RNPs), required for RNA transcription, replication and packaging. Within each RNP, the viral RNA is enclosed by individual NP molecules, which are attached to one another by depositing the ‘tail loop’ (residues 402-428) of one NP molecule (498 residues) within an adjacent NP molecule. RNPs are bound with the M1 protein in mature virions and during the assembly of newly formed RNPs which are ready for export from the host cell nucleus. NP oligomerization mediated by tail-loop binding is essential for the transcription and replication activity of RNPs (Rossman and Lamb, 2011, Du et al., 2012a).

The influenza virus also expresses three other proteins that are not incorporated into the mature virion. The matrix protein M1 imparts structure to the virion; combines interplay between the viral lipid membrane and the RNP core, thus forming the major driving force in virus budding. Nuclear export protein (NEP or NS2) mediates the export of viral RNPs from the host cell nucleus during virus replication. Non-structural protein 1 (NS1) is a multi-functional protein which protects viruses against the antiviral responses mediated by interferon α/β in infected cells (Rossman and Lamb, 2011, Du et al., 2012a).

1.8. INFLUENZA VIRUS REPLICATION

Influenza virus requires the host cell’s machinery in order to replicate. The life cycle of influenza virus can be divided into the following stages: viral entry into the host cell; release of vRNPs into the host cell nucleus; transcription and replication of viral RNA; export of vRNPs from the nucleus; viral assembly and budding from the host cell

Chapter 1 plasma membrane (Samji, 2009). A schematic representation of the life cycle of influenza virus is shown in Figure 1.3. ( Flint, 2009).

1.8.1. Viral entry into the host cell

As discussed earlier, the HA protein helps in the binding of the virus to the SA residues present at the surface of the host cell. As a result of receptor-mediated endocytosis, the virus enters the host cell in an endosome. The effect of pH plays a major role in the fusion of viral and endosomal membrane. The acidic environment in the endosome (pH around 5 to 6) enables HA to undergo conformational rearrangements exposing the HA2 fusion peptide, a hydrophobic segment of the HA molecule which is essential for viral fusion. Experimental studies have shown that fusion activity is maximal when the virus is bound to the target membrane before exposure to low pH. Also, the fusion process occurs only at low pH even after the exposure of the hydrophobic segment. Higher temperature is essential for fusion activity highlighting the importance of unfolding and unified action of HA trimers. Fusion activity was inactivated due to denaturation of HA at pH 4.8 and below, both at 0ºC and 37ºC. Insertion of the fusion peptide into the endosomal membrane brings the viral and endosomal membranes in close proximity, thereby causing fusion. Lower endosomal pH also opens up the M2 ion channel which acidifies the viral core due to the entry of protons into the virus. This process of viral core acidification releases vRNPs from M1 thereby allowing vRNP to enter the cytoplasm of the host cell (Stegmann et al., 1987, Ivanovic et al., 2012, Samji, 2009).

Chapter 1

Figure 1.3. Reproductive cycle of influenza virus

The replication of influenza virus requires a stable coaction of HA and NA while the functions of other viral proteins such as M1, M2, NP, NSI, NEP, PA, PB1, PB2, PB1-F2 are also vital for the completion of viral reproductive cycle, adapted from Flint, 2009.

Chapter 1

1.8.2. Release of vRNPs into the host cell nucleus

The vRNP released into the host cell must enter the nucleus in order to undergo transcription and replication. The nuclear localization signals (NLSs) present on the viral proteins that make up the vRNA, namely NP, PA, PB1 and PB2, help in binding to the cellular nuclear import machinery, which results in the release of vRNPs into the host cell nucleus (Samji, 2009).

1.8.3. Transcription and replication of the viral genome

One of the pre-requisites for the transcription of influenza viral genome is the conversion of negative-sense RNA into a positive-sense RNA that will serve as a template for the production of viral RNAs. RNA synthesis is initiated internally on the viral RNA by viral RNA-dependant RNA polymerase (RdRp). PB2, one of the three viral proteins (PB1, PB2 and PA) that make up the viral RdRp, binds to the 5΄ methylated caps of host cellular mRNAs and cleave the cellular mRNAs 10 to 15 nucleotides 3΄ to the cap structure which is later used by RdRp to prime viral transcription ( Samji, 2009).

In the event of transcription initiation, serine 5 on the C-terminal repeat domain (CTD) of cellular RNA polymerase II (Pol II), which binds to DNA and initiates transcription, is phosphorylated, resulting in the activation of cellular cap synthesis complex. Binding of RdRp to this form of Pol II may result in the “cap snatching” event through which the viral mRNAs acquire their 5΄ methylated cap. The virus uses the host cell’s splicing machinery to express NS1 and M1, which occur as spliced products of segments 7 and 8, respectively. The virus prevents the host cell from using its own cellular splicing machinery for processing the host cell mRNAs by the binding of NS1 to U6 small nuclear RNAs (snRNAs) and other splicing components which causes them to relocate to the nucleus of the infected cells (Samji, 2009).

Viral mRNAs lack the polyadenylation signal (AAUAAA) and the binding of viral RdRp to the 5΄ end of template viral RNA leads to steric blockage at the end of viral RNA synthesis. At approximately 17 nucleotides from the 5΄ end there is a stretch of five to seven U residues in each viral segment that serve as the basis for the viral polyadenylation signal. A stuttering mechanism, where RdRp moves back and forth over this stretch of U residues, results in the formation of a poly (A) tail. Binding of

Chapter 1

NS1 to the cleavage and polyadenylation specificity factor (CPSF) and poly(A) binding protein II (PABPII) prevents cellular mRNAs from being cleaved at the polyadenylation cleavage site, thus preventing the nuclear export of cellular mRNAs (Samji, 2009).

1.8.4. Export of vRNPs from the nucleus of the host cell

The chromosome region maintenance 1 (CRM1) dependent pathway appears to export vRNP out of the nucleus. Interaction of NP with CRM1 may be critical for the export of vRNPS. M1 protein interacts with the C-terminal end of the protein, whose N-terminal portion contains a NLS, essential for the import of vRNPs. The binding of N-terminal portions of M1 to NEP masks the NLS. NEP also binds to CRM1 along with GTP hydrolysis which generally occurs in a CRM1-dependent export pathway. It is hypothesized that M1 binds to both NEP and the negative-sense vRNPs. NEP also binds to CRM1 forming a “daisy-chain” complex through which the vRNPs are exported out of the nucleus. Live imaging of the influenza life cycle has shown that NP prefers to localize at the apical side of the infected nuclei, indicating polarized exit of the viral genome ( Samji, 2009, Huang et al., 2013).

1.8.5. Viral assembly and budding

The influenza virus uses the host cell’s plasma membrane to form mature viral particles. All viral proteins normally found within the viral lipid bilayer, such as HA, NA and M2, must be present to form a viral particle. These proteins are transported to the apical plasma membrane, since viral particles bud from the apical side of polarized cells. M2 is essential in the formation of viral particles. M1 and several host factors play a major role in closing and budding of the viral particle. Packaging signals identified in the 5΄ and 3΄ non-coding and coding regions of some of the viral segments indicate which segments are to be packaged into the virions, depending on the signals contained. NA removes the SA residues from glycoproteins and glycolipids which enables the newly made viral particles to leave the plasma membrane (Samji, 2009).

1.9. CLINICAL MANIFESTATIONS OF INFLUENZA VIRUSES

Influenza viruses spread primarily through person-to-person transmission via large- particle droplets that travel only a short distance through the air. Contacts with surfaces that are contaminated with respiratory droplets serve as another possible source of

Chapter 1 transmission. The incubation period for influenza is generally 1-4 days. Adults shed virus from the day before onset of symptoms up to 10 days after symptoms have ceased whereas children may shed influenza virus up to ten or more days after onset of symptoms. Severely immuno-compromised individuals can shed virus for weeks or even months (CDC, 2009).

A sudden onset of constitutional and respiratory abnormalities is a characteristic feature of uncomplicated influenza illness. Fever, headache, malaise, myalgia, non-productive cough, sore throat and rhinitis are common symptoms of the infection. Nausea and vomiting are also commonly reported with influenza illness. Though uncomplicated influenza illness usually resolves in 3-7 days, cough and malaise can persist for more than two weeks. Primary influenza viral pneumonia may be caused by influenza virus which worsens underlying medical conditions including pulmonary or cardiac disease. Severe influenza virus infections may also lead to secondary bacterial pneumonia, sinusitis and co-infections with other viral and bacterial pathogens (CDC, 2009).

1.10. PREVENTION AND TREATMENT OF INFLUENZA VIRUS INFECTION

Infections caused by the influenza virus are generally tackled with the help of vaccines and anti-influenza drugs. The best choice for reducing the effect of a pandemic virus remains vaccination, as vaccines reduce the number of susceptible people within a given population. Since current influenza vaccines protect only against closely related strains, anti-influenza drugs, which serve as a first line of defence in the event of a pandemic, work along with vaccines to fight against the infection (Robertson and Inglis, 2011).

1.10.1. Vaccines

Vaccines form the basis of preventive treatment for influenza. An influenza vaccine is a sterile, aqueous suspension made up of one or more strains of influenza virus: Type A or B, or a mixture of both types, which have been grown individually in embryonated chicken eggs or in mammalian cells, are contained in the vaccines. There are two types of influenza vaccines: an inactivated suspension of whole virus particles which is injected and an attenuated live influenza vaccine that is generally delivered through the nasal passage. Since 1990, there have been substantial modern developments in the field of influenza vaccines due to increased development of mammalian cell lines for vaccine 16

Chapter 1 production, experience in the use of adjuvants and reverse genetics technologies for the generation of vaccine viruses. Inactivated influenza vaccines mainly function by inducing IgG (immunoglobulin G) antibodies specific to the virus HAs, whereas attenuated vaccines also stimulate local and systemic immune protective mechanisms, including mucosal IgA antibodies and cellular immunity.

Inactivated vaccines are of three types: whole virus, split virus and subunit vaccines. Split vaccines contain virus particles that have been partially or completely disrupted by physicochemical means (detergent) whereas subunit vaccines contain HA and NA antigens which have been further purified by removal of other viral components. Adjuvants are included in some formulations and thiomersal, a preservative, is present in most multi-dose vaccine vials. In the case of live, attenuated influenza vaccines, temperature-sensitive variant virus strains that replicate well in the nasopharynx but poorly in the lower respiratory tract are used (WHO, 2013).

Although the best choice for extenuating the effect of a pandemic virus remains through vaccination, significant limitations in the usage of vaccines hamper the prophylaxis treatment. Close matching of vaccines and circulating virus strains is highly essential for vaccine efficacy. Development of influenza vaccines is hindered by two primary predicaments. Though the composition of influenza vaccines are not always altered, the continuously changing antigenic nature of influenza viruses demand regular checks for the close matching of circulating virus in the respective season. Also, the wild type viruses isolated from clinical samples needs to be modified in order to enhance its growth properties as they do not replicate well in embryonated eggs. These characteristics together present a major impediment to the manufacture of seasonal vaccines and are predominantly challenging in the event of a pandemic when access to large amounts of vaccine becomes vital to manage the situation. There are also well documented vaccine failures. Vaccine efficacy drops by 10% in immuno-competent young subjects, and a drastic loss of 50% efficiency is prevalent in the elderly due to suboptimal efficacy of vaccines in the high risk population where the infection can progress to very severe, complicated disease and even reach a fatal end (Robertson and Inglis, 2011, Wang et al., 2006, Fichera et al., 2009).

Chapter 1

1.10.2. Anti-influenza drugs

Current commercially available anti-influenza drugs target the viral matrix protein 2 and neuraminidase glycoprotein of the virus. Ion channel blockers (Amantadine and Rimantadine) and neuraminidase inhibitors (Oseltamivir and Zanamivir) have played a significant role in the treatment of seasonal influenza. Viral replication and spread are primary targets for these drugs which eventually lead to an early recovery from the symptoms of influenza (Nabeshima et al., 2012).

1.10.2.1. M2 inhibitors

The first generation of anti-influenza drugs targeted the M2 proton channels, present on the influenza virus. Amantadine (1-adamantanamine hydrochloride) (Symmetrel) and Rimantadine (alpha-methyl-1-adamantane methylamine hydrochloride) (Flumadine) are symmetric tricyclic amines that inhibit influenza A viruses at low concentrations (<1.0 mg/mL) but are inactive against Type B viruses. M2 inhibitors act as channel blockers by binding to the channel pore thereby preventing proton entry into the virion. Hence, viral structural changes that are essential for viral entry are blocked. The mechanism of action is exhibited through an “early antiviral effect”, where the dissociation of ribonucleoproteins from M1 is affected. M2 inhibitors, also cause a “late antiviral effect” of promoting early HA conformational change. However, the M2 gene is susceptible to mutations, resulting in drug resistant strains. Cross-resistance to Amantadine and Rimantadine is caused by single-amino-acid substitutions at multiple positions (residues 26, 27, 30, 31, or 34) within the trans-membrane domain of the M2 protein. As a result, H3N2 and H1N1 subtypes of influenza virus developed resistance against adamantanes. Moreover, the novel reassorted pandemic H1N1 variant from swine includes the M gene of European swine influenza A viruses which have been resistant to M2 inhibitors since 1989. Therefore, pandemic H1N1 infections cannot be treated with adamantanes. Several side effects associated with the central nervous system and gastrointestinal tract, along with the rapid emergence of antiviral resistance during therapy, have limited the usefulness of adamantanes in the treatment of influenza (Grienke et al., 2009, Ison, 2011, Uchide and Toyoda, 2008, Du et al., 2012a, Lee and Yen, 2012).

Chapter 1

1.10.2.2. Neuraminidase inhibitors (N AIs)

Effective inhibitors of the NA enzyme present in the influenza viral membrane were developed after the discovery of the three-dimensional structure of influenza neuraminidase. Thus, the second generation influenza drugs came into existence. Since NAIs are analogues of SAs, the mechanism of action is exerted through competition with the natural ligand, blocking the active site of NA. Thus, binding to the active site of NA, NAIs prevent NA from releasing viral progeny from the host cell. Hence, the virus is blocked from infecting other cells of the host. There are currently two NAIs approved worldwide, Oseltamivir and Zanamivir (Grienke et al., 2012). Since the enzyme site of NA is relatively conserved with only subtle structural differences between subtypes, NA inhibitors are effective against types A and B viruses, and even different NA subtypes (Lee and Yen, 2012).

Oseltamivir, is a prodrug that is converted by hepatic esterases to the active compound (Oseltamivir carboxylate) after being absorbed by the gastrointestinal tract. Approved as a capsule (30 mg, 45 mg or 75 mg doses) and as powder for suspension (12 mg/mL), the drug is generally well tolerated. The most common side effects include nausea, vomiting, neuropsychiatric events and abdominal pain in approximately 5-10% of patients. Zanamivir is available as a dry powder mixed with lactose (5 mg Zanamivir per 20 mg lactose) for inhalation as it has poor oral bioavailability due to rapid elimination by renal excretion. Zanamivir is also associated with side effects, some of which include oropharyngeal or facial oedema, diarrhoea, nausea, sinusitis, nasal signs and symptoms, bronchitis, cough, headache, dizziness, and ear, nose and throat infections. NAI drugs have been shown to possess enhanced tolerance compared to M2 inhibitors. Historically, NAIs rarely showed antiviral resistance until the 2007/2008 season when Oseltamivir-resistant H1N1 viruses spontaneously arose and spread globally in 2008. Limited supplies and high cost of NAI drugs together with rapid emergence of NAI resistant viruses present a major obstacle in the treatment of infections (Grienke et al., 2009, Ison, 2011, Dao et al., 2012, Nabeshima et al., 2012).

Chapter 1

1.10.2.3. Other investigational antiviral agents for influenza

As shown in Table 1.2., several antiviral agents for influenza virus are being developed. Laninamivir and Peramivir, two NAIs are approved in a few parts of Asia but under clinical trials elsewhere. Laninamivir octanoate (CS-8958), a prodrug that is converted to Laninamivir, has shown activity against wild type influenza A and B viruses and most Oseltamivir-resistant strains. The compound is approved in Japan with clinical studies under progress in the United States. Peramivir, structurally different from other NAIs, possesses substitutions that result in multiple binding interactions with the active site of the virus and fights against NAI-resistant strains. Peramivir is approved for use in Japan and South Korea. Also other investigational agents are under clinical trials (Ison, 2011).

Chapter 1

Table 1.2. Antiviral agents for influenza virus under development, adapted from Hayden. (2013) and Lee and Yen. (2012)

Class of antiviral agent Inhibitor Target for antiviral activity NA inhibitors Peramivir, Laninamivir NA enzyme activity

Nucleoside analogues Favipiravir (T-705) RNA elongation

Nucleozin and derivatives Nucleozin NP oligomer formation and nuclear transportation

Endonuclease inhibitor 4-Substituted, 2,4-dioxo-4-phenylbutanoic acid Cap-snatching endonuclease activity of PA

Fusion blocker Arbidol HA fusogenic conformational change

NS1 inhibitors NSC109834, NSC128164, NSC95676 NS1 protein NSC125044

Sialidse DAS181 Interaction between viral HA and SA receptors

Chapter 1

DAS181 (FluDase), a recombinant fusion protein that contains the catalytic domain from Actinomyces viscosus sialidase, linked with an epithelium-anchoring domain of human amphiregulin, removes both the human-like α2-6- and avian-like α2-3-linked SAs from cellular receptors. Therefore, DAS181 has an extensive range of activity for influenza viruses, including amino-adamantane and NAI-resistant strains (Lee and Yen, 2012).

Favipiravir (T-705; 6-Fluoro-3-hydroxypyrazine-2-carboxamide), a pyrazine derivative first identified in 2002, is a viral polymerase inhibitor that functions like a nucleoside. This unique mechanism of antiviral action renders activity against all influenza virus types (A, B, and C), including those that are resistant to amino-adamantanes and NAIs. Favipiravir’s mode of action is mediated through its processed forms, T-705 ribofuranosyl phosphates, including monophosphate and triphosphate forms. The triphosphate form is mistaken by the influenza virus polymerase for a purine nucleotide during RNA elongation, thereby inhibiting polymerase activity. The conserved C- terminus PA and N-terminus PB1 binding domain of the influenza polymerase complex has been explored as an antiviral target. Studies that provide basic steps in finding and designing small molecules which precisely disrupt protein-protein interactions have been published. The synthesis of viral mRNA requires host mRNA-derived 5’-caps. The cap-binding domain of the influenza polymerase has been found within PB2, while endonuclease cap-snatching activity is conferred by the N-terminus of PA. A series of 4-substituted 2, 4-dioxobutanoic acid compounds that inhibit endonuclease cap- snatching activity have also been reported. As a result of recent studies, that have solved the co-crystal structures of the PA endonuclease domain, new insights in the development of potential PA endonuclease inhibitors have been proposed (Lee and Yen, 2012, Tomassini et al., 1994).

Nucleozin, a small-molecule compound, and its analogues that inhibit NP transportation from the cytoplasm into the nucleus, have been identified. Several studies that support the concept of NP being a potential target for novel antivirals have been performed. Flufirvitide, a peptide that inhibits the fusogenic activity of HA has progressed into initial clinical development. Similarly, Arbidol [(ethyl-6-bromo-4- [(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3- carboxylate hydrochloride monohydrate)], a drug that acts via inhibition of the

Chapter 1 fusogenic conformational change of HA, has been licensed in Russia for the treatment of influenza A and B (Lee and Yen, 2012).

Other agents that are in the developmental phase as influenza antivirals are: Cyanovirin- N, a cyanobacterium-derived protein that binds to high-mannose structures on the HA to block entry; a peptide derived from a fibroblast growth-factor-4-signal sequence that acts against viral attachment to cells; Nitazoxanide which targets a post-translational stage, blocking HA maturation and transportation, and the high molecular-weight sulphated polysaccharide iota-carrageenan, obtained from red seaweed, that targets viral spread and entry (Lee and Yen, 2012).

Antibody therapies for influenza have received increased interests since the identification of conserved epitopes on the stem region of the influenza HA; recent studies have shown that naturally occurring human antibodies from vaccinated or exposed individuals can bind different HA subtypes. Antibodies that target conserved sites on the stem region prevent the conformational changes in HA that are essential for membrane fusion during virus replication. Since cross-protection can be achieved regardless of the subtype of the emerging strains, the use of adoptive immune-therapy may be considered for pandemic preparedness. Four NS1 inhibitors (Table 1.2.) were also found to inhibit influenza virus by reducing viral protein synthesis or viral RNA production (Hayden, 2013, Lee and Yen, 2012).

1.11. MUTATIONS THAT LEAD TO ANTIGENIC VARIATION OF INFLUENZA VIRUS

Antigenic variation, known as antigenic drift and antigenic shift, arises frequently and gives rise to new influenza viruses (Figure 1.4). A minor change such as an amino acid substitution in either HA and/or NA which paves way to an antigenic site change is referred to as antigenic drift. Antigenic shift refers to the formation of a new viral strain with combined HA and NA from diverse subtypes. Antigenic drift generally causes seasonal influenza while antigenic shift is the predominant reason for influenza pandemics. Amino acid substitutions alter the capacity of antibodies to neutralise the virus, through interference in antibody or receptor binding. Therefore, antisera generated for a specific viral strain has only limited activity against a newly emerging one (Pleschka et al., 2009, Robertson and Inglis, 2011). Thus, the virus acquires

Chapter 1 capacity to circumvent extant humoral immunity. Environmental selection pressure in Nature plays a vital role in electing antigenic changes in the antigen determining spots of HA, including parts that undergo adaptive evolution and substitution that alter the antigenicity of the virus. Hence, the virus resists host defence through co-evolution with the host (Chen and Deng, 2009).

The highly conserved structure of the NA active site among influenza A and B viruses present a striking target for NAIs in antiviral therapy. However, mutations in the NA may alter the shape of the NA catalytic site, thereby reducing the inhibitor binding potential. The NA must undergo rearrangements to accommodate drug binding, since Oseltamivir has a large hydrophobic side chain. The efficiency of Oseltamivir against influenza viruses may be lowered by mutations that affect the rearrangement enabling drug binding. In the N1 subtype, glutamic acid 276, a residue within the viral NA active site, is shown to be important for Oseltamivir carboxylate hydrophobic binding. This residue lies in close proximity to histidine 274. A single amino acid change, histidine to tyrosine at position 274 (H274Y; H275Y in N1 numbering system), confers Oseltamivir resistance to the virus. Oseltamivir reduced sensitivity has resulted from R292K and N294S mutations. During the 2007-2008 influenza seasons, higher rates of natural resistance to Oseltamivir were reported worldwide. Almost all H1N1 strains that were characterized in the subsequent season (2008-2009) were reported to contain the H274Y mutation. Oseltamivir resistant H3N2 (R292K mutation) have also been reported (Ives et al., 2002, Samson et al., 2013).

Unlike Oseltamivir, the molecular structure of Zanamivir includes a guanidino group instead of the hydrophobic group. This guanidino group interacts with the conserved E119 residue in the active centre pocket of NA. Mutations in the framework or catalytic residues of the NA protein that affect the binding between the enzyme and the inhibitor can lead to the development of resistance to Zanamivir. Resistance to Zanamivir is shown to be rare in the clinical setting for both seasonal and pandemic viruses compared to Oseltamivir due to the lower use of Zanamivir for treatment. Studies have also revealed the presence of A (H1N1) pdm09 viruses with I222R and I222K mutations that confer reduced susceptibility to Zanamivir. Since Peramivir contains both the hydrophobic chain and guanidino group, mutations that affect the activity of Oseltamivir and Zanamivir can also affect Peramivir activity. Also, mutations that

Chapter 1 confer resistance to Zanamivir are expected to confer resistance to Laninamivir since both drugs share similar binding properties with the NA protein (Samson et al., 2013).

Figure 1.4. Antigenic variations of influenza virus

New influenza viruses arise through antigenic drift and antigenic shift variations. Depending on the type of variation, seasonal influenza or influenza pandemics occur.

Reference: online at http://whatisswine-flu.blogspot.com.au/2009/05/pictorial-representation-of-swine- flu.html

1.12. THE NEED FOR THIRD GENERATION ANTI-INFLUENZA DRUGS

The development of third generation anti-influenza drugs that exhibit a distinct mode of action might be useful to fight against neuraminidase and adamantane drug resistant viruses. Developing an efficient antiviral drug base comprising of drugs with varied pharmacological properties is essential to defend against a new type of influenza virus (Matsubara et al., 2010). Even after thirteen years since the launch of Zanamivir and Oseltamivir, the hunt for unique lead structures still remains an area of intensive research (Grienke et al., 2012). Over the years, a better understanding of the virus envelope, viral nucleic acids and proteins that might serve as potential targets for antiviral activity has been achieved. Third generation anti-influenza drugs that target a different component of the virus, unlike neuraminidase inhibitors and adamantanes, along with combination therapies involving different antiviral drugs for influenza that

Chapter 1 attack several viral proteins, may be the best option to win the war against influenza (Matsubara et al., 2010, Hayden, 2013).

1.13. PLANTS - A POTENTIAL SOURCE FOR ANTIMICROBIAL COMPOUNDS

Healing illnesses with medicinal plants is as old as human history. Commonly referred to as “phytotherapy” in modern times, plant resources are utilized for the prevention and healing of various ailments, thereby promoting health and wellness. Plants have been used for basic health care by various cultures globally. Thus, traditional medicinal plants may be an excellent primary resource for the discovery of new antimicrobial compounds.

As a result of interactions among plants and the environment, plants produce secondary metabolites, also collectively known as phytochemicals, which encompass diverse chemical structures. Phytochemicals are essential for growth and maintenance of the plant, having a major role in survival and competitiveness. Some phytochemicals also serve as toxins, offering a natural protection system to safeguard against predators, and as pheromones to attract insects for pollination. Others act as phytoalexins offering protection against microbial infections and as allelochemicals which inhibit rival plants competing for soil and light. The spread and dominance of a species in an ecological surrounding greatly rely on the secondary metabolites produced. Medicinally useful phytochemicals (phytomedicines) perform diverse functions. Most of these are derivatives of a relatively small number of biosynthetic pathways that generally fit into few classes of chemical groups. Phytomedicines are specific to certain species/strain and possess varied organizations and bioactivities. The exploration of anti-microbial plant extracts or secondary metabolites might serve as an alternative approach to synthetic medication (Ameh et al., 2010, McRae et al., 2007, Mishra and Tiwari, 2011).

Natural compounds occupy a substantial portion in remedial treatment. The dominance of herbalism in medication is apparent from the significant amount of modern drugs which are derived from ethnobotanical therapy. Over 80% of the human population depends on phytochemicals for primary healthcare (Chattopadhyay and Naik, 2007). About 24% of the new drugs contain pharmacophores or functional groups with medicinal activity from natural products (Wink, 2012). Currently, herb and plant

Chapter 1 resources are unlimited for functional phytochemicals but these resources are dwindling rapidly due to deforestation and advancements of industrialization.

1.14. HISTORICAL EVIDENCES OF MEDICINAL PLANTS’ USAGE FROM SIMPLE BLENDS TO COMPOUND DRUGS

Plants have played an integral role in healing ailments throughout human history. Written documents, preserved monuments and even original plant medicines confirm the bond between humans and the search for drugs in nature. Early uses of medicinal plants were instinctive. Experience formed the basis of healing remedies, since information regarding the plant extracts and reasons for illness were limited. Understanding of medicinal plants is an outcome of the many years of struggle against diseases which led humans to track drugs in seeds, fruits, barks and other plant parts. Hence, the use of medicinal plants is generally based on explanatory evidences. Information regarding the therapeutic role of several medicinal plants has been conveyed to successive generations through verbal means. Plants have been the starting place for prevention and treatment until the introduction of iatrochemistry, a branch of science that provides chemical solutions to diseases and medical ailments (Petrovska, 2012).

The systems of herbalism were developed in the Middle East, Africa, Asia and Europe. The oldest written evidence that depicts the use of plants for the purpose of healing is a 5,000 years old clay slab from India making reference to 250 plants in drug preparations. The use of chaulmoogra oil for treating leprosy by Emperor Shen Nung of China (3000-2700 BC) is among the earliest reports of herbalism. The emperor also had a pharmacopoeia, which provided information regarding 365 drugs. The work, “De Materia Medica” of 77 AD describes 944 drugs, of which 657 are of plant origin and offers information regarding the external appearance, environment, type of collection, production of therapeutic preparations, and their beneficial effect. This constituted the basic materia medica until the late middle Ages and the Renaissance. Several medicinal plants were brought into Europe through Marco Polo’s expeditions (1254-1324) to Asia, China and Persia, the discovery of America (1492), and Vasco De Gama’s travel to India (1498). Botanical gardens were developed throughout Europe and efforts were made to farm domestic and imported medicinal plants (Petrovska, 2012).

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Medicinal plants were used as simple blends or decoctions up to the middle Ages but the requirement for compound drugs mounted between the 16th and 18th centuries. Isolation of alkaloids from poppy (1806), quinine (1820), pomegranate (1878) and glycosides from other plants acquired a remarkable spot in the separation of active compounds from medicinal plants. The advent of chemical methods enhanced the separation pathways and led to the isolation of tannins, saponosides, etheric oils, vitamins and hormones. Several authors wrote that drugs obtained from plant sources were inadequate and failing due to detrimental action of enzymes that generally cause primary changes during drying of plants. Hence, the remedial potential of medicinal plants was dependant on the mode of drying. The early 20th century marked the introduction of stabilization procedures for fresh medicinal plants, in particular those with labile medicinal constituents. Attempts were made to improve the state of manufacturing and farming therapeutic flora. Everlasting interest of people in relation to the healing properties of medicinal plants has paved the way for better chemical separation strategies and usage (Petrovska, 2012, Ameh et al., 2010).

1.15. NATURAL PRODUCTS IN DRUG DISCOVERY

Natural products drive the pharmaceutical industry towards drug discovery, since they serve as chief sources of starting materials for drug synthesis. In the past, pharmaceutical companies utilized plant extracts for the production of crude therapeutic formulations. With the development of antibiotics in the mid twentieth century, formulations of fairly purified compounds have been in use. Scaffold diversity of natural products may favour interaction with several targets within the cell, thereby increasing the potential of drug discovery (McRae et al., 2007, Mishra and Tiwari, 2011).

Tropical rain forests are regions of high biodiversity along with enormous chemodiversity. Being a haven to novel compounds, they attract pharmacologists for developing new leads in drug research. The first pharmaceuticals or quantified doses of therapeutic compounds were obtained from medicinal plants. Isolation of salicin from the bark of willow, Salix alba, which was traditionally used to alleviate pain and fever is a common example of medicinal compound isolation from plants. Conversion of salicin to salicylic acid and modification into aspirin reduced the side effects of the

Chapter 1 original compound. Anti-malarial quinine from Cinchona officinalis and the painkiller, morphine, from Papaver Somniferum are other renowned plant-derived pharmaceuticals (McRae et al., 2007).

Natural products have been a reliable source for the many FDA approved drugs. To name a few, Veregen TM, a defined mixture of catechins obtained from green tea was the first herbal medicine to receive FDA approval in 2006. Sativex® is the world’s first pharmaceutical prescription medicine derived from cannabis. Fumagillin, isolated from Aspergillus fumigatus, was approved for use against intestinal microsporidiosis, a disease caused by the parasite Enterocytozoon bieneusi. The therapeutic field of infectious diseases has greatly benefited from natural products. For example, ceftobiprole medocaril, a cephalosporin antibiotic with excellent activity against Penicillium aeruginosa and penicillin-resistant Streptococcus pneumoniae, and Tebipenem pivoxil, a broad spectrum antibiotic, have their roots in plants. Several antiviral, antifungal and antiparasitic agents have also been developed through research on natural products ( Mishra and Tiwari, 2011).

Very few businesses invest money and time in the screening and isolation of compounds from plants since it is hard to identify prospective compounds that match a pharmaceutical’s efficiency. Moreover, once a bioactive species is sited, the process of unravelling the diverse compounds within the extract can be very complex. This time- consuming process may not always result in a novel drug. Sometimes the compounds within a plant extract may only be active when working synergistically; therefore separation might dilute that bioactivity. Despite these barriers, the chemodiversity of species in biodiverse regions is definitely worth the attempt of exploration (McRae et al., 2007, Mishra and Tiwari, 2011).

Chapter 1

1.16. STRATEGIES FOR THE DISCOVERY OF ANTIMICROBIAL COMPOUNDS FROM PLANT SOURCES

There are over 500,000 plant species with varied phytochemical composition, with a diversity of chemical compounds, even within the same plant species, as the leaves, stem and roots vary in composition. Seasonal and geographical differences also alter the phytochemical make-up of plants. Therefore, discovery of an effective plant sample from a random screening strategy is often not productive (McRae et al., 2007).

Ethno botany offers the potential to overcome these hindrances. Exploiting the remarkable collection of information and insights of indigenous populations in relation to their ancient therapeutics and investigation of species that have been traditionally used as medicines for many generations may direct a constructive path in the discovery of active plant samples. Information regarding the season during which a particular plant species produces biologically active compounds, plant parts that possess biological activity, and the geographical location where the species is more effective are furnished by traditional knowledge of plants. The use of traditional medicine throughout human history indicates medical precision and is generally considered to be safe with the cytoprotective character being reported. The traditional use of plants and the presence of active compounds within a plant extract are linked to each other as reported by large-scale studies that explore the potential of medicinal flora. Antimicrobial testing often forms the first phase in the screening of plants since this path is speedy, ethical and economical. Exposing the specific organism to the plant extract and monitoring the presence or lack of growth after exposure is the principle of general antimicrobial testing. The technique is functional in detecting whether or not a plant extract should undergo further examination (McRae et al., 2007, Tayal et al., 2012).

1.17. ANTIVIRAL AGENTS FROM PLANT SOURCES

With respect to antiviral agents, 80% of 46 entities registered between 1981-2010 fall within the category of natural product-botanicals, natural product pharmacophores, synthetic natural product mimics, or a mixture of the latter two (Grienke et al., 2012). Screening of 288 plants against influenza A virus in embryonated eggs by Boots drug company at Nottingham, England, in 1952 marked the start of research interests for the

Chapter 1 development of antiviral agents from plant sources (Jassim and Naji, 2003). As shown in Tables 1.3., 1.4. and 1.5., several phytochemical and pharmacological studies on ethnomedical plants have rendered a range of active metabolites from diverse chemical groups (Sohail et al., 2011). In the last twenty five years, many broad-based screening programmes have been carried out in different parts of the globe to evaluate the antiviral activity of medicinal plants for in vitro and in vivo assays against viruses including herpes virus, human immunodeficiency virus (HIV), influenza virus and several other pathogens (Jassim and Naji, 2003).

The basic requirement of an effective antiviral drug is to prevent the completion of viral growth cycle in the infected cell without being toxic to the surrounding normal cells. The physical and functional incorporation of the virus into the host cell masks the distinction of unique biochemical features indispensable for selective encounter. Since molecular events that occur within the host cell are now better understood, the search for antiviral compounds is being made on a more rational basis. Generally, drugs obtained from plants follow two modes of viral inhibition, either acting on the virus directly or by inducing interferon and regulating immune function which eventually results in viral inhibition (Wang et al., 2006).

Chapter 1

Table 1.3. Some plants with reported antiviral activities against herpes virus, adapted from Sohail et al. (2011)

Plant Mode of action Aloe vera Prevents viral replication Andrographis paniculata Not known Bergenia ciliate Not known Clerodendrum inerme Reduction in viral cytopathic effect Conzya canadensis Not known Dianthus caryophyllus Not known Eichnacea purpurea Not known

Himantanthus phagedaenica Not known Hyssopus officinalis Not known Ocimum americanum, Ocimum basilicum, Not known Ocimum sanctum Ouratea castaneafolia, Ouratea semisrrata, Not known Ouratea spectabilis Rhus aromatica Prevents viral penetration

Chapter 1

Table 1.4. Antiviral activities of plants against HIV,

adapted from Sohail et al. (2011)

Plant Active compound(s) Antiviral target Calophyllum brasiliense Apetalic acid, Calanolides B Reverse transcriptase and C

Calophyllum cerasiferum Inophyllum, Calanolide A and Reverse transcriptase Coumarins

Calophyllum inophyllum Inophyllum B and P Not known

Daucus maritimus Not known Reverse transcriptase

Kadsura heteroclita Triterpenoid and lignans Not known

Monotes africanus Flavanoids Reverse transcriptase

Phaseolus vulgaris Lectin Reverse transcriptase

Prunella vulgaris Not known Early, post-virion binding events

Quillaja saponaria Triterpenoid saponins Not known

Terminalia chebula Not known Not known

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Table 1.5. Antiviral activities of plants against other viruses, adapted from Sohail et al. (2011)

Plant Virus Spirulina platensis, Hepatitis A virus Dianthus caryophyllus

Solanum nigrum, Acacia nilotica, Cannabis sativa, Hepatitis C virus Daucus maritimus

Narcissus tazetta, Schefflera heptaphylla Respiratory Syncytial Virus

Cicer arietum Parainfluenza-3 virus

Conyza canadensis Coxsackie B virus type 3

Daucas maritimus Dengue virus, West Nile virus

Hibiscus sabdariffa Measles virus

Prunus mume Human rhinovirus

Pterocaulom sphacelatum Poliovirus

Quillaja saponaria Reovirus

Sophora flavescens Coxsackievirus B3

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1.18. ANTIVIRAL ACTIVITY OF PLANTS AGAINST INFLUENZA VIRUSES

The search for plant-based antivirals against influenza is promising with several plants having been shown to possess anti-influenza activity (Table 1.6.). Oseltamivir, one of the currently available neuraminidase inhibitory drug for anti-influenza treatment, has its roots in nature: plant constituents, quinic acid and shikimic acid are the starting materials for the development of Oseltamivir (Grienke et al., 2012). Traditional medicines have played an important role in the health care area of the majority of Asian countries. Traditional Chinese herbal medicine (TCHM), an integral part of the traditional Chinese medicine system, which has been in use for a long time and is known for its multiple combinations of compounds in the form of processed natural products, has utilised several herbal drugs for the treatment of influenza virus infections (Table 1.7.) (Li and Peng, 2013).

Progress in the field of anti-influenza herbal medicines has provided alternative therapeutic measures for the treatment of influenza infections. For instance, two Japanese herbal medicines, Shahakusan and Hochuekkito, were shown to possess in vivo activity against the influenza virus (Dan et al., 2013, Hokari et al., 2012). On the other hand, studies on Jinchai, a capsule made of TCHM, indicated inhibitory activity against viral adsorption and cell membrane fusion, thereby blocking transcription and replication of the virus (Zhong et al., 2013). Also, Lianhuaqingwen capsule, a natural herbal medicine, was shown to have similar therapeutic effectiveness to that of Oseltamivir, in terms of reducing the duration of illness and shedding of Type A virus (Duan et al., 2011).

Chapter 1

Table 1.6. Antiviral activity of plants against influenza viruses

Plant (Reference) Active compound(s) Mode of action Agrimonia pilosa (Shin et al., 2010) Not known Viral membrane attack and inhibition of RNA synthesis Aronia melanocarp (Valcheva-Kuzmanova et al., 2006) Anthocyanin Not known Bergenia ciliate (Rajbhandari et al., 2009) Not known Not known Camellia sinensis (Song et al., 2005) Catechin derivatives Viral replication, hemagglutination is inhibited Cistus incanus (Kalus et al., 2009) Polyphenol, CYSTUS052 Prevention of viral entry Clinacanthus siamensis (Wirotesangthong et al., 2009) Not known Production of antibodies against the virus

Commelina communis (Bing et al., 2009) Alkaloids Viral growth inhibition Echinacea purpurea (Pleschka et al., 2009) Not known Inhibition of receptor binding and replication Geranium sanguineum (Sokmen et al., 2005) Polyphenols Inhibition of viral proteins’ expression Narcissus tazetta (Ooi et al., 2010) Protein Not known Toddalia asiatica (Li et al., 2005) Not known Not known

Chapter 1

Table 1.6. Antiviral activity of plants against influenza viruses (continued)

Plant (Reference) Active compound(s) Mode of action

Thuja orientalis, Aster spathulifolius, Pinus thunbergia Not known Inhibition of virus replication (Won et al., 2013) Allium fistulosum (Lee et al., 2012) Polysaccharide Enhancement of host immune system

Sambucus nigra (Kinoshita et al., 2012) Not known Stimulation of host immune response

Psidium guajava (Sriwilaijaroen et al., 2012) Tannin Sialidase and hemagglutinin inhibition

Chapter 1

Table 1.7. Herbal drugs for the treatment of influenza,

adapted from Li and Peng. (2013)

Herbs Botanical name Trade name Radix bupleuri Bupleurum chinense, Xiao-chai-hu capsule, Bupleurum corzonerifolium Zheng-chai-hu-yin granule

Fructus forsythiae Forsythia suspensa Yin-qiao-jie-du-wan (granule, tablet), Yin-qiao-san

Flos lonicerae; Lonicera japonica; Shuang-huang-lian-he-ji (granule, capsule, tablet), Radix scutellariae Scutellaria baicalensis Yin-huang granule (tablet)

Radix isatidis Isatis tinctoria, Ban-lan-gen granule, Isatis indigotica, Li-zhu (Chuan-fang), Baphicacanthus cusia kang-bing-du granule

Spica prunellae; Prunella vulgaris; Xia-sang-ju granule, Flos chrysanthemi Chrysanthemum indicum, Guang-yao-xing-qun-xiasang-ju Indici; Folium mori Chrysanthemum boreale, Chrysanthemum lavandulaefolium; Morus alba

Chapter 1

1.19. CHEMICAL CLASSES OF PLANT METABOLITES SHOWING ANTI- INFLUENZA ACTIVITY

Pharmacological research on medicinal plants has yielded bioactive compounds that inhibit influenza virus. A range of phenolic compounds, saponins, glucosides and alkaloids isolated from medicinal plants have been studied extensively due to their ability to prevent adsorption, penetration and multiplication during the course of viral spread (Grienke et al., 2012, Nagai et al., 2001, Serkedjieva and Velcheva, 2003b, Wang et al., 2006).

1.19.1. Phenolic compounds

Flavanoid compounds target the activity of neuraminidase and membrane fusion. Flavanoids or Phenyl-benzopyranes are widespread in the plant kingdom. The biological activities of the family Fabaceae has been studied intensively. In regard to drug likeness, flavanoids represent a remarkable class of low-molecular weight chemical units associated with good bioavailability. Biflavanoids like ginkgetin, isolated from Ginkgo biloba and Cephalotaxus harringtonia, hinokiflavone, 4′-O- methylochnaflavone, fourteen carbon methylated flavanoids from Cleistocalyx operculatus, flavones such as apigenin, apiin and luteolin, flavanols isolated from the roots of Rhodiola rose, quercetin, gossypetin, herbacetin, isoflavones including daidzein and genistein, oligostilbenes obtained from Vitis amurensis and Gnetum pendulum, have all been identified as potential NAIs. Naringenin 7-O-(2′,6′-di-O-alpha- rhamnopyranosyl)-beta-glucopyranoside,hesperetin7-O-(2′,6′-di- Oalpharhamnopyranosyl)-beta-glucopyranoside, hesperidin and narirutin, isolated from the fruits of Citrus junos v. Tanaka, Rutaceae have been shown to inhibit influenza A virus. Flavonoids obtained from the aerial part of Buple-urum chinense inhibited influenza B virus. On the other hand, the synergistic effects of anthocyanin pigments present in Solanum tuberosum result in the inactivation of both influenza Type A and Type B viruses.

Polyphenol compounds have been shown to inhibit viral protein or RNA synthesis and viral adsorption. Plant polyphenols have also been shown to possess antioxidant and radical scavenging activity. A polyphenolic complex isolated from Geranium

Chapter 1 sanguineum L., high polymeric procyanidins and hydroxycinnamic derivatives obtained from Chinese quince (Cydonia oblonga Mill.), demonstrated anti-influenza activity (Wang et al., 2006). Methanolic extracts of the roots of Glycyrrhiza uralensis demonstrated NA inhibition as a result of 18 polyphenols comprised of chalcones, flavonoids, coumarins and phenylbenzofuran (Grienke et al., 2012).

1.19.2. Saponins

Saponins may target the interaction of viral HA and SA receptor on the surface of the host cell (Ding et al., 2012). Four onjisaponins isolated from hot water extracts of Polygala tenuifolia showed an increase in the serum HI antibody titers of mice that were inoculated intranasally along with influenza vaccine. HI antibody titres of mice were 3-14 times greater than control mice group treated with vaccine only (Nagai et al., 2001).

1.19.3. Alkaloids

An indole alkaloid isolated from Uncaria rhynchophylla demonstrated potent inhibitory activity against influenza A virus in vitro (Wang et al., 2006). The pavine alkaloid (-)- thalimonine (Thl) isolated from Thalictrum simplex was shown to inhibit the replication of influenza viruses A/Ger-many/27, str. Weybridge (H7N7) and A/Germany/34, str. Rostock (H7N1). Virus-induced cytopathic effects, infectious virus yields and expression of viral glycoproteins HA and NA, and nucleoprotein (NP) on the surface of infected cells were shown to be reduced (Serkedjieva and Velcheva, 2003a, Serkedjieva and Velcheva, 2003b).

1.19.4. Other compounds

The major components of the volatile oil from Cynanchum stauntonii which exhibited activity against the influenza virus contained (E, E)-2, 4-Decadienal, 3-efhyl-4- methypentanol, 5-pentyl-3H-furan-2-one, (E, Z)-2, 4-decadienal and 2(3H)-furanone, dihydro-5-pentyl (Zai-Chang et al., 2005a). The anti-influenza component, 9S,12S,13S- trihydroxy-10E-octadecenoic acid (pinellic acid), obtained from the tuber of Pinellia ternat, increased antiviral IgA antibody titers when administered along with influenza vaccine. Hypericin, a phytochemical extracted from Hypericum perforatum, Arctigenin, a lignanoid compound in Arctium lappa and Chinonin, the major component of mango

Chapter 1 leaf Mangifera indica, have all been reported to possess anti-influenza activity (Wang et al., 2006). Amide compounds have been shown to affect influenza viral endonuclease (Carcelli et al., 2013, Hao et al., 2012). Favipiravir (T-705; 6-fluoro-3-hydroxy-2- pyrazinecarboxamide), an anti-influenza drug that is currently under trials also contains amide group in its structure and affects the influenza viral RNA polymerase (Baranovich et al., 2013, Furuta et al., 2009). Fatty acids have also been shown to demonstrate activity against influenza viruses. Since they are easily metabolized in the human body, fatty acid derivatives which are not easily metabolized may be a good candidate in the development of anti-influenza agents (Kim et al., 2003).

1.20. AIMS OF THIS STUDY

The long-term use of herbal medicines by humans and the promising results obtained through the investigation of plants used for anti-influenza activity were taken into account while framing this study. The emergence of drug resistant variants of the influenza virus has led to an urgent need to establish novel and effective antiviral agents to confront the impending risk of influenza pandemics. As a sustainable alternative to synthetic drugs, the consolidation of empirical knowledge with ethnopharmacological evidence of medicinal plants offers a productive platform for the development of safe and effective antiviral drugs.

The aim of this study was to verify the anti-viral properties of plant extracts with purported activity against the influenza virus. In this perspective, extracts of fifty medicinal plants, originating from the tropical rainforests of Borneo and used as herbal medicines by traditional healers to treat flu-like symptoms, were tested against the H1N1 and H3N1 subtypes of the virus. The aims of this study included, identification of plant extracts with anti-influenza activity, examining the modes of action on the virus, chemical characterization of the anti-influenza extracts and potential isolation of the active compound(s).

Chapter 2

CHAPTER 2 MATERIALS AND METHODS

2.1. CELLS

Madin Darby Canine Kidney (MDCK) cells, obtained from the American Type Culture

Collection (Manassas, VA) were grown at 37ºC with 5% CO2 in Roswell Park Memorial Institute medium (RPMI; Invitrogen, No: 22400-105) supplemented with 10% foetal bovine serum (FBS; Invitrogen, No: 16140-071) and 1% Penicillin- Streptomycin (Invitrogen, No: 15140-122), 75cm2 flasks (In Vitro, No: COR430641) were used to grow the cells. Before adding the compounds or the virus, or when quantifying the results, the monolayers were thoroughly washed twice with phosphate buffered-saline (PBS, pH 7.4 at room temperature). In all experiments, the following controls were included: cell control (cells that were not infected with the virus or treated with the plant extracts), virus control (cells that were infected only with the virus but not treated with the plant extracts in the antiviral assays), and the positive controls (virus-infected cells treated with Zanamivir or Oseltamivir, currently available NAIs).

2.2. VIRUSES

Type A influenza virus strains (Table 2.1.), “Mem-Bel” reassortant (H3N1), a reassortant of A/Memphis/1/71 (H3N2) × A/Bellamy/42 (H1N1), containing the HA of A/Memphis/1/71 and the remaining gene segments of A/Bellamy/42 and A/Puerto Rico/8/34 (H1N1) "PR8" were provided by Professor Lorena Brown, Department of Microbiology and Immunology, The University of Melbourne, Australia. Six influenza viruses (Table 2.1.) that were isolated, plaque purified and cultured in MDCK cells were provided by Dr. Aeron Hurt, WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory (VIDRL), Melbourne, Australia. Virus stocks were grown in MDCK cells using RPMI medium supplemented with 4 µg/mL trypsin (Sigma, No: T1426) at 37ºC in 5% CO2 for three days as described elsewhere (Oh et al., 2008). Supernatants containing virus were collected after cytopathic effects (CPE) were noted and antiviral titres were determined using 50% Tissue Culture Infectious Dose, according to Reed and Muench’s endpoint method (Reed and Muench, 1938), and a colorimetric endpoint to obtain quantitative results (Pourianfar et al., 2012a). All aliquots of virus stocks were stored at -80ºC until use. 42

Chapter 2

Table 2.1. List of influenza viruses that were employed to perform antiviral assays

Viruses Description H3N1 “Mem-Bel” reassortant (H3N1), a reassortant of A/Memphis/1/71 (H3N2) × A/Bellamy/42 (H1N1), containing the HA of A/Memphis/1/71 and the remaining gene segments of A/Bellamy/42 H1N1 "PR8", A/Puerto Rico/8/34 (H1N1) Seasonal A (H1N1) A/MISSISSIPPI/3/2001 wild-type virus (former seasonal H1N1; A/New Caledonia/20/99-like) - carrying histidine at position 275 (275H) of the neuraminidase glycoprotein Seasonal A (H1N1) (H275Y) A/MISSISSIPPI/3/2001 variant virus (former seasonal H1N1; A/New Caledonia/20/99-like) – carrying tyrosine at position 275 (275Y) of the neuraminidase glycoprotein – i.e. a H275Y substitution Pandemic A(H1N1) A/PERTH/265/2009 wild-type virus (H1N1pdm09; A/California/7/2009-like) - carrying histidine at position 275 (275H) of the neuraminidase glycoprotein Pandemic A(H1N1) (H275Y) A/PERTH/261/2009 variant virus (H1N1pdm09; A/California/7/2009-like) - carrying tyrosine at position 275 (275Y) of the neuraminidase glycoprotein – i.e. a H275Y substitution Type B B/PERTH/211/2001 wild-type virus (B/Sichuan/379/99-like) – carrying aspartic acid at position 197 (197D) of the neuraminidase glycoprotein Type B (D197E) B/PERTH/211/2001 variant virus (B/Sichuan/379/99-like) – carrying glutamic acid at position 197 (197E) of the neuraminidase glycoprotein – i.e. a D197E substitution

Chapter 2

2.3. PLANT EXTRACTS

Fifty medicinal plant extracts, collected from the tropical rainforests of Borneo, Sarawak, Malaysia, were selected on the basis of their traditional use in healing various diseases, including symptoms of influenza such as cough and sore throat. The medicinal plants were initially dried at 45ºC in an oven for three days or until completely dried. The plant materials were cleansed with water prior to drying and placed in different trays depending on the plant part. The resulting dried plant parts were cut to fine pieces with a cutting mill and stored in Ziploc® bags. Approximately 60 mL of dichloromethane and methanol in a volume ratio 1:1 was dispensed into a conical flask containing approximately 6 g of ground sample and shaken overnight in an orbital shaker at 150 rpm. The sample was filtered using filter paper and concentrated in a rotary evaporator at 7 rpm at 40-45º C. The process of filtration was repeated five times until the filtrate was clear, ensuring maximum extraction from the plants. The concentrated extract was transferred to an amber bottle for drying in the desiccator at room temperature. The dried extract was then weighed and stored at 4°C.

As shown in Table 2.2., the mass of extract obtained was dependent upon the part of the plant used for extraction. Whole plants and leaves yielded more extracts compared to stems and roots. Prior to use, the extracts were reconstituted in PBS with 10% dimethyl sulfoxide (DMSO, Sigma No: D 5879) and filtered using a 0.45 µm filter (Sartorius Stedium Australia No: 16533K).

Table 2.2. Extracts obtained from plant materials

Plant parts Weight of dried material (g) Weight of extract obtained (g) Whole plant 1.00 0.05-0.10 Leaves 1.00 0.05-0.10 Stem 1.50 0.01-0.05 Roots 1.00 0.01-0.05

Chapter 2

2.4. CYTOTOXICITY STUDIES OF EXTRACTS

Briefly, MDCK cells were seeded into 96-well flat-bottomed microtitre plates (Costar) at 4×103 cells per well. Following overnight incubation, the media were aspirated, followed by addition of 100 L of plant extract solution diluted in RPMI medium (two- fold dilutions, ranging from 0.78-100 µg/mL) and another 100 L of growth medium

(supplemented RPMI) were then added to each well. After incubation at 37ºC/ 5% CO2 for a further 3 days, the results were quantified using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT, Invitrogen, No: M-6494) as per the manufacturer’s instructions. The optical density (OD) was measured at 540 nm using a Bio-Rad iMark TM microplate reader. The percentages of cell viability were based on the amount of living cells in compound-treated cells relative to cell controls (defined as 100% viability). Cytotoxicity graphs were then generated by plotting the percentage of cell viability versus the concentration of extracts and the 50% cytotoxic concentrations

(CC50) were estimated by regression analysis of the cytotoxicity curves using Microsoft Excel. A trend line that best suited the curve was selected and the corresponding equation was used to calculate 50% cytotoxic concentrations (CC50) (Pourianfar et al., 2012b).

2.5. IN VITRO MICRO-INHIBITION ASSAY

The method used to assess the activities of the plant extracts against influenza viruses was adapted from a previously described one (Pourianfar et al., 2012b). Briefly, 96-well 4 plates were seeded with 3×10 cells/well and incubated for 24 h at 37º C with 5% CO2 until a confluent monolayer was attained. The cells were washed twice with PBS, and two-fold serial dilutions of plant extracts (0.78-100 µg/mL) in RPMI medium were challenged with 100 TCID50 of either of the two virus strains. To all wells, 100 µL of RPMI medium supplemented with 2µg/mL trypsin (virus growth medium) were added.

After incubation for three days at 37ºC/ 5% CO2, the results were quantified as previously described. The antiviral activity curve was then generated by plotting percentages of virus inhibition against concentrations of extracts. IC50, the concentration of extract essential to reduce virus-induced CPE by 50%, was expressed relative to the virus control employing dose-response curves. Using regression analysis

Chapter 2 of antiviral activity curves, a trend line that best suited the curve was selected and the corresponding equation was used to calculate IC50 values (Pourianfar et al., 2012b).

2.6. TIME-OF-ADDITION ASSAY

The antiviral effects of extracts were evaluated at different times of viral infection as described elsewhere (Chiang et al., 2002). Briefly, 100 L/well of each plant extract, serially diluted in RPMI at four concentrations (0.78, 12.5, 25 and 50 µg/mL), was added to 80% confluent MDCK cells at either 1 or 2 hours prior to infection (-1 and -2, respectively), at the time of infection (0), or 1 or 2 hours after infection (+1 and +2, respectively). Infection was performed by adding 100 µL/well of either H1N1 or H3N1

2.7. VIRUS BINDING (ATTACHMENT) ASSAY

To assess the activity of the compounds in inhibiting viral binding, an attachment assay adapted from De Logu et al (De Logu et al., 2000), was performed. Briefly, 80% confluent cells were chilled at 4ºC for 1 hour followed by infection with 50 µL/well of

H1N1 or H3N1 (200 TCID50) and simultaneous supplementation with 100 µL/well of each plant extract at four concentrations (0.78, 12.5, 25, 50 µg/mL). All plates were held at 4ºC for a further 3 h, after which the supernatant was removed; the cells were washed twice with ice-cold PBS and the medium was replaced with an equal volume of RPMI and virus growth medium, and incubated for a further three days at 37ºC/5%

CO2. MTT was employed to evaluate cell viability and the percentage of viral inhibition was calculated in relation to the virus control wells.

2.8. PENETRATION ASSAY

The effect of plant extracts on viral penetration was studied according to a method described elsewhere (Albin et al., 1997). Briefly, 80% confluent cells were chilled at

4ºC for 1 hour prior to infection with H1N1 or H3N1 (200 TCID50) in virus growth medium and held at 4ºC for further three hours. After the incubation period, specific

Chapter 2 concentrations of extracts (0.78, 12.5, 25 or 50 µg/mL) were added in triplicates to the wells with virus. The activity was studied at three time intervals (30, 60 and 120 min), employing one plate per interval, at 37ºC/5% CO2. After the specified time interval, the supernatant was removed and treated with acidic PBS (pH 3) for 1 min to inactivate unpenetrated virus (Stegmann et al., 1987), and finally treated with alkaline PBS (pH 11) for neutralization. Cells were washed once with PBS (pH 7.4) and overlaid with an equal volume of RPMI and virus growth media. After three days incubation at 37ºC/5%

CO2, cell viability was evaluated using MTT.

2.9. NEURAMINIDASE (NA) INHIBITION ASSAY

The NA-Fluor™ Influenza Neuraminidase Assay Kit (Life Technologies, No: 4457091) was employed to test the effects of extracts on the viral NA of influenza viruses as per the manufacturer’s instructions. The virus stock was titrated by performing NA activity assay and the optimum virus dilution for the NA inhibition assay was selected. Two- fold serial dilutions of plant extracts (0.3-25 µg/mL) were tested for NAI activity. Zanamivir and Oseltamivir were included as positive controls in the assay and tested at nanomolar concentrations (10-2 to 104 nM). Fluorescence was measured using a POLARstar Omega fluorescence polarization microplate reader (excitation 355 nm, emission 460 nm). IC50 values were determined from dose-response data using sigmoidal curve-fitting generated and analysed using GraphPad Prism Software.

2.10. HEMAGGLUTINATION INHIBITION (HI) TEST

An HI assay was used to determine the effect of extracts on virus adsorption (Chen et al., 2010). Briefly, two-fold serial dilutions of the extract (0.78-100 µg/mL) were prepared in PBS and an equal volume (25µL/well containing 4HAU) of the virus stock was added to each well in a round-bottomed 96-well microtitre plate in triplicate. Subsequently, 50 µL of 5% chicken red blood cells (CRBC) were added to all wells and mixed. The following controls were included in every plate (i) CRBC without virus, (ii) CRBC with virus devoid of extract and (iii) CRBC with extracts devoid of virus (iv) non-commercial anti-HA monoclonal antibody (MAb) for H3N1 with an antibody titre of 80 and anti-HA MAb for H1N1 with antibody titre of 200 (provided by Professor Lorena Brown, Department of Microbiology and Immunology, The University of

Chapter 2

Melbourne), 1:8 dilution of either of the two antibodies in PBS were included in the assay. The hemagglutination reactions were observed after 30 minutes incubation at room temperature.

2.11. RDE TREATMENT

The effect of Receptor Destroying Enzyme (RDE, Denka Seiken Co. Ltd., Tokyo, Japan) treatment upon the antiviral activity of the extracts was studied through in vitro micro inhibition and HI assays. The extracts were added to RDE solutions in the ratio 1:3 and incubated at 37ºC for 20 hours according to the manufacturer’s recommendations. This was intended to eliminate compounds that may possess SA-like structures which mimic the receptors of RBC and compete for HA (WHO, 2002) . The extract and RDE mixture was inactivated at 56ºC for 60 minutes and then subjected to the assays.

2.12. TRYPSIN TREATMENT

The effect of trypsin (Sigma, No: T1426) upon the antiviral activity of the extracts was studied through an in vitro micro inhibition assay and HI assays. The plant extracts were treated with 4 µL of trypsin (1 mg/mL in 1% acetic acid), incubated at 37ºC for 24 hours and followed by incubation at 56ºC for 60 minutes before performing the assays. Plant extracts subjected to the same temperature without trypsin and extracts that were neither subjected to temperature nor trypsin treatments were included as controls.

2.13. ANALYSIS OF CRUDE EXTRACT AND FRACTIONS WITH INFLUENZA

VIRUS USING TRANSMISSION ELECTRON MICROSCOPE (TEM)

An attempt to visualize the effect of extract on virus using TEM was included in the study. Extract 8 and fractions of the same extract (100 µL) at a concentration of 1µg/µL were mixed with 100µL of either Mem-Bel or PR8 viruses (1 x 107 pfu). Aliquots of the virus and extract sample (20 µL) were placed onto parafilm on a flat glass plate. Pioloform coated 100 mesh TEM grids (Agar scientific, No: AGS1341) were incubated film side down onto virus droplets for 60 min at room temperature. After the incubation period, excess sample was removed by blotting the grids with filter paper. The grids were then placed film side down onto 20 µL droplets of 2% uranyl acetate (Polysciences, No: 6159-44-0) for 3 min. Excess uranyl acetate was removed by 48

Chapter 2 blotting with filter paper. The grids were allowed to dry for 24 hours before imaging with CM-120 Bio Twin TEM (Philips Electronics, Netherlands) operating at 120 kV. This standard protocol of sample preparation for TEM imaging was developed and performed by Dr. Simon Crawford (Electron Microscopy Unit, School of Botany, University of Melbourne).

2.14. PLAQUE NEUTRALISATION ASSAY

The plaque neutralisation assay utilises virus plaque formation in MDCK cells. The assay was performed according to a method described previously with some modifications (Ng et al., 2010, Tannock et al., 1984). Briefly, MDCK cells were seeded into 6-well tissue culture plates (TPP, CSL Ltd) at 1.2 × 106 cells in 3 ml of RPMI medium containing 10% foetal calf serum in each well. Following overnight incubation at 37ºC in 5% CO2, the monolayers were washed with medium containing antibiotics before adding 135µL of virus-plant extract mixture to each well, in duplicate. The virus- plant extract mixtures comprised two-fold dilutions of extract or fractions (3.13-12.5 µg/mL) in 200µL mixed with an equal volume of either Mem-Bel or PR8 influenza virus (5 × 105 pfu / mL) and incubated for 30 min at 37ºC. The virus and plant extract or fraction mixtures were allowed to adsorb onto MDCK monolayers for 45 min, with shaking the plates at 15 min intervals of time.

Then 3 ml warmed (45ºC) overlay medium consisting of Leibovitz L-15 with glutamine at pH 6.8 (Gibco Invitrogen Corporation, Victoria, Australia) supplemented with 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid buffer (0.01M at pH6.8), 0.028% (w/v) NaHCO3 (APS Finechem, NSW, Australia), 100 IU per ml penicillin (CSL Ltd), 100mg per ml streptomycin (CSL Ltd), 0.1% (w/v) TPCK-treated trypsin (Worthington Biochemical Corp., Lakewood, NJ, USA) and 0.9% (w/v) agarose (Sigma Chemicals Co.) were added to each well. The plates were then incubated for 3 days at 37ºC in 5%

CO2 and plaques on the monolayers were counted without staining.

The virus neutralization titre was expressed using the following formula:

Percentage virus neutralization = Number of plaques in the virus control sample - Number of plaques in the virus sample treated with extract or fraction / Number of plaques in the virus control sample *100

Chapter 2

2.15. SOLID PHASE EXTRACTION (SPE)

Extract 8 was fractionated using SPE columns (Phenomenex Australia Pty Ltd, No: 8B- S001-KDG) with decreasing polarity of acetonitrile (ACN) (Merck Millipore, No: 1000304004). Compressed air was employed to force the solvent through the SPE in order to fractionate the plant extract. Elution time and volume of eluent were set to 20 min and 20 ml respectively. After conditioning the column by elution with 20 mL distilled water which was then discarded, 100 mg of Extract 8 was loaded on the SPE and eluted with the following solvents consecutively; Milli Q water, 10% ACN, 25% ACN, 40% ACN, 55% ACN, 70% ACN, 85% ACN and 100% ACN. Each eluate was collected after elution with the solvent in the above mentioned order and was concentrated using a rotational vacuum concentrator (Christ ®; RVC 2-18) at 40ºC for 4-48 h, depending on the rate of evaporation for each solvent. Blanks containing corresponding ACN solvent only, were used as balance in the instrument. After removal of the solvent, the concentrate was taken up in 1 ml of water and subjected to sonication in order to promote solubility. The solution was then filtered using a 0.45µm filter (Sartorius Stedium Australia No: 16533K). Blanks were also subjected to the same process. The weight of extract that was eluted in the fractions could not be detected accurate, hence a theoretical estimate, deeming that 100% of the active compound(s) ended in the respective fractions tested was used to arrive at the concentration of extract present in the fractions. Since 100 mg of plant extract that was shown to demonstrate antiviral activity was fractionated, the active component(s) concentration was also deemed to be 100 mg. Each fraction obtained through elution with the respective solvent was classified to contain the entire amount of the active component(s) (100 mg). All the fractions were subjected to in vitro micro-inhibition assay; blanks were also included in the bioassay.

2.16. GAS CHROMATOGRAPH MASS SPECTROMETER (GC-MS) ANALYSIS

OF EXTRACT AND FRACTIONS

Crude Extract 8 and eight fractions that were obtained through SPE were analysed using a gas chromatograph coupled to a mass spectrometer as detector (GCMS-QP2010 Ultra, Shimadzu), equipped with an Rxi®-5SIL-MS column. Helium gas was used as a carrier gas with total flow, column flow and purge flow set to 7.5 mL/min, 1.50 mL/min and

Chapter 2

3.0 mL/min respectively; 2 µL of the sample was injected. Injector temperature was set to 200ºC and split injection mode (1:2) was selected at pressure 88.9 kPa. The column oven temperature was programmed as follows; held at 50ºC for the first two minutes and ramping at a rate of 20ºC/min to a final temperature of 250ºC , then held for eight minutes. Total GC run time was 20 min. The chromatogram obtained after the run was used to generate a qualitative table of the 50 most abundant compounds using peak integrate option. A similarity search was then performed to determine the possible structural units. The compounds were identified by comparison with the NIST and Wiley 8.0 MS libraries. Only peaks with a match greater than 75% and area % greater than 0.5% were reported.

2.17. STATISTICAL ANALYSIS

All treatments were performed in triplicate s and each experiment was independently repeated at least twice. The data were expressed as mean ± standard error of the mean (SEM). The results of the antiviral activity assays were analysed with a one-way ANOVA (Analysis of Variance) test and a significance level (p value) of 0.05 or 0.01 was considered to compare the means.

Chapter 3

CHAPTER 3 CYTOTOXICITY AND ANTIVIRAL SCREENING OF PLANT EXTRACTS

3.1. INTRODUCTION

Influenza viruses are highly infective and constitute a major causative agent for the recurrent epidemics and pandemics (McCaughey, 2010). More generally, the viruses cause acute respiratory infections referred to as “flu” and presents a considerable financial burden upon the health and wellness of the world population.

In the eventuality of a pandemic infection with a new strain, antiviral drugs represent the first line of defence (Uchide and Toyoda, 2008). Side effects associated with the central nervous system and the gastrointestinal tract, along with the rapid emergence of antiviral resistance during therapy, have limited the usefulness of adamantanes in the prevention and treatment of influenza (Grienke et al., 2009, Ison, 2011). The spread of Oseltamivir-resistant H1N1 viruses in 2008 further indicate the need for anti-influenza drugs that exhibit a different mode of action ( Uchide and Toyoda, 2008).

Since plants have been used throughout human history, exploiting the traditional knowledge of plants is a promising pathway to developing anti-influenza drugs. Tropical rainforests with immense biodiversity and chemodiversity attract pharmacologists since they harbour a prosperous supply of compounds with complex structures, that may point the way to developing new leads in drug research (Grienke et al., 2012, McRae et al., 2007).

In ancient times, treatment was based on symptoms of illness rather than the causative agent. Plants that are linked to an ethnomedical framework are generally considered to be safe since they have been used for many generations in the symptomatic treatment of various types of illnesses. Moreover, several plants have been shown to exhibit cytoprotective effect, where the components contained within the plant extract provide protection to the cell against harmful agents (Srivastava et al., 2012, Tayal et al., 2012). The property of compounds being toxic to cells is termed cytotoxicity. Cell toxicity may result in necrosis, in which cell membrane integrity is lost, affecting growth and multiplication that eventually leads to cell death. Cytotoxicity forms the basis of evaluating the safety of a potential antiviral compound. Since influenza viruses replicate

Chapter 3 within the host cell, any agent that targets the virus should preferentially inhibit the virus only, causing minimal or no damage to the host cell. Hence, cytotoxicity screening of plant extracts were studied in the initial stages of this research work; the effects of extracts upon the cell were observed. In this way, the safety aspect is first explored before examining the plant extracts for antiviral activity.

Generally plaque reduction assays have been used to test the activities of compounds against influenza viruses. In this phenotypic assay, the multiplication of each infectious virus particle results in a localized area of infected cells or ‘plaque’. Though plaque reduction assays are highly reliable, an in vitro micro inhibition assay was initially used to determine the antiviral activity of plant extracts against influenza viruses, since this method is an accepted screening tool for testing the antiviral efficacy of numerous compounds. Due to the large number and limited volume of plant extracts used in this study, the in vitro micro inhibition assay was the preferred method for initial antiviral screening of extracts (Smee et al., 2002, Mosmann, 1983).

Since influenza viruses cause extensive cell lysis that detaches the destroyed cells and debris from the plate, MTT, a colorimetric indicator of cell viability, was used to quantify cell damage or cell viability in the cytotoxicity and in vitro micro-inhibition assays. MTT measures the activities of cellular enzymes that reduce the tetrazolium dye to an insoluble purple coloured product, formazan, in living cells. Cellular metabolic activity is measured through NAD (P) H (nicotinamide adenine dinucleotide phosphate, reduced form) - dependent cellular oxidoreductase enzymes (Mosmann, 1983, Smee et al., 2002).

In this work, a cytotoxicity assay was initially employed to study the effects of extracts on cells, thereby evaluating the safety aspects, followed by an in vitro micro inhibition assay to determine the antiviral activity of plant extracts against Type A influenza viruses. Plant extracts that showed antiviral activity were then subjected to the next level of investigation, comprising a range of bioassays to study the modes of antiviral action. This study aims to validate the ethno pharmacological evidence of crude plant extracts from the tropical rainforests of Borneo.

Chapter 3

3.2. RESULTS

3.2.1. Cytotoxicity studies of all fifty plant extracts

The effects of plant extracts on MDCK cells were studied in a cytotoxicity assay. As detailed in Table 3.1, the concentrations of the plants extracts required to reduce the number of viable cells by 50% (CC50) relative to control wells without test compound were more than 100 µg/mL for 28 extracts, whereas the CC50 of the remaining extracts ranged between 7.5-96 µg/mL. Although, certain plant extracts were shown to contain

CC50 less than 100 µg/mL, there is a possibility that the extracts may preferentially act on the virus rather than cells, hence toxicity exerted on the cells may be reduced. Therefore, all extracts were subjected to in vitro-micro inhibition assays at concentrations ranging from 0.78-100 µg/mL, similar to the cytotoxicity assay.

The cytotoxicity curves of selected plant extracts that demonstrated antiviral activity against influenza viruses are reported in Figure 3.1. An interesting trend was noted in the assays for extracts 37 and 38 where even higher concentration of plant extract were not cytotoxic with cell viability close to 100%, indicating the safety aspect of these crude extracts. Zanamivir and Oseltamivir on the other hand demonstrated minimal cytotoxicity between 50-100 µg/mL, but concentrations less than 50 µg/mL did not show any cytotoxic effects.

The concentration associated with 50% cytotoxicity (CC50), estimated using regression analysis, was greater than the highest tested concentration (>100 µg/mL) for 28 extracts. In this case, the CC50 was an estimated theoretical value obtained by extrapolation of the results.

Chapter 3

Table 3.1. Cellular toxicity concentrations of all extracts tested

Extract CC50 (µg/mL) Extract CC50 (µg/mL) 1 194.4±6.0 26 7.5±1.4 2 103.2±1.4 27 92.8±8.5 3 89.7±5.0 28 20.3±3.2 4 105.3±0.7 29 157.2±9.6 5 117.6 0.7 30 130.0±3.5 6 159.9±7.7 31 106.7±2.1 7 85.5±11.7 32 35.1±1.8 8 133.0±14.2 33 13.7±1.6 9 111.3±1.6 34 53.0±2.4 10 139.2±6.5 35 134.5±9.7 11 126.2±2.8 36 68.7±3.0 12 126.6±15.5 37 149.0±7.1 13 130.0±3.5 38 165.0±10.0 14 136.3±8.2 39 128.6±12.6 15 161.0±1.8 40 55.8±9.1 16 53.8±3.5 41 140.7±0.9 17 90.7±1.1 42 140.7±0.9 18 43.4±0.4 43 109.8±10.1 19 128.7±7.6 44 18.4±3.7 20 152.2±16.7 45 94.63±3.15 21 42.5±3.7 46 129.4±7.3 22 82.8±3.8 47 10.5±5.1 23 163.3±15.4 48 129.5±17.9 24 94.1±0.4 49 88.0±1.9 25 23.2±0.3 50 48.2±6.4 Zanamivir 124.8±4.9 Oseltamivir 123.0±2.6

CC50 represents the concentration of plant extract required to reduce the number of viable cells by 50% relative to control wells without test compound, calculated from dose–response data

Chapter 3

Cell viabilityCell (%)

Concentration (µg/mL) Figure 3.1. Cytotoxicity effects of plant extracts Cells were seeded at 4x103 cells per well into 96-well flat-bottomed microtitre plates and left for overnight incubation. The media were then aspirated and overlaid with 100 µL of two-fold serial dilutions of plant extract (0.78-100 µg/mL) with an additional 100 µL of growth medium (supplemented RPMI). After three days incubation, cell viability was evaluated using MTT and percentage cell viability calculated relative to cell control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

Chapter 3

3.2.2. Inhibitory effects of plant extracts on influenza virus

The plant extracts were then subjected to a high throughput in vitro micro-inhibition screening assay to determine the antiviral activity against Mem-Bel (H3N1) and PR8 (H1N1) strains of influenza virus. Those demonstrating more than 50% viral inhibition were deemed to have anti-influenza activity. A number of plant extracts have been shown to exhibit inhibitory activity against the H3N1 strain. However, only eleven extracts consistently reduced viral infectivity by greater than 50% (Figure 3.2.). Hence, only these eleven extracts were tested for antiviral activity against the H1N1 strain. As shown in Figure 3.3., all eleven extracts were shown to demonstrate virus inhibition against the H1N1 strain.

Chapter 3

Viral inhibition (%) inhibition Viral

Concentration (µg/mL) Figure 3.2. Inhibitory effects of plant extracts on H3N1 virus

Cells at 80% confluence were treated with two-fold serial dilutions of plant extract (0.78-100 µg/mL) and

100 TCID50 of H3N1 simultaneously. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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Viral inhibitionViral (%)

Concentration (µg/mL)

Figure 3.3. Inhibitory effects of plant extracts on H1N1 influenza virus

Cells at 80% confluence were treated with two-fold serial dilutions of plant extract (0.78-100 µg/mL) and 100 TCID50 of H1N1 simultaneously. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

Chapter 3

As detailed in Table 3.2, there were duplicate samples among the plant extracts; extracts 13 and 30 were obtained from two sources of the same species, collected in the same location at different times, while extracts 41 and 42 were obtained from different parts of the same plant. More details of the plants that demonstrated antiviral activity against influenza viruses are provided in Appendix I.

Table 3.2. Medicinal plant extracts from Sarawak demonstrating antiviral activity against H3N1 and H1N1 strains

Specimen No Plant part Botanical name (Family) Medicinal use* no. Conjunctivitis, 8 SABC 0782 Whole plant Mussaenda elmeri (Rubiaceae) headache Trigonopleura malayana 13 SABC 1753 Leaves Cough (Euphorbiaceae) 14 SABC 1768 Whole plant Santiria apiculata (Burseraceae) Flu, headache Anisophyllea disticha 29 SABC 1984 Stems Fever (Anisophylleaceae) Trigonopleura malayana 30 SABC 1996 Leaves Cough (Euphorbiaceae) 31 SABC 1988 Roots Trivalvaria macrophylla (Annonaceae) Flu, headache

37 SABC 3970 Stems Baccaurea angulata (Euphorbiaceae) Conjunctivitis

38 SABC 3809 Leaves Tetracera macrophylla (Dilleniaceae) Cough Potential to treat 41 SABC 1528 Whole plant Calophyllum lanigerum (Clusiaceae) AIDS Potential to treat 42 SABC 1528 Stems Calophyllum lanigerum (Clusiaceae) AIDS 43 SABC 4492 Stems Albizia corniculata (Fabaceae) Sore throat

*Information obtained from; Chai 2006 (Chai, 2006), Salleh 2002 (Salleh et al., 2002), Yaacob 2009 (Yaacob et al., 2009) , Maji 2010 (Maji et al., 2010), Focho 2010 (Focho et al., 2010)

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The optimal concentration for antiviral inhibition was found to be different for each extract, but 12.5 to 50 µg/mL was the most active range for all extracts except Extract 29, which was active between 50-100 µg/mL. There were differences in the trends of antiviral inhibition followed by these extracts depending on the influenza virus strain. For instance, Extract 8 caused more than 90% viral inhibition against H1N1 but the viral inhibition was only close to 85% in the case of H3N1. Although only 70% to 80% cells were viable in the cytotoxicity assay, about 90% of the virus was inhibited by

Extract 8. Given that its CC50 value was similar to the commercial NAIs, Extract 8 seems to be a potential candidate for the generation of safe anti-influenza compounds.

Around 70% of H3N1 virus was inhibited by Extract 14 between 12.5 to 50 µg/mL of the extract following a concentration-independent viral inhibition, where the difference in concentration did not alter the percentage viral inhibition. Against H1N1, Extract 14 was more active with 80% viral inhibition between 12.5-25 µg/mL compared to 55% to 64% viral inhibition exhibited at 50 µg/mL concentration.

As shown in Table 3.3, the IC50 values of plant extracts were greater than 0.78 µg/mL (with the exception of Extract 8), unlike the positive controls Zanamivir and

Oseltamivir. Extract 8 and Extract 43 both showed lower IC50 values, less than 0.78 and

1.5 µg/mL respectively, against the H1N1 virus. Extract 29 exhibited the highest IC50, 39.3 and 37.5 µg/mL against H3N1 and H1N1, respectively. On the other hand, as expected for pure drugs, Zanamivir and Oseltamivir inhibited influenza virus at all concentrations tested (0.78 to 100 µg/mL).

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Table 3.3. Inhibitory concentrations of extracts against H3N1 and H1N1 strains

IC50 (µg/mL) against IC50 (µg/mL) against Extract H3N1 H1N1 8 19.4±4.5 <0.78

13/30 15.5±2.8/6.8±0.2 17.0±0.3/13.1±0.5

14 7.3±2.7 9.3±1.5

29 39.3±6.9 37.5±1.5

31 15.6±1.5 6.3±2.0

37 27.2±1.6 17.6±0.6

38 14.3±1.7 2.8±1.8

41/42 10.8±3.6/2.0±1.3 6.3±2.4/9.9±0.2

43 6.7±0.5 1.5±0.7

Zanamivir <0.78 <0.78

Oseltamivir <0.78 <0.78

IC50 represents the concentration of plant extracts needed to reduce the viral inhibition by 50% relative to the virus control wells without test compound, calculated from dose–response data of virus inhibition.

Plant extracts (0.78-100 µg/mL) in RPMI medium were challenged with 100 TCID50 of either of the two virus strains.

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3.3. DISCUSSION

Growing trends in the use of natural products have resulted in more detailed research on medicinal plants. Currently, there is an inevitable loss of traditional knowledge of medicinal plants, as not all cultures have preserved the important medicinal facts about flora in written form. Documenting results obtained through scientific studies on medicinal plants may aid to channel drug discovery towards a promising path for the generation of antimicrobials. Prior to modern scientific testing, efficacies of plants were determined by directly subjecting them to humans. Therefore, those plants that were used by several generations in the treatment of infections may serve as a primary source for the development of drugs. This could be the predominant reason behind the assumed safety of drugs derived from an ethnomedical background. Though plants synthesize these chemicals as a preventive measure against oxidative stress, several diversified pharmacological and physiological effects have been demonstrated by several studies (Srivastava et al., 2012, Tayal et al., 2012).

Out of the 50 extracts tested in this study, 28 showed similar CC50 to that of the commercial NAIs. Therefore, these extracts might serve as potential candidates for the development of safe drugs. CC50 values within the range of 7.5 to 18.4 µg/mL were observed in extracts 26, 33, 44 and 47. Despite the effect of these plant extracts upon the viability of MDCK cells, there exists a possibility for this cytotoxic effect to be reduced or diminished in the presence of a virus where the extracts may preferentially act against the cytopathic effects caused by the virus in an antiviral screening assay rather than affecting the host cells.

The cytotoxicity studies of plant extracts indicated the cytoprotective effects of medicinal plants. The chemical components present in these extracts may also have a positive effect on the growth or viability of cells as reports indicate the presence of growth promoting factors in plants. The presence of phosphates, amino acids, vitamins and sugars that constitute the growth medium of cells are known to be present in plants (Bansode and Chavan, 2013, Benmehdi et al., 2013, Bhuvaneswari and Periyanayagam, 2012, Velraj et al., 2013). Dichloromethane and methanol, the solvents that were used for plant extract preparation cover a range of polarity; dichloromethane being a polar aprotic solvent and methanol, a polar protic solvent, hence supporting the extraction of

Chapter 3 a variety of compounds with different polarity (Lowry and Richardson, 1987). Therefore, these components may have promoted the cell viability as observed in extracts 29, 37, and 38 in the cytotoxicity assay.

Since the plant material subjected to the assays are crude extracts, those that showed lower IC50 may contain higher amount of the active principle, responsible for causing virus inhibition or potent active components that exert a larger response. Chemical characterization of the active components present in the plant extracts may lead to concentration-independent virus inhibition like that of Zanamivir and Oseltamivir, where the difference in the amount of drug used for virus inhibition did not seem to alter the antiviral activity curve. The amount of active components present in the plant extracts and their efficiency in preventing virus inhibition play a major role in demonstrating antiviral activity.

An extract potentially contains various chemical components such as flavonoids, indoles, isoflavones, alkaloids, aminoacids, isothiocyanate, phytosterols, carotenoids, chlorophyll derivatives (Srivastava et al., 2012). Hence, any component present in the extract may have anti-influenza potential. Therefore, isolation of the active component(s) responsible for the antiviral activity is essential for characterising the compound. However, synergistic effects of the mixture of bioactive constituents in the plant extracts may be required for their effectiveness (Wagner and Ulrich-Merzenich, 2009). Since synergism is a key factor for traditional medicines, the plant extracts were not subjected to thorough chemical analyses at this stage.

A better understanding of the various proteins present in the influenza virus has been achieved through intense virological studies (Du et al., 2012a). Thus, the mechanism through which the extracts exert antiviral inhibition could be studied in a detailed manner. The study of plant extracts’ anti-influenza efficiency against the transmembrane proteins, hemagglutinin (HA) and neuraminidase (NA), supported by various bioassays that determine the antiviral activity of extracts against the binding and penetration of the viruses forms the next stage of this project.

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CHAPTER 4 MODE OF ACTION STUDIES ON ANTI-INFLUENZA MEDICINAL PLANT EXTRACTS

4.1. INTRODUCTION

An anti-influenza agent may have the potential to affect any part of the virus, including transmembrane proteins, which in turn affect viral replication and spread. Hence, effective viral inhibition is initiated by the anti-influenza compound. Testing for activity against HA and NA virion glycoproteins forms the next level of screening for extracts that demonstrated activity against influenza viruses. Other proteins present in the virus such as polymerase complex proteins, polymerase acidic subunit, nucleoproteins, ribonucleoprotein particles (RNPs), matrix proteins, nuclear export proteins and non- structural proteins (Du et al., 2012b) may also serve as potential targets for activity against influenza viruses. In the event of evaluating antiviral efficacy of a potential anti- influenza agent, processes such as viral attachment, penetration and release of viral progeny should also be taken into account, since assessing antiviral efficacy against these vital processes might support the demonstrated mode of action.

Although other proteins present in the influenza virus serve as targets for antiviral agents, the influenza NA is an established primary target for treating influenza infections. Zanamivir and Oseltamivir are the only NAI drugs that are approved worldwide, while Laninamivir and Peramivir are approved in some parts of Asia. These drugs prevent the release of vial progeny from infecting the nearby cells, thereby blocking viral infection. The present narrow drug profile for the prophylaxis and treatment of flu infection has developed an active quest for NAI drugs. The highly conserved structure of the viral NA active site was the predominant reason for the lack of viral resistance during the initial years of NAI drug application. However, the emergence of Oseltamivir resistant H1N1 strains has urged the need for more antivirals to treat the infection. NAIs are still, the preferred choice of extenuating the infection due to the drug tolerance in humans compared to M2 ion channel inhibitors. Thus, the search for new NAIs still remains an area of active research (Grienke et al., 2012).

At present, functional NA inhibition assays are widely accepted in NAI susceptibility examinations, as they are highly standardized and approved in primary screening for novel NAIs. Functional NA enzyme inhibition assays are based on either fluorescence 65

Chapter 4

(FL) or chemiluminescence (CL). The virus type, subtype and strain, as well as the NAIs tested, have an influence on the 50% inhibitory concentrations indicated by the results from NAI susceptibility surveillance studies with FL and CL-based NA- inhibitory assays. FL assays were preferred over CL assays since IC50 values generated by CL assays are lower and phenol red interference with the light signal emission might affect the quality of the assay. Though in-house FL assays are flexible and inexpensive, the lack of a standard universal protocol supported the use of the NA-FluorTM kit (WHO, 2012). The substrate for FL assays, 4-methylumbelliferyl-N-acetyl-α-D- neuraminic acid (MUNANA), is cleaved by viral NA to release the fluorescent compound 4-methylumbelliferone (MU). An antiviral agent that targets the viral NA may prevent the conversion of MUNANA to MU. Therefore, any lack in the relative fluorescence unit indicates NAI activity (Grienke et al., 2012).

The influenza viral HA derives its name from the hemagglutination process. The property of influenza HA to agglutinate erythrocytes through the process of hemagglutination presents a simple test to assess potential antiviral activity of extracts against viral HA. Specific attachment of compounds to the antigenic sites on the HA molecule interferes with the binding between the viral HA and receptors on the erythrocytes. Thus, visualizing the loss of hemagglutination activity of viral HA in the presence of a potential antiviral compound indicates the presence of hemagglutination inhibition (HI) activity. The HI test is extremely reliable, and widely used as an antiviral screening assay to classify the specific mode of action for influenza antivirals. As the currently available drugs, namely Zanamivir, Oseltamivir, Laninamivir and Peramivir, inhibit the NA activity of the virus, an agent that inhibits viral HA may be considered novel. Such an anti-influenza agent may have the potential to evolve into a third generation anti-influenza drug. In theory, an HA inhibitor could be a more effective anti-influenza approach, since NAIs, in the case of Oseltamivir, are not effective during the later stages of viral infection as the virus builds up in number in the host body (Chang et al., 2011b).

Development of protocols to test the antiviral efficacy of potential extracts against specific stages of viral life cycle such as viral binding and penetration aids to further support the mode of action assay studies. The extracts that may inhibit viral HA would also demonstrate inhibitory activity against both viral binding (attachment) and

Chapter 4 penetration of the virus. Since recent studies have demonstrated ample evidence for the role played by NA in viral entry, this study also aims to compare the level of inhibitory activity exhibited by extracts demonstrating NAI activity with those extracts that possess HI or both modes of action. A time of addition assay was also included to assess the antiviral activity at different time points of viral inoculation.

Receptor destroying enzyme (RDE) treatment of extracts was included in the assays to identify extracts that possess SA-like components that interfere with the active sites of viral HA. Any loss in the HI activity of extracts after RDE treatment may be the results of the elimination of compounds that contain a structure similar to the host cell SA receptor. Therefore, RDE treatment aims to give an indication regarding the structural features of potential anti-influenza compounds (WHO, 2002). Thus, the framed set of assays serves to assess the mode by which the extracts act against influenza viruses.

4.2. RESULTS

4.2.1. Anti-influenza extracts studied with a time-of-addition assay

The antiviral potential of eleven extracts that demonstrated antiviral activity against the H3N1 and H1N1 strains were tested at different times (-1h, -2h, 0h, +1h, and +2h) relative to virus inoculation. As shown in Table 4.1 and Figure 4.1, all extracts inhibited the viruses by more than 50% in at least one concentration in all time points against the H3N1 strain. As shown in Figure 4.1, all extracts, except 13/30 at +1h, Extract 14 at +2h and -2h, and Extract 41/42 at -2h, were active at 50 µg/mL against the H3N1 strain. Extract 14 was the only extract that was inactive in the -2h time point against the H1N1 strain (Figure 4.2). As shown in Figure 4.2, all extracts were capable of inhibiting the PR8 (H1N1) except Extract 14 which was inactive at 50 µg/mL at -2h and -1h, however efficiently inhibiting the virus at 12.5 µg/mL at -1h time point (Table 4.2). At 0.78 µg/mL, none of the extracts demonstrated inhibitory activity against both the viruses.

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Table 4.1. Inhibitory effects of plant extracts against H3N1 at various time points of viral inoculation

Viral inhibition (%) of extract (concentration in µg/mL) against H3N1 strain

Extract -2h -1h 0h +1h +2h

25 12.5 25 12.5 25 12.5 25 12.5 25 12.5

8 - - 55.0±12.8 60.3±3.9 93.4±0.2 93.9±0.2 55.2±7.3 64.5±2.9 - - 13/30 66.3±1.0 66.2±2.3 76.6± 1.0 68.5±0.3 91.5±0.4 89.9±0.3 64.3±5.8 - 55.5±6.5 51.7±6.3

14 87.8±4.5 78.8±7.6 - 63.3 ±2.3 83.1±2.1 91.8±1.2 76.8±9.3 52.6±1.2 59.0±9.4 -

29 - 62.4±1.8 - 56.4±1.8 ------31 85.3 ± 0.1 91.3±0.4 71.9±1.3 88.7±0.5 89.7±0.7 92.1±0.3 63.6±5.2 53.8±3.3 77.5±2.0 72.9±6.0

37 - - - - 66.4±0.4 79.0±1.3 - 61.2±15.0 - -

38 77.7±1.2 94.7±0.2 78.8±2.0 94.9±0.2 87.1±1.1 74.6±9.5 89.4±1.6 75.3±1.3 80.9±1.2 91.2±0.8

41/42 56.3±1.8 73.6±0.9 - 63.0±1.3 72.8±2.7 75.3±4.2 55.7±2.2 74.0±1.3 - 61.3±3.7

43 - - - 53.3±5.8 74.5±3.1 80.2±2.2 - - - -

Zanamivir 83.6±0.5 87.8±0.6 86.9±0.7 88.9±0.4 69.4±0.7 70.5±0.3 74.7±2.3 80.6±0.5 60.5±0.6 78.1±0.6

Oseltamivir 78.6±4.6 81.8±1.0 83.2±6.2 82.8±1.4 70.8±0.8 69.9±0.3 59.0±2.1 50.7±0.5 - -

The activity of extracts at 25 and 12.5 µg/ml concentrations against H3N1 (100 TCID50) influenza virus at various time points of viral inoculation are shown along with standard errors. A dash (-) indicates lack of anti-influenza activity

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Figure 4.1. Inhibitory effects of plant extracts against H3N1 at various time points of viral inoculation Briefly, 100 L/well of each plant extract, at 50 µg/mL concentration, were added to 80% confluent MDCK cells at either 1 or 2 hours prior to infection (-1 and -2, respectively), at the time of infection (0), or 1 or 2 hours after viral infection (+1 and +2, respectively). The infection was performed by adding 100 µL/well of H3N1

(100 TCID50). The various time points (-1, -2, 0, +1, +2) were tested independently in separate plates. 100 µL of virus growth medium was added to each well and the plates were then incubated for three days at 37ºC/5% CO2, after which the virus inhibition was quantified as described above. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p value < 0.05 (one way ANOVA). 69

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Figure 4.2. Inhibitory effects of plant extracts against H1N1 at various time points of viral inoculation

This experiment is the same as explained in Figure 4.1, except that another viral strain, H1N1 (100 TCID50) was employed. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p value < 0.05 (one way ANOVA).

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Table 4.2. Inhibitory effects of plant extracts against H1N1 at various time points of viral inoculation

Antiviral activity of extract (concentration in µg/mL) against H1N1 strain

Extract -2h -1h 0h +1h +2h

25 12.5 25 12.5 25 12.5 25 12.5 25 12.5

8 58.1±3.5 57.4±2.3 94.3±0.8 86.8±2.6 75.4±0.8 80.5±0.9 79.2±1.3 78.9±1.7 82.4±4.3 75.0±4.6 13/30 92.0±0.4 84.1±2.1 89.0±0.6 91.5±0.7 87.3±2.9 80.9±8.2 90.9±0.1 78.9±1.1 85.1±0.2 87.8±0.5 14 - - - 91.9±0.6 55.5±1.5 74.2±0.2 50.1±2.6 76.5±6.9 93.1±0.1 84.9±0.6 29 - - 55.8±10.5 69.5±0.2 - - - 56.5±5.7 - 66.1±3.2 31 56.8 ± 0.4 88.6±0.6 63.8±3.5 83.9±0.8 87.0±0.5 88.3±0.3 80.2±2.8 88.8±1.5 70.6±3.0 90.1±0.3 37 - 52.9±2.5 - 50.0±7.1 - 63.5±2.6 67.0±0.8 76.1±0.7 - 69.6±3.3 38 79.1±9.3 93.0±0.4 54.9±11.7 90.3±1.3 92.1±0.6 87.8±1.7 93.7±0.4 93.9±0.2 81.8±0.7 88.6±1.0 41/42 54.7±3.9 82.3±0.7 89.4±0.4 94.2±0.3 72.9±0.6 76.4±0.4 - 52.2±3.7 90.2±0.5 92.3±0.1 43 89.8±1.9 94.6±0.5 91.5±2.1 71.1±1.0 74.5±0.8 78.3±0.3 68.9±1.3 76.7±2.6 69.8±0.4 72.9±4.6 Zanamivir 83.8±0.9 87.5±0.4 88.0±1.0 88.2±0.2 80.3±2.0 80.6±0.1 84.9±1.2 85.2±0.3 75.1±3.8 81.3±1.3 Oseltamivir 77.7±4.1 79.8±0.7 80.5±1.3 78.0±4.4 75.1±2.0 76.5±2.5 - - - 62.3±3.3

The activity of extracts at 25 and 12.5 µg/ml concentrations against H1N1 (100 TCID50) influenza virus at various time points of viral inoculation are shown along with standard errors. A negative sign (-) indicates lack of anti-influenza activity

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4.2.2. Inhibitory effects of extracts on the attachment of H3N1 and H1N1

Plant extracts were tested for their ability to inhibit viral attachment using a virus binding assay. As shown in Figure 4.3, nine out of eleven extracts demonstrated more than 50% viral inhibition against both H3N1 and H1N1 at 25 µg/mL. The results evidenced significant inhibitory effects of extracts 8, 13, 14, 30, 31, 38, 41, 42 and 43 on the binding of influenza virus. As expected, the established NAIs, Zanamivir and Oseltamivir, did not inhibit virus binding. As shown in Table 4.3., plant extracts inhibited the binding of H3N1 and H1N1 viruses depending on the concentration used in the assay. The virus inhibition percentages of the wells that received higher concentrations of the plant extracts (50 or 12.5 µg/mL) were greater than the wells treated with lower concentrations (0.78 µg/mL). Only extracts 14 and 42 inhibited binding of the H3N1 strain at lower concentrations, the other extracts were inactive at 0.78 µg/mL.

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Figure 4.3. Inhibitory effects of plant extracts on the binding of H3N1 and H1N1 virus

Cells that are 80% confluent and pre-chilled at 4ºC for an hour were infected with 200 TCID50 of H1N1 or H3N1 followed by supplementation with plant extract at 25 µg/mL concentration. After 3 h incubation at 4ºC, cells were washed twice with ice-cold PBS and overlaid with RPMI and virus growth medium. Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. The effects of plant extracts on virus binding at a concentration of 25 µg/mL are shown. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p value < 0.05 (one way ANOVA).

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Table 4.3. Inhibitory effects of anti-influenza extracts on the binding of H3N1 and H1N1 strains

Percentage viral inhibition against Percentage viral inhibition against the binding of H3N1 strain the binding of H1N1 strain Extract Concentration (µg/mL) Concentration (µg/mL) 50 12.5 0.78 50 12.5 0.78 8 92.7±0.8 95.5±0.9 - 88.8±1.0 89.3±0.7 - 13 88.8±2.5 56.1±8.5 - 89.5±0.1 56.4±1.6 - 14 - 69.2±1.3 65.0±0.3 - 89.3±3.7 - 29 ------30 88.3±2.4 62.8±1.1 - 76.0±3.0 - - 31 91.0±0.1 90.4±0.7 - 62.4±1.0 61.9±1.0 - 37 ------38 69.2±4.5 63.2±3.5 - 79.0±3.0 82.6±0.43 - 41 95.4±0.1 87.9±2.0 - 89.7±1.3 88.1±0.2 - 42 90.3±0.3 90.1±0.2 57.9±6.5 86.8±0.2 88.1±0.6 - 43 93.5±0.1 93.0±2.8 - 83.0±1.5 87.2±0.4 - Zanamivir ------Oseltamivir ------

The activity of extracts against the binding of influenza virus at 50, 12.5 and 0.78 µg/ml is shown along

with standard errors. Plant extracts were challenged with 200 TCID50 of either of the two virus strains. A negative sign indicates lack of anti-influenza activity.

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4.2.3. Inhibitory effects of extracts on the penetration of influenza virus

As shown in Table 4.4., all extracts were able to prevent viral penetration, with the exception of Extract 29 which was ineffective at all the time points and Extract 37 which was active only against the H3N1 strain. Figure 4.4, shows the effects (25 µg/ml) of extracts against the H3N1 and H1N1 strains at 60 min. Four plant extracts (8, 30, 31 and 38) demonstrated virus inhibition at all three time points, including the effect of 50 and 12.5 µg/ml of plant extracts at 60 min (Appendix II). For three HI extracts (41, 42 and 43), inhibition of virus penetration increased over time as the antiviral activity of the extract at 60 and 120 min was greater than that observed at 30 min against the H3N1 strain. Of interest, some extracts that lacked HI activity (13/30, 14, 31, 37, 38) were also shown to inhibit virus penetration. Although Zanamivir and Oseltamivir should normally act against virus release and not virus penetration, surprisingly, 50 µg/ml Zanamivir showed 68% viral inhibition against H3N1 at 60 min (Appendix II). As expected, Oseltamivir was inactive against both viruses in the assay.

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Table 4.4. Inhibitory effects of anti-influenza extracts on the penetration of H3N1 and H1N1 strains at 30 and 120 min

Antiviral activity of extract (concentration in µg/mL) Antiviral activity of extract (concentration in µg/mL) against against the penetration of H3N1 strain the penetration of H1N1 strain Extract 30 min 120 min 30 min 120 min 50 25 50 25 50 25 50 25 8 81.7±0.7 - 90.4±3.6 85.3±7.3 89.8±3.3 80.5±9.7 90.1±0.8 89.5±0.1 13/30 60.7±1.1 - 63.5±4.5 - 76.8±1.7 67.9±8.7 85.6±1.9 64.6±2.5 14 85.7±0.5 64.2±1.2 71.8±0.8 64.1±1.6 53.8±1.9 - - - 29 ------31 92.8±0.3 81.8±9.2 78.3±2.6 56.1±14.1 58.2±3.2 65.2±7.9 86.7±1.5 87.4±0.6 37 73.7±1.9 - 59.4±2.4 - - - - - 38 88.2±4.8 65.0±2.7 51.4±1.4 52.7±3.1 54.2±9.6 64.5±1.8 82.2±4.6 91.8±0.6 41 73.4±5.2 - 91.9±1.8 68.7±16 79.9±1.1 73.5±12.2 79.8±0.9 79.3±0.3 42 83.7±2.3 58.0±7.7 85.8±2.7 65.8±0.8 57.1±6.2 - 60.9±7.6 54.0±1.8 43 76.0±9.0 - 86.5±1.2 91.2±1.0 71.3±1.0 68.2±1.8 - 65.4±0.3 Zanamivir ------Oseltamivir ------

The activity of extracts against the penetration of influenza virus (200 TCID50) at 50 and 25µg/mL is shown along with standard errors. The threshold concentration of active components that prevent virus penetration appears between 25-50 µg/mL of plant extracts. A negative sign indicates lack of anti-influenza activity.

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Figure 4.4. Inhibitory effects of anti-influenza extracts on the penetration of H3N1 and H1N1 strains at 60 min

Monolayers of MDCK cells (80% confluent) were chilled at 4ºC for an hour and then incubated with 200

TCID50 of H3N1 or H1N1 viruses at 4ºC for 3h. Plant extracts (25 µg/ml in RPMI medium) were then added in triplicate and incubated for 60 minutes at 37ºC/5% CO2. Following inactivation and neutralization of unpenetrated virus using acidic and alkaline PBS, respectively, cells were washed with PBS and overlaid with RPMI medium and virus growth medium in equal proportion. Cell viability was evaluated using MTT after three days of incubation at 37ºC/5% CO2. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p value < 0.05 (one way ANOVA).

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4.2.4. NAI effects of plant extracts

The virus NA glycoprotein has sialidase activity and mediates the release of viral progeny from the infected cell, thus promoting virus transmission and spread (Wirotesangthong et al., 2009). In addition, the viral NA removes SA from glycans expressed by the viral HA glycoprotien, thereby preventing self-aggregation of the virions (Song et al., 2005). All eleven extracts were tested for NA inhibitory activity against the H3N1 and H1N1 strains using the NA- Fluor™ Influenza Neuraminidase Assay Kit. Increasing concentrations of plant extracts were associated with decreased relative fluorescence (Table 4.5.) , consistent with inhibition of NA activity. IC50 values indicated that extracts 8 and 43 reduced NA activity at much lesser concentration than the other extracts. It should be noted that crude plant extracts were tested and so results should not be compared directly with Zanamivir and Oseltamivir, as these commercially available drugs were tested at nanomolar concentrations (Table 4.6).

Table 4.5. NAI activity of extracts

Extract IC50 (µg/ml) against H3N1 IC50 (µg/ml) against H1N1

8 1.15±0.03 0.59±0.10 14 7.81±0.81 4.33±0.12 29 4.19±1.26 4.57±0.35 13/30 3.87±0.07 2.46±0.47 31 5.51±0.21 2.21±0.18 37 6.70±1.35 9.91±0.30 38 3.12±0.04 0.52±0.11 41/42 4.47±0.15/6.35±0.87 5.76±0.05/5.34±0.11 43 0.43±0.01 1.38±0.07

The plant extracts’ NA inhibitory activity was measured at concentrations ranging between 0.3 to 25 µg/mL. The optimum virus dilution for the NA inhibition assay was selected by titration of virus stock in an NA activity assay; 1:8 dilution of either of the viruses was selected to perform the NAI assay

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Table 4.6. NAI assay using commercial drugs

NAIs IC50 (nM) against H3N1 IC50 (nM) against H1N1

Zanamivir 6.00±0.58 7.12±0.80 Oseltamivir 6.90±3.10 16.10±0.30

The controls Zanamivir and Oseltamivir were assayed at 0.01 to 10,000 nM as recommended by the manufacturer. The optimum virus dilution for the NAI assay was selected by titration of virus stock in an NA activity assay; 1:8 dilution of either of the viruses was selected to perform the NAI assay

4.2.5. Inhibitory effects of plant extracts on influenza virus-induced hemagglutination

The influenza virus HA mediates attachment to SA residues expressed by host cell glycoproteins and glycolipids, which is a critical first step in the initiation of infection (Chang et al., 2011a). Similarly, the viral HA binds to SAs expressed on the surface of erythrocytes resulting in hemagglutination. Thus, the ability of plant extracts to inhibit virus-induced hemagglutination using a hemagglutination inhibition (HI) assay was examined. As shown in Figure 4.5., four out of eleven extracts mediated HI activity against the H3N1 and H1N1 viruses at specific concentrations. Extract controls were included to study the direct effect of extracts on chicken red blood cells in the absence of viruses. All four HI extracts exhibited hemolysis above 25 µg/mL in the absence of viruses. Some hemolytic concentrations of extract control resulted in HI when the virus was included, suggesting that extract components preferentially attach to the virus rather than the erythrocytes. Plant extracts that mediated HI activity against both viral strains were active in preventing virus-induced hemagglutination at concentrations ranging between 6.25-25 µg/mL.

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Concentration of E81 E82 E83 E41/421 E41/422 E41/423 E431 E432 E433 extract (µg/mL) 100

12.5

6.25

3.13

1.56

0.78

Virus control for H3N1 Virus control for H1N1 Monoclonal antibody Monoclonal antibody Cell control against H1N1 against H3N1

Figure 4.5. Inhibitory effects of plant extracts on influenza virus-induced hemagglutination

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HI activities of four extracts (0.78-100 µg/mL) against 4HAU/25µL of virus are shown.

E - Extract 1 - Extract control 2 – HI activity of extract against H3N1 3 – HI activity of extract against H1N1 The following controls were included on each plate:

(i) extract controls with extract and chicken red blood cells (CRBC) only, (ii) virus controls containing virus and CRBC, and (iii) cell controls containing only CRBC

Monoclonal antibody against the HA of either H3N1 or H1N1 strains were included as a positive control. The antibody titres for monoclonal antibody against H3N1 and H1N1 were 80 and 200, respectively; 1:8 dilutions of either of the two antibodies in PBS were employed in the assay. Data are shown from one of three independent experiments, each performed in triplicate.

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4.2.6. Effect of RDE treatment on the antiviral activity of extracts

Four extracts which were shown to interfere with hemagglutination of CRBC, were treated with RDE in order to eliminate compounds that might contain SA mimics that compete with the RBC receptors for virus hemagglutinin. An HI assay was performed with RDE-treated extracts which were originally able to prevent hemagglutination. As shown in Figure 4.6., RDE treatment removed HI activity originally exhibited by all four extracts.

HI against H3N1 RDE Treated HI HI against H1N1 RDE Treated HI Extract extracts against extracts against H3N1 H1N1

41/42

Virus control

MAb

Cell Control Figure 4.6. Effect of RDE treatment on plant extracts’ activity

HI activities of four extracts (25 µg/mL) treated with RDE against 4HAU/ 25µL of either virus are shown. (i) Virus controls containing virus and CRBC and (ii) cell controls receiving CRBC only are shown. Corresponding RDE treated monoclonal antibody which acts against the HA of H3N1and H1N1 and extracts that mediate HI activity without RDE treatment were included in all plates as positive controls. The experiment was performed in triplicate.

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Further, an in vitro micro-inhibition assay was performed with the RDE-treated extracts against H3N1 and H1N1 viral strains. As shown in Figure 4.7, there was a significant reduction in the antiviral efficacy of extracts with HI potential whereas non-HI extract 38, included as a negative control, did not show any significant difference in viral inhibition before and after RDE treatment.

Figure 4.7. Effect of RDE treatment on the antiviral activity of plant extracts against H3N1

An in vitro micro-inhibition assay was used to assess the ability of plant extracts (25 µg/mL) to inhibit

H3N1 (100TCID50) influenza virus. Extracts were either treated with RDE as per the manufacturer’s instructions or left in their native form without RDE treatment. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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RDE treatment was also shown to reduce the antiviral activity which was originally demonstrated against H1N1 virus (Figure 4.8).

Figure 4.8. Effect of RDE treatment on the antiviral activity of plant extracts against H1N1

This experiment is essentially the same as explained in Figure 4.7, except for the virus; H1N1

(100TCID50) influenza virus was studied. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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4.2.7. Effect of Trypsin treatment on the antiviral activity of extracts

Plant extracts that were shown to prevent virus-induced hemagglutination were treated with trypsin in order to denature any protein that might be the cause of such inhibition. An in vitro micro-inhibition assay was initially performed to determine the activity of trypsin-treated extracts and controls against H3N1 and H1N1 strains. As shown in Figure 4.9., the antiviral activity of plant extracts against the H3N1 virus was not altered by either trypsin treatment or temperature (without trypsin). The trypsin-treated plant extracts and controls were then subjected to an HI assay. As shown in Figure 4.9, HI activity of plant extracts was exhibited against H3N1 despite trypsin treatment or temperature change. A similar pattern in the percentage viral inhibition and HI activity was observed with the H1N1 strain exposed to trypsin-treated extracts (Appendix III).

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(a) (b)

HI against H3N1 Trypsin Extract Treatment on HI extracts

Virus control Cell Control

Figure 4.9. Effect of trypsin treatment on the antiviral activity of plant extracts

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(a) Inhibitory effect of plant extracts on the hemagglutination of H3N1 viral strain

HI activities of three extracts (50 µg/mL), from a different batch than those that were discussed in section 4.2.5. and section 4.2.6, treated with trypsin against 4HAU/ 25µL of virus is shown. (i) Virus controls containing virus and CRBC and (ii) cell controls receiving CRBC only are shown. Extracts that mediate HI activity without trypsin treatment were included in all plates as positive controls. The experiment was performed in triplicate.

(b) Antiviral inhibition of HI extracts against H3N1 strain

An in vitro micro-inhibition assay was used to assess the ability of plant extracts to inhibit H3N1 (100

TCID50) influenza virus. Extracts (50 µg/mL) were treated with trypsin for 24 hours at 37ºC, followed by incubation at 56ºC for 60 minutes or subjected to temperature treatment without trypsin. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

4.2.8. TEM study of Extract 8

In order to determine if exposure to the plant extracts results in any morphological changes to the virus, the effect of one medicinal extract (Extract 8) was visualized by Transmission electron microscope (TEM). Examples of mock-treated PR8 virus and PR8 treated with Extract 8 are shown in Figures 4.10 and 4.11 respectively. As shown in Figure 4.11, there was no significant change in the morphology of the virus, although virus treated with extract stained darker than mock-treated virus. There were also more virus particles present on the grids of mock-treated samples relative to those of virus treated with the extract or fractions, although this observation could not be formally quantitated. Aggregation of virus particles was also evidenced.

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Figure 4.10. TEM image of PR8 virus

Pioloform coated 100 mesh TEM grids were incubated film side down onto 20 µL virus droplets for 60 min at room temperature. After excess sample was removed by blotting with filter paper, the grids were placed film side down onto 20 µL droplets of 2% uranyl acetate for 3 min. Excess uranyl acetate was removed by blotting with filter paper. The grids were allowed to dry for 24 hours before imaging with CM-120 Bio Twin TEM operating at 120 kV.

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Figure 4.11. TEM image of PR8 virus treated with crude Extract 8

Pioloform coated 100 mesh TEM grids were incubated film side down onto 20 µL virus with extract droplets (equal volume of extract (1µg/µL) and viral stock) for 60 min at room temperature. After excess sample was removed by blotting with filter paper, the grids were placed film side down onto 20 µL droplets of 2% uranyl acetate for 3 min. Excess uranyl acetate was removed by blotting with filter paper. The grids were allowed to dry for 24 hours before imaging with CM-120 Bio Twin TEM operating at 120 kV.

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4.3. DISCUSSION

The mode of action study of the extracts that inhibited influenza viruses allows a better understanding of the effects of these extracts upon the viral proteins and stages of virus replication. All plant extracts tested, exhibited NAI activity. Extracts 8, 38 and 43 demonstrated the lowest IC50 range, indicating a higher amount of NAI in the given plant extract or the presence of potent NAI component(s) that may exert a larger response even at lower concentrations. Extracts were tested at µg/mL concentrations, unlike Zanamivir and Oseltamivir which were tested at nanomolar levels. Selection of the µg/mL concentration range in NAI assay relies on the likelihood that anti-influenza activity may not be detected at very low concentrations as none of the plant extracts showed activity at concentrations less than 3.13 µg/mL in the in vitro micro inhibition assays. As suggested in the literature, the chemical components in the extracts might have combined with the viral membrane to modify the physical properties of viral NA (Shin et al., 2010). The results also indicated that the concentration and time required by the extracts to inhibit viruses is critical and different for each.

Since drug-resistant viruses appear frequently, it is important to identify drugs with a different mode of action to the one observed with the conventional drugs used today (NAI and adamantanes). A sialylated molecule that can block virus attachment to cellular receptors might act to limit the initial stages of virus infection, compared to NA inhibition that is believed to act largely through preventing release of new virions from virus-infected cells (Chang et al., 2011b). Four of the extracts mediated HI activity within a specific range of concentrations. Previous work has shown that some plant extracts can cause hemolysis at higher concentrations in HI assays (Hsieh et al., 2012a).

Hemolysis caused by the extracts in the controls may be attributed to the presence of other compounds apart from those with anti-influenza activity. It is also possible that more than one anti-influenza component could be present in the extracts that showed multiple modes of action. The stable interaction between HA and NA, which is vital for the effective entry and release of influenza virus, may have been disrupted by the anti- influenza component(s) present in the extracts. This could also be considered a new anti-influenza pathway as dual action drugs have not previously been used.

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The inhibitory effects demonstrated against viral binding and penetration further suggested the HI mode of action of extracts 8, 41, 42 and 43. Interestingly, non-HI extracts 13, 14, 30, 31, 37 and 38 which exhibited NAI activity showed significant inhibition in the viral binding and penetration assays. Although, the major functions of sialidase activity of influenza NA are to facilitate the release of viral progeny from infected cells and enable viral spread, desialylation of HA and masking of glycoconjugates carried by the host cell surface proteins and lipids by NA help to promote viral entry (Hsieh et al., 2012a , Matrosovich et al., 2004, Su et al., 2009b, Su et al., 2009a).The significant effects against viral binding and penetration exhibited by extracts that demonstrated only NAI activity may be attributed to the NA inhibitory component(s) which might have inhibited the viral NA that helps to promote viral entry. Similar results have been obtained in previous studies (Hsieh et al., 2012a). The inhibitory effect of Zanamivir at the 60 minutes time point in the penetration assay might have also resulted from the NAI pathway.

The loss of HI activity of the extracts following RDE treatment suggests that the component(s) causing HI may possess SA-like structures that mimic the receptors of CRBC, thereby competing for viral hemagglutinin. RDE-treated extracts showed less than 50% virus inhibition in in vitro micro-inhibition assay at 25 µg/mL whereas native extracts which were not treated with RDE showed significant antiviral activity at the same concentration. All the non RDE-treated HI extracts were able to prevent HI at 25 µg/mL. Deactivation of SA mimics that were originally present in the extract may be the reason for this significant drop in virus inhibition. Thus, SA mimics could be potential compounds that target the viral HA. Since anti-influenza activity was observed at other concentrations tested, potential synergistic effects of components in the plant extracts, apart from those that are HI active, may be present. Further studies to investigate the synergistic effects of NAI activity and HI exhibited by the plant extracts will need to be performed.

The observation that antiviral efficacy was not affected by temperature or trypsin treatment suggests that the compound(s) of interest may not be proteinaceous. It is worthwhile noting that minor variations were evident with the activity of extracts for different batches; this could possibly result from seasonal changes in the composition of

Chapter 4 plant extracts collected at different times or through the collection and extraction processes. For instance, Extract 8 did not demonstrate HI activity in the most recently acquired batch that was used to study the effects of trypsin treatment upon the antiviral activity of extracts. Also, the concentration at which HI activity was shown to be present for extracts 41/42 and 43 were different for the most recent batch unlike the results presented in Figure 4.5. HI activity was present between 50-100 µg/mL for the most recent batch of 41/42 and 43 and the effect was being lost at concentrations less than 50 µg/mL. This phenomenon needs to be further examined.

The anti-influenza effects of extracts showing HI activity have not been published previously, although some plants belonging to the same genus have been reported to show antimicrobial activity (Ali et al., 2001, Basavaraja et al., 2011, Kaur and Kharb, 2011). As the morphology of the virus was not affected by treatment with extract in the TEM study (Figure 4.10. and 4.11.), this further suggests the specific activity of extract (against viral NA and/ or HA). The darker staining of virus upon extract treatment may be due to the reaction between the extract components with the uranyl acetate used in the staining process of grids. Since fewer viruses were present in grids containing virus and extract compared to the virus control grids, further studies to confirm the effect by quantitative determination of virus is essential.

This study suggests that plants with reported medicinal properties could be a potential source for new antiviral drugs against influenza. The plant extracts investigated could serve as promising candidates for the development of third generation anti-influenza drugs, thereby challenging the NAI drug resistant viruses in an attempt to safeguard human health and the global economy.

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CHAPTER 5 ANTIVIRAL ACTIVITY OF SELECTED MEDICINAL PLANT EXTRACTS AGAINST TYPE A AND B INFLUENZA VIRUSES INCLUDING NAI RESISTANT STRAINS

5.1. INTRODUCTION

New influenza viruses arise through variations called antigenic drift and antigenic shift. Antigenic drift refers to a minor change such as an amino acid substitution in either HA and/or NA that causes an antigenic site change. The majority of seasonal influenza infections are caused by antigenic drift. On the other hand, the predominant reason for influenza pandemics is antigenic shift which results in the formation of new viral strains, combining HA and NA from diverse subtypes (Chen and Deng, 2009, Pleschka et al., 2009, Robertson and Inglis, 2011). Since 2010, when the Centers for Disease Control and Prevention (CDC) strongly advised against the use of adamantanes due to the adverse effects and rapid emergence of drug resistance, the NAIs have formed the only class of antivirals recommended by the WHO for the treatment and prophylaxis of influenza A and B infections ( Samson et al., 2013).

Although the structure of the NA active site is highly conserved, making it a prominent target for NAIs in antiviral therapy, mutations in the NA may alter the shape of the catalytic site, thereby reducing the inhibitor binding potential. Due to differences in the chemical structures of the inhibitors, many of the mutations do not confer reduced sensitivity to all the NAIs. Oseltamivir, an NAI drug, has a hydrophobic chain; therefore NA must undergo rearrangements to accommodate drug binding; the efficiency of Oseltamivir may be lowered by mutations that affect this rearrangement. Oseltamivir carboxylate hydrophobic binding relies on the glutamic acid 276 residue present within the viral NA active site. This residue is in close proximity to histidine 274. Oseltamivir resistance is conferred to the virus by a single amino acid change: histidine to tyrosine at position 274 (H274Y). H274Y mutation (H275Y in N1 numbering) confers resistance in only N1 subtype viruses. Similarly, resistance to Zanamivir may result from mutations in framework or catalytic residues of the NA, thereby affecting the binding affinity between the enzyme and the inhibitor (Ives et al., 2002, Samson et al., 2013).

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A novel influenza A (H1N1) that was first detected in 2 children in California was also found to circulate in Mexico and later spread worldwide causing a pandemic infection. Known as pandemic (H1N1) 2009, the virus contains a unique arrangement of RNA segments from North American and Eurasian swine lineages and is efficient in human- to-human transmission. Over 296,000 people were infected by pandemic (H1N1) 2009 virus throughout the world. Although the virus caused a mild-to-moderate illness, severe and fatal cases were also described, with 3486 deaths reported. The major difference between seasonal influenza and pandemic (H1N1) 2009 virus is the ability of the latter to extensively infect the lower respiratory system, in addition to infecting the tracheobronchial epithelium as seen with seasonal influenza. Initial isolates of pandemic (H1N1) 2009 virus were susceptible to NA inhibitors; Oseltamivir was extensively used in the treatment and prophylaxis of pandemic influenza and strains resistant to the drug emerged occasionally in pandemic (H1N1) 2009 viruses. However, seasonal H1N1 influenza viruses from the 2007-2008 seasons onwards were mostly resistant to Oseltamivir with resistance conferred by the H275Y mutation in the viral NA (Chen and Shih, 2009, Goldsmith et al., 2011, Brookes et al., 2011).

Type B viruses are often responsible for a significant proportion of seasonal influenza infections. Oseltamivir has been reported to have lower clinical efficacy in children infected with influenza B compared with those infected with the Type A, and this is consistent with the observation of higher IC50 values in enzyme assays. The D197E mutation in the Type B virus confers cross-resistance to all NA inhibitors. A higher than

10-50 fold change in IC50 values compared with the wild type virus is evident with Oseltamivir and Peramivir, affecting the drug’s potential at a medium rate whereas only a less than 10-fold change in the NAI potential of Zanamivir is shown against Type B virus with D197E mutation (McKimm-Breschkin, 2013). In addition to NA mutations, NAI resistance could also emerge in vitro due to mutations in or near the HA receptor binding site, reflecting the importance of functional balance between HA and NA (Ginting et al., 2012).

With respect to the current study, an anti-influenza agent with demonstrated activity against a wide range of influenza viruses may be considered a more promising target before attempting to fractionate each plant extract. Hence, the plant extracts 8, 41, 42 and 43, that were shown to act through the HI and NAI pathways were selected for

Chapter 5 further testing against types A and B viruses, including Oseltamivir resistant seasonal and pandemic H1N1 viruses and a Type B virus with the D197E mutation. Wild type viruses corresponding to the mutant strains that are susceptible to the NAI inhibitors were also included in the study. In addition to studying the effects of RDE upon the demonstrated HI activity of extracts, the effect of RDE treatment on the NAI activity of extracts was also included to determine whether the antiviral sialic acid (SA) mimics exhibit activities against viral NA. This study may also help in the understanding of whether the synergistic effects of components in the extracts are responsible for the multiple modes of action exhibited.

5.2. RESULTS

5.2.1. Inhibitory effects of plant extracts against seasonal Type A H1N1 virus and Oseltamivir resistant seasonal Type A H1N1 virus with H275Y mutation

The plant extracts 8, 41, 42 and 43 were active against both the wild type seasonal A

(H1N1) and mutant seasonal A (H1N1) (H275Y) viruses. As shown in Table 5.1, IC50 values for Extract 8 were less than 3.13 µg/mL against both viruses. Extracts 41 and 42, obtained from different parts of the same plant, showed similar IC50 values of 14.86

µg/mL and 14.46 µg/mL, respectively, against the wild type virus, while higher IC50 values (22.87 µg/mL and 21.06 µg/mL) were obtained against the mutant virus. Extract

43 was shown to have an IC50 of 16.45 µg/mL against the wild type, while 27.63 µg/mL of Extract 43 was required to reduce the activity of mutant virus to 50%. Among the NAI drugs that were included as controls, Zanamivir showed antiviral inhibition against both viruses while Oseltamivir was active only against the wild type and showed an

IC50 value greater than 100 µg/mL against mutant seasonal A (H1N1) (H275Y) virus.

As shown in Figure 5.1, Extract 8 demonstrated similar activity against both the wild type seasonal A (H1N1) and mutant seasonal A (H1N1) (H275Y) virus between 25-100 µg/mL, but the activity of the extract against the mutant strain showed a lower percentage of viral inhibition compared to the wild type virus at concentrations less than 12.5 µg/mL. Extracts 41, 42 and 43 also showed a similar trend in viral inhibition against both wild type and mutant seasonal Type A viruses however inhibition against the mutant strain was relatively lower than that demonstrated against the wild type.

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Table 5.1. Inhibitory concentration of anti-influenza extracts

IC50 (µg/ml)

Extract Seasonal A Seasonal A Pandemic A Pandemic A Type B Type B (H1N1) (H1N1) (H1N1) (H1N1) (D197E) (H275Y) (H275Y)

8 <3.13 <3.13 <3.13 <3.13 <3.13 <3.13 41/ 14.86±2.47/ 22.87±3.33/ <3.13/ 9.99±2.02/ 11.16±5.88/ 11.29±4.2/ 42 14.46±6.89 21.06±5.79 <3.13 <3.13 4.6±2.13 <3.13

43 16.45±6.00 27.63±4.67 <3.13 12.2±4.99 13.55±1.40 9.19±3.06 Zanamivir <3.13 96.62±23.18 <3.13 <3.13 <3.13 <3.13 Oseltamivir <3.13 >100 <3.13 >100 6.2±0.1 22.67±6.89

IC50 represents the concentration of plant extract needed to reduce the viral inhibition by 50% relative to virus control wells without test compound, calculated from dose–response data of virus inhibition. Plant extracts (3.13-100 µg/mL) in RPMI medium were challenged with 100 TCID50 of influenza viruses.

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Viral inhibitionViral (%)

Concentration (µg/mL)

Seasonal A(H1N1) Seasonal A (H1N1) (H275Y)

Figure 5.1. Inhibitory effects of plant extracts against seasonal H1N1 viruses

Cells at 80% confluence were treated with two-fold serial dilutions of plant extracts (3.13-100 µg/mL) and 100 TCID50 of either seasonal H1N1 or mutant seasonal H1N1 (H275Y) type A influenza viruses. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA) and the differences among means for each concentration were statistically significant with p < 0.05. 97

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5.2.2. Inhibitory effects of plant extracts against pandemic Type A H1N1 virus and Oseltamivir resistant pandemic Type A H1N1 virus with H275Y mutation

The plant extracts 8, 41, 42 and 43 showed antiviral activity against both the wild type pandemic A (H1N1) and mutant pandemic A (H1N1) (H275Y) viruses. As shown in Figure 5.2, Extract 8 showed similar activities against the wild type and mutant pandemic viruses with a demonstrated IC50 value of less than 3.13 µg/mL. Though similar trends in virus inhibition were shown by extracts 41, 42 and 43, a lower percentage viral inhibition was demonstrated against the mutant virus at concentrations less than 6.25 µg/mL in relation to the activity of extracts against the wild type virus.

As shown in Table 5.1, IC50 values were less than 3.13 µg/mL against the wild type virus for all the extracts while 9.99 µg/mL and 12.2 µg/mL were obtained for extracts 41 and 43, respectively, against the mutant virus. Even though extracts 41 and 42 were obtained from the same plant, the IC50 value against the mutant virus was less than 3.13 µg/mL for Extract 42 while, 9.99 µg/mL was obtained for Extract 41; the differences between means for each concentration were statistically significant with p < 0.05.

Since the H275Y mutation confers Oseltamivir resistance, the NAI inhibitor

Oseltamivir was only active against the wild type virus with an IC50 value less than 3.13

µg/mL while the IC50 was greater than 100 µg/mL against the Oseltamivir resistant mutant virus. Zanamivir was shown to be active against both viruses with IC50 values less than 3.13 µg/mL.

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Viral inhibitionViral (%)

Concentration (µg/mL)

Pandemic A(H1N1) Pandemic A(H1N1)(H275Y)

Figure 5.2. Inhibitory effects of plant extracts against pandemic H1N1 viruses

The experiment was performed similar to that explained in Figure 5.1., except that pandemic H1N1 and mutant pandemic H1N1 (H275Y) Type A viruses were employed. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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5.2.3. Inhibitory effects of plant extracts against Type B virus and Type B virus with D197E mutation

Extracts 8, 41, 42 and 43 were also shown to be active against both the wild Type B and mutant Type B (D197E) viruses. As shown in Figure 5.3, the effects of Extract 8 showed similar trends in viral inhibition against both the wild type and mutant Type B viruses, this behaviour was also observed with extracts 41 and 43. However, Extract 42 was active between 6.25-100 µg/mL against both viruses, while the antiviral activity against the wild type virus was only 17% at 3.13 µg/mL of extract. As shown in Table

5.1, Extract 8 demonstrated an IC50 less than 3.13 µg/mL against both the viruses;

Extract 43 showed IC50 values of 13.55 µg/mL and 9.19 µg/mL against the wild type and mutant viruses, respectively. Extract 41 demonstrated a similar IC50, 11.16 µg/mL and 11.29 µg/mL against the wild type and mutant viruses, respectively, while Extract

42 showed 4.6 µg/mL and less than 3.13 µg/mL IC50 values for the same.

Since the Type B virus with D197E mutation confers resistance against all NA inhibitors, both Oseltamivir and Zanamivir were shown to demonstrate significantly lower antiviral activity against the mutant virus relative to the wild type (Figure 5.3), but the IC50 value of Zanamivir showed no significant change for both viruses, even though the D197E mutation affects the NAI potential of the drug in the order of less than 10 fold. However, the IC50 value of Oseltamivir, which is known to be affected by the D197E mutation in the order of 10-50 fold or higher, was shown to have a calculated IC50 value of 22.67 µg/mL against the mutant virus while that against the wild-type virus was 6.23 µg/mL.

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Viral inhibitionViral (%)

Concentration (µg/mL)

Type B Type B(D197E)

Figure 5.3. Inhibitory effects of plant extracts against Type B influenza virus

The experiment was performed similar to that, as explained in Figure 5.1., except that Type B or mutant Type B (D197E) influenza viruses were employed in the assay. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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5.2.4. NAI effects of plant extracts

Influenza NA serves as a principal drug target for the treatment of influenza (Grienke et al., 2012). The plant extracts 8, 41, 42 and 43 were tested for NA inhibitory activity against the six influenza viruses included in the study. Increasing concentrations of extracts were associated with decreased relative fluorescence, consistent with the inhibition of NA activity (Table 5.2.). Extract 8 reduced the NA activity of the six viruses at lower concentrations (3.14 -7.99 µg/mL) compared to other extracts. NAI potential of Extracts 41 and 42 collected from the same plant source were not similar.

As shown in Table 5.2, Extract 41 showed lower IC50 values than Extract 42 against wild type and mutant seasonal A (H1N1), pandemic A (H1N1) (H275Y) and Type B viruses, while Extract 42 showed lower IC50 values than 41 against pandemic A (H1N1) and Type B (D197E) viruses. The IC50 value for Extract 43 ranged between 4.90-10.02 µg/mL against the viruses that were tested.

Zanamivir and Oseltamivir were included as positive controls in the assay and tested at nanomolar concentrations, as recommended by the manufacturer; therefore the results should not be directly compared with the plant extracts that were tested at µg/mL concentrations. IC50 values obtained in our studies for the NAIs were in agreement with those reported in the literature except for minor variations in the NAI potential of Oseltamivir against pandemic A (H1N1), Type B (D197E) virus and Zanamivir against seasonal A (H1N1) (Appendix IV).

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Table 5.2. NAI activity of anti-influenza extracts

IC50 (µg/mL)

Seasonal A Seasonal A Pandemic A Pandemic A Type B Type B (D197E) Extract (H1N1) (H1N1) (H1N1) (H1N1) (H275Y) (H275Y)

8 7.26±0.16 6.21±0.31 3.14±0.04 5.54±0.14 7.99±0.48 4.95±3.60 6.33±2.08/ 6.28±0.23/ 6.96±0.07/ 8.19±0.26/ 8.79±0.43/ 7.69±0.10/ 41/42 9.64±0.50 10.57±0.07 5.15±0.25 9.78±0.16 11.36±2.04 6.43±0.81 43 9.28±0.68 10.02±1.13 6.74±0.05 9.363±0.86 9.26±1.37 4.90±1.50

NAI drugs IC50 (nM) Oseltamivir 4.52±1.14 1517±93.31 11.92±0.08 1413.5±197.28 22.03±0.10 1335.5±234.0 Zanamivir 5.152± 0.78 1.88±0.12 1.07±0.01 1.01±1.41 4.56±0.10 50.18±2.93

NAI activity of the plant extracts was measured at concentrations ranging between 0.3 to 25 µg/mL, while the controls, Zanamivir and Oseltamivir were measured at 0.01 to 10,000 nM, as recommended by the manufacturer. The optimum virus dilution for the NAI assay was selected by titration of virus stock in an NA activity assay; 1:4 dilutions of the virus strains were selected in the NA activity assay to perform NAI assay.

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5.2.5. Inhibitory effects of plant extracts on viral HA

The critical step in the initiation of an influenza infection is the binding of viral HA to the SA residues expressed by host cell glycoproteins and glycolipids (Chang et al., 2011a). The viral HA also binds to SAs expressed on the surface of erythrocytes, resulting in hemagglutination. The ability of plant extracts to inhibit influenza virus- induced hemagglutination was studied in a HI assay.

As shown in Figure 5.4, Extract 8 did not demonstrate HI activity against any of the six viruses studied. Extract 41 and 42, which were collected from the same plant, inhibited virus-induced hemagglutination against both the wild type and mutant strains of pandemic A (H1N1) at a concentration ranging between 50-100 µg/mL. Although Extracts 41 and 42 demonstrated HI activity against Type B virus only at 100 µg/mL, both the extracts demonstrated effective HI activity against Type B (D197E) virus between 50-100 µg/mL; however no HI activity was evidenced against the seasonal A (H1N1) viruses. Extract 43 inhibited viral HA of both wild type and mutant pandemic A (H1N1) viruses at 100 µg/mL while exhibiting HI activity only against Type B (D197E) at the same concentration. Nevertheless, Extract 43 did not exhibit HI activity against wild type and mutant seasonal A (H1N1) and wild Type B influenza viruses. The effects of plant extracts on the CRBC did not cause hemolysis at 12.5-100 µg/mL for the batch of extracts that were used to study the HI activity.

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Viruses Virus Concentration of extract (µg/mL) Control Extract 8 Extract 41 Extract 42 Extract 43 100 50 25 12.5 100 50 25 12.5 100 50 25 12.5 100 50 25 12.5 Seasonal A

(H1N1) Seasonal A

(H1N1) (H275Y) Pandemic A (H1N1)

Pandemic

A (H1N1) (H275Y) Type B

Type B

(D197E) Cell control

Figure 5.4. Inhibitory effects of plant extracts on influenza virus-induced hemagglutination

HI activities of four extracts (12.5-100 µg/mL) against 4HAU/25 µL of virus are shown. The following controls were included on each plate; (i) extract controls with extract and CRBC only, (ii) virus controls containing virus and CRBC and (iii) cell controls containing only CRBC. Since extract controls were similar to cell controls, they are not included in the figure. Data are shown from one of three independent experiments, each performed in triplicate.

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5.2.6. Effect of RDE treatment on the antiviral activity of plant extracts

Extracts 8, 41 and 42 and 43 were treated with RDE in order to eliminate SA mimics that may demonstrate antiviral potential against the viruses similar to the results obtained against Mem-Bel and PR8 viruses (Section 4.2.6.). An in vitro micro- inhibition assay against all the six viruses was performed with the RDE-treated extracts. As shown in Figure 5.5, antiviral activities originally exhibited by all four extracts were either eliminated or reduced following RDE treatment. Upon RDE treatment, the antiviral activity of Extract 8 was suppressed against all viruses except mutant pandemic A (H1N1) (H275Y) and Type B viruses where viral inhibition of 51% and 57% respectively, were exhibited. RDE-treated extracts 41 and 42 showed loss of antiviral activity against all the six viruses. On the contrary, the antiviral activity of RDE-treated extract 43 was reduced by only 14% against pandemic A (H1N1) (H275Y) virus (down to 78% virus inhibition), 22% against Type B (45% virus inhibition) and 30% against Type B (D197E) virus (48% virus inhibition). However, the activity against Type B viruses was considered to be inactive since the 50% virus inhibition threshold was not reached. There was no evidence of antiviral activity against either the wild type or the mutant seasonal A virus and pandemic A (H1N1) viruses.

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Viral inhibitionViral (%)

Native extract RDE treated extract

Figure 5.5. Effects of RDE treatment on the antiviral activity of plant extracts An in vitro micro-inhibition assay was used to assess the ability of plant extracts (25 µg/mL, optimum concentration for antiviral activity) to inhibit influenza viruses (100 TCID50). Extracts were either treated with RDE as per the manufacturer’s instructions or left in their native form without RDE treatment. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p value < 0.05 (one way ANOVA). 107

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5.2.7. Effect of RDE treatment on the HI activity of plant extracts

Extracts 41 and 42, which demonstrated HI activity against both the wild type and mutant pandemic A (H1N1), Type B viruses, and Extract 43, which was shown to inhibit virus-induced hemagglutination caused by both the wild type and mutant pandemic A (H1N1) viruses and Type B virus only, were treated with RDE in order to eliminate SA-like components that may compete with the CRBC receptors for viral HA. An HI assay was then performed with RDE-treated extracts against wild type and mutant strains of pandemic A (H1N1) and Type B viruses. As shown in Figure 5.6, RDE treatment suppressed the HI activity originally exhibited by extracts 41, 42 and 43.

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Virus Concentration of extract (µg/mL) Viruses Control Extract 41 RDE-treated Extract 42 RDE-treated Extract 43 RDE-treated Extract 41 Extract 42 Extract 43 100 50 100 50 100 50 100 50 100 50 100 50 Pandemic A (H1N1) Pandemic A (H1N1) (H275Y) Type B

Type B (D197E)

Cell control

Figure 5.6. Effect of RDE treatment on the HI activity of plant extracts

HI activities of three extracts (50-100 µg/mL) treated with RDE against 4HAU/25 µL pandemic A (H1N1), pandemic A (H1N1) (H275Y), Type B and Type B (D197E) influenza viruses are shown. (i) Virus controls containing virus and CRBC and (ii) cell controls receiving CRBC only are shown. Extracts that mediate HI activity without RDE treatment were included in all plates as positive controls. The experiment was performed in triplicate. 109

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5.2.8. Effects of RDE treatment on the NAI activity of plant extracts

RDE-treated extracts were also studied in an NAI assay to determine whether NAI sialic acid (SA) mimics were present in the extracts; all four extracts were studied against the six influenza viruses. As shown in Figure 5.7, the NAI activity that was originally present in the extracts (Table 5.2.) was also affected by RDE treatment. The NA activity of the virus-only wells were set to 100% and the loss in the NA activity of the virus by treatment with plant extracts in the native form and those that were treated with RDE is shown in Figure 5.7. In its native form, Extract 8 was shown to significantly reduce the NA activity to less than 30% against all viruses except Type B where the extract reduced the NA activity to 43%, while RDE-treated Extract 8 showed decreased NAI potential. Similarly, RDE treatment decreased the NAI potential of extracts 41 and 42; NA activity of virus in wells treated with RDE treated extracts was close to 100%, like that of the virus only wells. Extract 43 also showed a similar trend in the loss of NAI activity upon RDE treatment. Only with Type B virus, the difference in the NAI activity of extracts 8, 41 and 42 in native form and those that were treated with RDE was less than 25%. RDE treated extracts were shown to contain relatively lowered NAI potential than native extracts; the differences among means for each concentration were statistically significant with p < 0.05.

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Neuraminidase activity Neuraminidase activity (%)

Native extract RDE treated extract

Figure 5.7 Effect of RDE treatment on the NAI activity of plant extracts The NA activity of virus only wells were set to 100% and the NA activity of virus in the presence of native extracts (25 µg/mL) and those that were treated with RDE were calculated relative to the virus control. The optimum virus dilution for the NAI assay was selected by titration of virus stock in an NA activity assay; 1:4 dilutions of either of the viruses were selected in the NA activity assay to perform the NAI assay. Representatives of two independent experiments are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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5.3. DISCUSSION

The antiviral activity of Extracts 8, 41, 42 and 43 against recent influenza viruses indicates that these extracts are a promising source for anti-influenza drugs. Since the extracts were shown to act against the NAI drug resistant Type A and Type B influenza viruses, they might serve as potential candidates to treat infections caused by drug resistant viruses.

Extract 8 was shown to be the most potent among the plant extracts as indicated by its

IC50 values less than 3.13 µg/mL against all six viruses, irrespective of the type of strain (wild type or mutant). Extract 8 was shown to demonstrate similar antiviral activity against seasonal A viruses between 25-100 µg/mL, however the antiviral potential against seasonal A (H1N1) (H275Y) was relatively less than that against seasonal A (H1N1). This may have resulted due to the presence of multiple active components at different concentrations and the decrease in potential may be attributed to the decrease in the synergism exhibited by the actives contained within the extract. Similarly, Extract 42 demonstrated antiviral activity at 3.13 µg/mL against pandemic A (H1N1) while being inactive against pandemic A (H1N1) (H275Y), hence synergy between the active components may be lost at lower concentration due to the decreased concentration of actives contained in the extract. Extract 8 was also shown to demonstrate similar activity against both the wild type and mutant strains of pandemic A and Type B viruses indicating that similar active components in the extract may affect the viruses as reflected by the alikeness in the trends of antiviral inhibition.

Being collected from different parts of the same plant, Extract 41 (whole plant) and Extract 42 (stems) showed similar activity in antiviral inhibition against both wild type and mutant seasonal A viruses and pandemic A (H1N1) virus. However, Extract 42 was shown to be relatively more potent than 41 in inhibiting pandemic A (H1N1) (H275Y) and Type B viruses as reflected by the low IC50 (Table 5.1). Hence, the stems of the plant may be a better source containing either higher amounts or more potent antiviral compound(s) against influenza virus than the whole plant from which Extract 41 was obtained. Extract 43 was shown to be more potent against the wild type A viruses as indicated by the lower IC50, relative to the mutant strains. On the contrary, the same extract was less potent against the Type B virus than Type B (D197E) mutant virus.

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Hence, the type of virus and the plant part, from which the extract is obtained, play a crucial role in determining the antiviral potential of extracts. Generally, the extracts demonstrated higher antiviral activity against the wild type viruses than against the mutant strains, indicating that amino acid changes in the virus alter the inhibitory potential of plant extracts and a higher concentration of extracts may be essential to inhibit the mutant viruses compared to wild type strains.

The antiviral potential of Oseltamivir was affected by viruses with H275Y and D197E mutations as indicated by an IC50 greater than 100 µg/mL. Although the D197E mutation in the Type B virus affects Zanamivir by a 10-fold change or less in the IC50 (McKimm-Breschkin, 2013), the antiviral potential of the drug showed only 5% loss in the viral inhibition against Type B (D197E) virus in comparison to the activity against the wild Type B virus (Figure 5.3.). However, the NAI potential of Zanamivir showed around 45 µg/mL increase in the IC50 against Type B (D197E) compared to the activity against the wild Type B virus (Table 5.2.). This result was within the IC50 range reported elsewhere (ISIRV, 2012) (Appendix IV). Hence, the effects of the D197E mutation on the activity of Zanamivir are more evident in the NAI assay rather than the in vitro micro inhibition assay.

Though all plant extracts were shown to affect the viral NA activity in the NAI assay, Extract 8 demonstrated higher NAI potential compared to other extracts indicating the presence of compound(s) with potent NAI activity. Since NAIs are well tolerated compared to ion channel blockers, the first approved anti-influenza drugs and their activity against types A and B viruses supports the research on compounds demonstrating NAI activity (Grienke et al., 2012). The variations in the IC50 values obtained for extracts 41 and 42 in the NAI assay further suggests the difference in the antiviral potential of extracts collected from different parts of the same plant. The diversity in the chemical make-up of whole plant (Extract 41) and stems (Extract 42) from which the extracts were obtained may contribute to the minor variations that were evident in the antiviral assays.

Since drugs with a unique mode of action, unlike the currently available NAIs, hold much promise in the fight against drug-resistant viruses, compounds demonstrating HI activity are particularly important agents for thorough research. Though Extracts 8, 41,

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42 and 43 were subjected to this study based on their HI activity in the previous experiments against Mem-Bel and PR8 viruses, only extracts 41, 42 and 43 demonstrated HI activities (Figure 5.4.), indicating the presence of HI component(s) in the extract . Extract 43 showed HI activity against Type B (D197E) virus, but lacked any HI effect against wild Type B virus. Similarly, the antiviral potential of Extract 43 was also shown to be higher against Type B (D197E) virus than the wild Type B virus (Table 5.1.); hence a higher concentration of active components may be essential to obtain similar antiviral activity against Type B as exhibited against Type B (D197E) virus. Provided that stable concerted actions of HA and NA are essential for viral replication (Grienke et al., 2012), a single amino acid change in NA may affect the antiviral potential or HI activity of the plant extract, as NA also plays a critical role in viral entry (Hsieh et al., 2012a, Matrosovich et al., 2004, Su et al., 2009b, Su et al., 2009a). The lack of HI activity against seasonal type A (H1N1) viruses may have resulted from the insufficiency of appropriate concentration of active component(s) in extracts 41, 42 and 43, since HI activity was previously detected against PR8 (H1N1) virus. The concentration of HI active component(s) in the plant extract is also crucial in exhibiting the visible effects in the HI assay. Hence, chemical fractionation and isolation of the active components responsible for affecting the viral HA may be essential to support the demonstrated HI activity of extracts.

The antiviral potential of the extracts against both the seasonal (H1N1) viruses (wild type and mutant) were suppressed by RDE treatment (Figure 5.5.). These results indicate that SA mimics are responsible for the demonstrated activity against seasonal (H1N1) viruses. Although RDE treatments suppressed the antiviral activities of extracts against wild type pandemic A (H1N1), extracts 8 and 43 demonstrated antiviral activity against pandemic A (H1N1) (H275Y) indicating the presence of antiviral non SA-like components; also, the components that affect the wild type and mutant viruses of pandemic A may be different. This result was also reflected in the NAI potential of RDE-treated Extract 8, where the extract retained the ability to reduce NA activity of pandemic A (H1N1) (H275Y) similar to the native extract (Figure 5.7.), although the native extract was shown to demonstrate relatively greater NAI potential. This indicates the presence of non SA-like NAI components in Extract 8. However, RDE-treated Extract 43 did not show NAI activity like that of native extract 43 against pandemic A

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(H1N1) (H275Y) (Figure 5.7), but demonstrated activity in the in vitro micro-inhibition assay (Figure 5.5.). Hence, Extract 43 may contain non SA-like component(s) that affect other viral proteins or replication stages, while SA mimics act against HA and NA.

The antiviral activity of RDE-treated extracts 8 and 43 against Type B virus (Figure 5.5.) was also reflected in the NAI assay (Figure 5.7.). The difference in the NAI potential between RDE-treated and native Extract 42 showed minor variations (<10%) relative to the difference in the NA activity as exhibited by other native and RDE- treated extracts (Figure 5.7). However, the extract failed to demonstrate antiviral activity in the in vitro micro-inhibition assay (Figure 5.5.), nor any HI activity upon RDE treatment (Figure 5.6.); therefore the antiviral activity derived from the SA-like components is essential for the efficient functioning of extract against the virus to obtain detectable levels (more than 50% virus inhibition) of antiviral activity although some non SA-like components may affect the viral NA. None of the extracts exhibited antiviral activity against Type B (D197E) virus upon RDE treatment (Figure 5.5.); this result was reflected in both HI (Figure 5.6.) and NAI (Figure 5.7.) assays where the antiviral potential of extracts was suppressed upon RDE treatment.

The suppression of HI activity by RDE treatment suggests that SA-like components in the extract may be responsible for the demonstrated HI activity (Figure 5.6.). SA mimics affecting the HA of influenza virus have been reported previously (Guo et al., 2002 , Matsubara et al., 2010). The loss in NAI potential of extracts upon treatment with RDE further supports the presence of NAI sialic acid-like components (Figure 5.7.). The two successful NAI inhibitors, Zanamivir and Oseltamivir, are structural mimics of SA that bind to the active site of the NA enzyme (Racaniello, 2013). As such, the results of the current studies are promising for the development of more sialic acid-like NAI inhibitors as it is apparent that the plant extracts investigated also contain NAI sialic acid mimics.

Collectively, the plant extracts investigated contain both SA mimics and non SA-like components which appear to work in synergy against viruses. Although components bearing structural similarity to SA are alone responsible for the demonstrated HI activity of all the plant extracts, NAI components may contain both SA-like and non

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SA-like components, both being responsible for the antiviral potential of the plant extracts. Hence, the results further support the need to study the chemical properties of the medicinal extracts. The activity of these extracts against the NA drug resistant strains following the HI pathway indicates that they could serve as promising candidates for the development of third generation anti-influenza drugs.

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CHAPTER 6 STUDIES TO DETERMINE THE CHEMICAL PROPERTIES OF MEDICINAL EXTRACT 8

6.1. INTRODUCTION

The search for drugs in nature and the utilization of plants to treat various diseases shows the bond between mankind and phytomedicines (Petrovska, 2012). Since any plant extract may contain several chemical components, the effect of different chemical procedures on the activity of plant extracts may help to determine the chemical properties of the components contained therein and serve as a basis for further chemical characterization studies to isolate the active ingredients. Once active compounds are isolated, modern techniques could be applied to chemically synthesize the compounds which aids in the large-scale manufacture of drugs.

Although there have recently been substantial developments in extraction and separation techniques, the isolation of natural products (NPs) from plants is still a challenging process and involves various studies to determine the chemical nature of the samples before reaching the final step of isolating the active compound. The conventional way for isolating NPs commences with the identification, collection and drying of the plant materials, followed by extraction with solvents of differing polarity. Several fractionation procedures often carried out by (semi-) preparative high- performance liquid chromatography (HPLC) or liquid-liquid chromatographic techniques may help to remove most of the unwanted matrix prior to the isolation of pure compounds and in studying the chemical properties of the extracts. Bioassay- guided fractionation approaches based on inhibitory activities in in vitro bioassays may reduce the time for hit compound discovery. The majority of studies involving NPs use a wide range of chromatography methods like vacuum liquid chromatography (VLC), medium-pressure liquid chromatography (MPLC) and HPLC (Bucar et al., 2013).

Most of the fractionation procedures still utilize simple extraction processes with organic solvents of different polarity or water, or a combination of both. Solid-phase extraction is increasingly recognized as a tool for the rapid fractionation of crude extracts prior to HPLC or GC analyses, and is also used for trapping pure compounds eluted after HPLC. GC-MS remains the method of choice for the analysis of biological products containing volatile constituents like essential oils (Bucar et al., 2013). 117

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Although the main purpose of the chemical fractionation of the extracts is to study the chemical properties of individual active components, it is important to keep in mind that medicinal plants are generally taken as a mixture to treat symptoms of illnesses, and the use of single constituent for therapeutic needs was rather limited in the past. The possibilities of synergistic action in herbal drug combinations used in traditional medicine must not be ignored. Therefore, chemical fractionation processes may cause a broad spread of the active component(s) across the multiple fractions thus affecting the bioactivity. The utilization of drug combinations in traditional medicine exerts the need to discover the grounds for such use, since combinations are therapeutically superior in comparison to single constituents (Wagner and Ulrich-Merzenich, 2009).

With regard to the extracts with demonstrated antiviral activities against influenza, synergism or potentiation, where the effect of the combination of active components is greater than the sum of the individual effects, may be present. The mechanisms of synergy effects generally fall into the following categories: synergistic multi-target effects where the individual constituents of a single extract affect several targets, thus cooperating in an agonistic, synergistic way; in phyto-pharmacology, associated compounds in an extract e.g. polyphenols or saponins that often do not possess specific pharmacological effects by themselves may aid to increase the solubility and/or resorption rate of the majority of constituents in the extract thereby increasing its bioactivity; the associated components may tend to reduce the toxicity coupled with the original active component(s), thus enhancing the safety aspect (Wagner and Ulrich- Merzenich, 2009, Rasoanaivo et al., 2011).

Among the four plant extracts that were chosen for this study, Extract 8 was selected for chemical fractionation, since the extract was shown to demonstrate relatively higher antiviral activity against influenza, as indicated by the lower IC50 obtained in the in vitro assays. Bioactivity-guided fractionation was employed in this study; Extract 8 was fractionated by Solid Phase Extraction (SPE) and each fraction was studied in an in vitro micro inhibition assay. Fractions that showed antiviral activity against Mem-Bel and PR8 strains were subjected to further screening against six influenza viruses; wild type and mutant strains of seasonal A (H1N1) and pandemic A (H1N1), and Type B. Mode of action studies to determine the effects of HI and NAI activities of the extract were initially performed against Mem-Bel and PR8 strains, and only those that were

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The effects of RDE treatment on the antiviral activity of the various fractions against the eight viruses were also investigated in this study. Irrespective of the activity, GC- MS analyses were performed to determine the chemical profile of the crude extract and its fractions. Since plaque reduction assays were not included in the early stages due to the high number of plant extracts, this reliable phenotypic assay was included in the study, as a confirmatory step to support the antiviral activity of the fractions.

Simultaneously, all the fractions of Extract 8 obtained through the chemical fractionation technique were studied by GC-MS to identify their main chemical constituents. This entire study serves as a starting point for the chemical analyses of plant extracts, identified as promising candidates and possessing novel activity against influenza, including NAI drug resistant strains.

6.2. RESULTS

6.2.1. Bioactivity-guided fractionation of Extract 8

All eight fractions of Extract 8 were tested for antiviral activity against Mem-Bel and

PR8 influenza viruses (100 TCID50). As shown in Figure 6.1, the 40% ACN and 100% ACN fractions exhibited antiviral activity similar to the crude extract (RAW) in inhibiting Mem-Bel and PR8 viruses. The activity of the 100% water fraction was close to 50% virus inhibition against PR8 virus, while less than 50% was shown against Mem-Bel. Hence, the two fractions, 40% ACN and 100% ACN, were chosen for further studies against both the wild type and mutant seasonal A (H1N1) and pandemic A (H1N1) viruses and Type B influenza viruses. For each fraction, a control blank, as detailed in Section 2.15, was included. As anticipated, the blanks showed no antiviral activity.

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Figure 6.1. Inhibitory activities of Extract 8 fractions

Cells at 80% confluence were treated with plant extract or fractions (100 µg/mL) and 100 TCID50 of either Mem-Bel (H3N1) or PR8 (H1N1) simultaneously. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and the percentage of viral inhibition calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that the data are significant with p < 0.05 (one way ANOVA).

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6.2.2. Inhibitory activities of Extract 8 and its fractions

The potential of the crude extract and its fractions to inhibit Mem-Bel and PR8 viruses was determined using a plaque neutralisation assay. As concentrations above 12.5 µg/mL resulted in complete inhibition of virus (no visible plaques), only concentrations ranging from 3.13-12.5 µg/mL were investigated. As shown in Table 6.1, the extract and its fractions were shown to inhibit Mem-Bel and PR8 viral plaques. At 6.25-12.5 µg/mL of either extract or fractions, plaque formation of both viruses was inhibited by more than 97%.

It is noteworthy that at a lower concentration of 3.13 µg/mL, both the 40% ACN and 100% ACN fractions showed a decrease in viral inhibition for Mem-Bel to 88.5% and 86.3%, respectively, while no major effect was observed for PR8. Similarly, the crude extract was shown to reduce viral plaques of Mem-Bel and PR8 by 88.5% and 93.7%, respectively, at 3.13 µg/mL. The reductions in the viral plaques upon treatment with crude extract and fractions are shown in Figure 6.2.

Table 6.1. Inhibitory activities of crude extract and fractions in reducing Mem-Bel and PR8 viral plaques

Concentration Extract 8 40% ACN 100% ACN (µg/mL) Mem-Bel Pr8 Mem-Bel Pr8 Mem-Bel Pr8 12.5 99.9±0.1 99.7±0.1 99.7±1.4 99.7±0.1 99.9±0.1 99.97±0.1

6.25 97.8±0.6 99.0±1.1 98.6±1.1 99.8±0.1 98.0±1.8 99.7±0.1

3.13 88.5±1.5 92.5±8.5 88.5±1.6 98.5±2.0 86.3±4.4 98.6±1.1

The percentage of virus neutralization was calculated relative to the mock-treated virus control of either Mem-Bel or PR8 influenza virus (5 × 105 pfu/mL). Representatives of two independent experiments performed in duplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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Concentration (µg/mL)

Figure 6.2. Inhibition of viral plaques by Extract 8 and its fractions

The number of viral plaques in the virus samples treated with extract or fraction and virus control samples are shown in the figure. The virus neutralization percentage was calculated relative to the mock-treated virus control of either Mem-Bel or PR8 influenza virus (5 × 105 pfu/mL). Representatives of two independent experiments performed in duplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

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6.2.3. Inhibitory effects of Extract 8 fractions against six influenza viruses

As shown in Figure 6.3, both fractions of Extract 8 demonstrated antiviral activities against the six viruses that were included in the study. The 40% ACN and 100% ACN fractions were relatively more potent against Seasonal A (H1N1) than the corresponding mutant strain. On the contrary, the fractions showed similar activity to that of the crude extract against mutant pandemic A (H1N1) (H275Y) and the wild type virus. However, the fractions were relatively more potent against Type B virus than the mutant strain, Type B (D197E). The differences between means for each concentration were statistically significant with p<0.05. Since the concentrations of active components in the fractions are only theoretical, the calculations of IC50 values have been omitted.

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Viral inhibitionViral (%)

Figure 6.3. Inhibitory activities of Extract 8 fractions against influenza viruses Cells at 80% confluence were treated with two-fold serial dilutions of plant extract (50-100 µg/mL) and

100 TCID50 of any of the six viruses simultaneously. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and the percentages of viral inhibition were calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA). 124

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6.2.4. Effects of plant extract against influenza virus-induced hemagglutination

The batch of Extract 8 used for fractionation did not inhibit virus-induced hemagglutination, although HI assays with other batches of the extract demonstrated HI activity against Mem-Bel and PR8 viruses. Despite the lack of HI activity, this batch of Extract 8 was potent in virus inhibition (as explained in Chapter 5). An HI assay was performed on the fractions 40% and 100% ACN against Mem-Bel and PR8 strains with an aim of determining whether any HI-active component(s) had been eluted from the crude extract through SPE. As shown in Figure 6.4, both the fractions did not inhibit HA of either Mem-Bel or PR8 viruses, hence the effects of fractions against the six other viruses were not included in the study.

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Concentration Extract Fraction Fraction Mem-Bel (H3N1) PR8 (H1N1) (µg/mL) control control control Extract 8 40% ACN 100% ACN Extract 8 40% ACN 100% ACN 8 40% ACN 100% ACN 100

12.5

6.25

3.13

Virus control for H3N1 Virus control for H1N1 Monoclonal antibody against Monoclonal antibody against Cell control H1N1 H3N1

Figure 6.4. Effects of fractions against influenza virus-induced hemagglutination

HI activities of crude Extract 8 and two fractions (40% ACN and 100% ACN) of Extract 8 (3.13 -100 µg/mL) against 4HAU/25 µL of virus are shown. The following controls were included on each plate; (i) fraction controls with fraction and CRBC only, (ii) virus controls containing virus and CRBC and (iii) cell controls containing only CRBC. Monoclonal antibody against the HA of either H3N1 or H1N1 strains were included as a positive control. The antibody titres for monoclonal antibody against H3N1 and H1N1 were 80 and 200, respectively; 1:8 dilutions of either of the two antibodies in PBS were employed in the assay. Data are shown from one of three independent experiments, each performed in triplicate.

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6.2.5. NA inhibitory effects of plant extract fractions

The 40% ACN and 100% ACN fractions of the plant extract were tested in NAI assays to determine whether the demonstrated antiviral activity resulted from their effect on the viral NA. As shown in Figure 6.5, the 40% ACN fraction was shown to reduce the NA activity of both Mem-Bel and PR8 viruses. As the concentration of fraction increased, the NA activity of the viruses decreased from 100% to 7% and 32% for Mem-Bel and PR8 strains, respectively. Similarly, NA activity of both the wild type and mutant seasonal A (H1N1) viruses decreased from 100% to 38% and 42%, respectively. The 40% ACN fraction was relatively more potent against the mutant pandemic A virus than the wild type, as no NA activity was shown at concentrations greater than 3.13 µg/mL, while the NA activity was only reduced to 26% for the wild type. A similar trend of NA inhibition was shown against Type B viruses, where the NA activity of the wild type was reduced to 60% whereas that of the mutant Type B (D197E) was reduced to 29%. The NAI effects of the 40% ACN fraction followed a similar trend to that of crude Extract 8, but the activity of the latter was in general more effective against viral NA than the 40% ACN fraction. The differences between means for each concentration were statistically significant with p<0.05.

On the contrary, the 100% ACN fraction showed NAI effect only against the PR8 virus, reducing the NA activity of the virus to 31% at the highest concentration of the fraction while no NAI activity was observed against the Mem-Bel virus (Figure 6.5). Since NAI activity of the fraction was demonstrated against one of the viruses in the initial analysis, the fraction was subjected to the next set of six influenza viruses, but did not show any NAI effect against any of these viruses. The NA activity of the viruses was close to 100% even after incubation with the 100% ACN fraction.

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(%) Neuraminidase activity virus Neuraminidase activity of

Concentration (µg/mL) Figure 6.5. NAI effects of the 40% ACN fraction and Extract 8

The effects of 40% ACN, 100% ACN and crude Extract 8 on viral NA were measured in an NAI assay. The NA activity of virus only wells was set to 100% and the NA activity of virus in the presence of plant samples (0.78 to 25 µg/mL) were calculated relative to the virus control. The optimum virus dilution for the NAI assay was selected by titration of the virus stock in an NA activity assay; 1:4 dilutions of the viruses were selected in the NA activity assay to perform the NAI assay except for Mem-Bel and PR8 viruses where 1:8 dilutions of the virus stock were chosen. Representatives of two independent experiments are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA). 128

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6.2.6. Effects of RDE treatment on the antiviral activity of Extract 8 and its fractions

Although no activity against the HA of influenza viruses was observed with the batch of Extract 8 under investigation, it was nevertheless treated with RDE to determine whether the observed antiviral activities could be the result of SA mimics through a different mode of action. As shown in Figure 6.6, RDE treatment affected the antiviral potential of the 40% ACN and 100% ACN fractions against Mem-Bel and PR8 strains. On the contrary, the antiviral activity of 100% fraction treated with RDE was similar to the native one against seasonal A (H1N1) while the antiviral activity was affected by RDE treatment for the 40% ACN fraction. Also, against the mutant seasonal A (H1N1) (H275Y) virus, RDE treatments of the fractions were shown to affect the antiviral activity that was originally demonstrated. Though RDE-treated fractions did not demonstrate inhibition against wild type pandemic A (H1N1), both the fractions showed inhibitory activities against mutant pandemic A (H1N1) (H275Y) similar to the native fractions. With respect to Type B virus, RDE treatment of the 40% ACN fraction was shown to decrease antiviral activity by 20% in comparison to the native fraction, while the RDE-treated 100% ACN fraction was not active against the viruses.

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Viral inhibitionViral (%)

Native RDE treated Figure 6.6. Effect of RDE treatment on the inhibitory activity of plant fractions An in vitro micro-inhibition assay was used to assess the ability of plant extracts to inhibit either of the following viruses; Mem-Bel, PR8, wild type and mutant strains of seasonal A (H1N1), pandemic A

(H1N1) and Type B influenza viruses (100TCID50). Extracts (100 µg/mL) were either treated with RDE as per the manufacturer’s instructions or left in their native form without RDE treatment. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p value < 0.05 (one way ANOVA). 130

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6.2.7. GC-MS analyses of crude extract and fractions

The crude extract and fractions were analysed using GC-MS in order to determine their chemical profile. Several components were present in both crude and fractions of Extract 8 (Appendix V). The first 50 peaks present in the fractions were identified using NIST and Wiley 8.0 MS libraries based on the area (%), however, only compounds that showed greater than 75% matching and 0.5% area are reported. Since the crude extract and fractions demonstrated the presence of several components with multiple peaks of minor area, 50 peaks were chosen for each sample in order to determine the chemical nature. The reason for choosing 50 peaks was to ensure that compounds which contributed only to a minor area were not neglected; hence compounds that contribute to the antiviral potential of the extract through synergy may be detected.

The criteria for selecting the reported compounds are discussed in Appendix VI. As shown in Figure 6.7., cyclitols (Quinic acid - 24.11% and mome inositol - 6.09%) were the major components of the crude extract, along with amino ketones (8.5%), esters (7.88%), phenolic compounds (5.94%), alcohols (5.72%) and several other components (Appendix V). However, 33.24% of the compounds present in the extract did not show similarity of greater than 75% to the compounds in the NIST and Wiley 8.0 MS library. The 100% water fraction showed a similar distribution of components; cyclitols (32.5%), amides (17%), esters (9%), and aldehydes (5%). On the contrary, amides (21%) formed the major constituents of the 10% ACN fraction along with cyclitols (18%), cyclic acids (3.47%) and alcohols (4%). The 25% ACN fraction showed the highest amount of amides (70%).

As shown in Figure 6.8, the 40% ACN fraction showed amides (49%), esters (10%), alkanes (8.35%) and cyclic acids (6.69%) along with several other components including phenolic compounds (3.63%), alcohols (3.6%), aldehydes (3.7%) and flavonoids (1.55%) ; 91.63% of the compounds were identitified by NIST and Wiley 8.0 MS library. The 55%, 70% and 85% ACN fractions were shown to contain amides at 28%, 16%, 40%, and cyclohexane groups at 6%, 7% and 4%, respectively (Appendix V). As shown in Figure 6.9, esters (methyl palmitate – 20.96%, methyl esters of fatty acids – 5.7%), amides (24.16%), fatty acids (17.73) were the predominant constituents of the 100% ACN fraction along with ketones (4.04%), carboxylic acids (1.84%) and aldehydes (1.44%). 131

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30.2 33.24

0.77 8.5 1.02 2.19 7.88 5.72 2.19 2.35 5.94

Cyclitol Amino ketone Ester Phenolic Alcohol Aldehyde Fatty acid Amide Alkaloid Flavanoid Unidentified compounds

Figure 6.7. Components of Extract 8

Extract 8 was analysed using a gas chromatograph coupled to a mass spectrometer as detector (GCMS- QP2010 Ultra, Shimadzu), equipped with an Rxi-5SIL-MS column. Helium gas was used as a carrier gas with a total GC run time of 20 min. The compounds were identified by comparison with the NIST and Wiley 8.0 MS library. Components present (area %) in Extract 8 are shown in the figure; 66.76% of compounds showed similarity of more than 75% match to the compounds in the library. The remaining 33.24% constituted several compounds that showed less than 75% match with the library of compounds.

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0.85 0.86 8.37 1.11 1.55 2.26 3.7 3.6 3.63 49.03

6.69

8.35

Amide Ester Alkane Cyclic acid Phenolic Alcohol Aldehyde Flavanoid Fatty acid Ketone Nitrile Ether Unidentified compounds

Figure 6.8. Components of 40% ACN fraction of Extract 8

Extract 8 was fractionated using SPE columns with decreasing polarity of acetonitrile (ACN) solvent. All the fractions obtained through bio-activity guided fractionation were analysed similar to Extract 8 as explained in Figure 6.7. Components present (area %) in 40% ACN fraction of Extract 8 are shown in the figure; 91.63% of compounds showed similarity of more than 75% to the compounds in the library. The remaining 8.37% constituted several compounds that showed less than 75% match with the library of compounds.

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24.13 26.66

1.44 1.84 4.04

24.16 17.73

Ester Amide Fatty acid Ketone Cyclic acid Aldehyde Unidentified compounds

Figure 6.9. Components of 100% ACN fraction of Extract 8

Extract 8 was fractionated using SPE columns with decreasing polarity of acetonitrile (ACN) solvent. All the fractions obtained through bio-activity guided fractionation were analysed similar to Extract 8 as explained in Figure 6.7. Components present (area %) in 100% ACN fraction of Extract 8 are shown in the figure; 75.87% of compounds showed similarity of more than 75% match to the compounds in the library. The remaining 24.13% constituted several compounds that showed less than 75% match with the library of compounds.

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6.3. DISCUSSION

Chemical fractionation of the extract was performed in order to study the chemical nature of the active components that may be responsible for the inhibition of the influenza virus. The reason for having chosen eight solvents of differing polarity was to isolate components spanning a large range of polarity. Although the 100% water fraction of Extract 8 inhibited 50% of the virus, only the 40% and 100% ACN fractions demonstrated similar antiviral inhibitions to the crude extract (Figure 6.1), thereby indicating that the active component(s) may be present in the 100% water fraction but in much smaller amounts than in the 40% and 100% ACN fractions. It is also possible that a completely different component is present in the fractions. Since 97% of the viruses were neutralised by all three samples in the plaque neutralisation assay (Table 6.1), the results further indicated the antiviral potential of the medicinal extract. Concentrations above 12.5 µg/mL of the extract showed 100% virus neutralisation with no visible plaques.

One of the most striking features of Figure 6.3., is the behaviour of crude Extract 8 which shows significant viral inhibition irrespective of the strain under investigation. This contrasts sharply with both the 40% ACN and 100% ACN fractions which showed variations in their actions against the viruses, with the exception of pandemic A (H1N1) (H275Y) and Type B viruses. This was particularly obvious with seasonal A (H1N1) with a significant drop in the antiviral activity when the concentration was reduced to 50 µg/mL. The most likely explanation for this observation is that fractionation has caused a decrease in concentration of the active ingredient. On the other hand, the effects of the 40% ACN fraction upon both wild type and mutant pandemic A viruses indicates the possibility of the same active ingredients affecting the virus differently according to the amino acid composition of viral NA. It is also interesting to note that with the pandemic A (H1N1) (H275Y), the fractions exhibited similar activities to the crude extract and all three seem to be independent of the concentration. This may be an indication that the change in the amino acid composition of the viral NA has resulted in an increase in the affinity of the active components for the virus.

Although the batch of Extract 8 used for chemical fractionation did not exhibit HI activity, the initial batches tested did demonstrate HI effect. Hence, HI assays were

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Chapter 6 performed upon the fractions to determine whether or not the fractionation process was able to isolate HI active component(s), from the crude extract. Preliminary studies showed the presence of HI activity in the 40% ACN fraction but the result was not consistent with further experiments. Hence, the fractionation process was not shown to isolate HI component(s) from the extract (Figure 6.4.). The 100% ACN fraction was also shown to not contain HI activity.

Similar to the TEM results of Extract 8 (section 4.2.8), the 40% and 100% ACN fractions did not affect the morphology of the virus, supporting the demonstrated specific inhibitory effects on the virus rather than non-specific inhibition. NAI activity against all the viruses included in the study was evidenced by the 40% ACN fraction (Figure 6.5.); hence NAI active components that affect viral NA were eluted in the 40% ACN fraction of Extract 8. However, the 100% ACN fraction was active only against PR8 virus. This observation suggests that the antiviral activity demonstrated by the 100% ACN fraction against other viruses studied may be due to its effects on other proteins [polymerase complex proteins, polymerase acidic subunits, nucleoproteins, ribonucleoprotein particles (RNPs), matrix proteins, nuclear export proteins and non- structural proteins (Du et al., 2012a )] or stages of viral replication.

RDE treatments of both fractions were shown to suppress the antiviral activities originally demonstrated against Mem-Bel and PR8 viruses (Figure 6.6), suggesting that sialic acid mimics may be the active ingredients. However, RDE treatments did not affect the antiviral activity of the 100% ACN fraction against seasonal A (H1N1) while the inhibitory effect of the 40% ACN fraction was completely suppressed. Hence, non- sialic acid like component(s) may be present in 100% ACN that are inhibitory against seasonal A (H1N1) virus. Another important observation is related to the chemical nature of the active components present in the 100% ACN fraction which are most likely to be highly non-polar compounds.

With regard to pandemic A (H1N1), it is obvious from Figure 6.6 that RDE treatments of the crude extract and its fractions have affected the antiviral potential against the wild type while the activity against the mutant strain was similar to that of native fractions. It can thus be suggested that the demonstrated antiviral activity against mutant pandemic A (H1N1) (H275Y) may predominantly result from non-sialic acid-like components

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Chapter 6 while on the other hand, the silaic acid mimics may be responsible for the activity against the wild type. Hence, the fractions of Extract 8 may contain both sialic acid mimics as well as non-sialic acid like component(s) that exert influenza inhibitory effect against different strains of the virus.

GC-MS studies of crude Extract 8 and its subsequent fractions allowed the determination of its chemical profile. Several compounds such as cyclohexane carboxylic acids, amides, fatty acids, esters and polyols were present in most of the fractions (Appendix V), indicating that the fractionation process may have diluted the components between different fractions which might affect the synergism contained within the extract or diluted the anti-influenza activity, as shown in Figure 6.5 where NA activity of the crude extract was greater than that of the fractions.

Quinic acid, a cyclitol, was shown to be the dominant compound in the crude extract, the 100% water fraction and present in significant amounts in several fractions, including the 40% ACN which was shown to inhibit influenza virus. Quinic acid is known for its anti-influenza activity; it was once used to prepare the NAI drug, Oseltamivir, but was dropped in favour of shikimic acid (Federspiel et al., 1999). Thus, cyclitols in the crude extract may be responsible for the inhibition of influenza A and B viruses. Amides have been recently studied for their anti-influenza activity affecting influenza viral endonuclease (Carcelli et al., 2013, Hao et al., 2012). Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide), an anti-influenza drug that is currently under trials, also contains an amide group that affects the influenza viral RNA polymerase (Baranovich et al., 2013, Furuta et al., 2009). Other studies have reported that fatty acids also demonstrate anti-influenza activity. Since they are easily metabolized in the human body, fatty acid derivatives which are not easily metabolized may be good candidates in the development of anti-influenza agents. Methyl palmitate, that constitutes 20.96% of the 100% ACN fraction, was shown to have specific NAI activity (Kim et al., 2003). Chemical constituents like those found in crude Extract 8 (furanones, alkanes and esters) have been reported in other plants with anti-influenza activity (Zai-Chang et al., 2005b).

In order to determine whether inhibitory activity is decreased by the distribution of active component(s) in more than one fraction, the 40% ACN fraction (that originally

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Chapter 6 demonstrated anti-influenza activity) was mixed with an equal volume of the 55% ACN fraction and the 100% ACN fraction was mixed with the 85% ACN fraction. The mixtures tested for inhibitory effect against Mem-Bel and PR8 viruses (Appendix VII). The mixing of the 40% and 55% ACN fractions reduced anti-influenza activity against Mem-Bel virus to below 50% and against PR8 by 20%. On the contrary, mixing of the 85% ACN with 100% ACN fractions removed anti-influenza activity that was originally demonstrated by the 100% ACN fraction, thus exhibiting less than 50% virus inhibition. Though these results indicate that the 40% ACN and 100% ACN fractions may contain active anti-influenza compound(s), concurrent studies involving other fractionation processes are essential for efficient active compound isolation, since the NA activity of fractions are relatively less potent than that exhibited by crude extract (Figure 6.5.)

Although compounds with groups associated with known anti-influenza activity were distributed throughout the fractions, anti-influenza activity was demonstrated only in the 40% and 100% ACN fractions. This may have been due to the presence of compounds at the appropriate concentrations to show activity in the in vitro assays. Overall, the results support the presence of antiviral activity associated with chemical groups such as cyclic polyols, fatty acids and amides. This chapter of experimental findings serve as a starting point to chemically explore the medicinal extract using the wide range of analytical instruments currently available, coupled with bioactivity assays.

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CHAPTER 7 SUMMARY AND CONCLUSIONS

Flu is an acute respiratory illness caused by types A and B influenza viruses, the former being known to cause frequent epidemics resulting in significant human morbidity and mortality; influenza pandemics are also caused by Type A viruses. Moreover, currently circulating H5N1 viruses that cause avian flu are extremely virulent in humans, but have not yet acquired the ability to undergo efficient human- to-human transmission (Lee et al., 2012). However, in rare circumstances, limited person-to-person spread is thought to have occurred, with the outbreak of human infections caused by a new avian influenza virus, H7N9, in China during April 2013, in which 44 people died as reported by the WHO (CDC, 2013). Hence, novel approaches are essential for the prevention and cure of influenza infections. Utilization of traditional knowledge of medicinal plants might serve as a potential base that could lead to the identification and isolation of novel active compounds from plant sources, with proven activities against the viruses. New drugs targeting different viral structural components and stages of virus replication may serve to overcome the challenges of drug resistant viruses.

7.1. CYTOTOXICITY AND ANTIVIRAL SCREENING

Drugs with an ethnomedical background are generally considered safe since they have been used for several generations in the treatment of infections. Fifty medicinal plant extracts that were traditionally used to treat various ailments, including symptoms of influenza such as sore throat, cough, cold and fever, were provided and studied in a number of bioactivity assays. Cytotoxicity studies were performed prior to antiviral screening assays and out of 50 plants, 28 demonstrated similar CC50 to that of the commercial NAIs, indicating the non-toxic nature of the compounds. In some cases, cytoprotective activities were also noted and this property has previously been reported in plants (Tayal et al., 2012). In the initial in vitro micro-inhibition assays, eleven extracts were shown to demonstrate antiviral activity against Mem-Bel (H3N1) and PR8 (H1N1) viruses. The consistency of the bioassay was demonstrated by the observation that positive hits were acquired from extracts that were obtained from the whole plant or different parts of the same plant, i.e. extracts 13 and 30 from Trigonopleura malayana leaves, and extracts 41 and 42, from Calophyllum lanigerum whole plant and stems, respectively. Although the active extracts were potent in inhibiting the virus es within

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Chapter 7 certain ranges of concentrations unique for each extract, a concentration-independent viral inhibition, similar to the commercial NAI drugs, may be demonstrated by the active compound.

With respect to the IC50, extracts that demonstrated relatively low values may contain higher amounts of the active principle or be intrinsically more active. Any single plant extract may contain a wide variety of chemical components (Srivastava et al., 2012), hence, the isolation of individual active components is essential. As synergism is a key factor with respect to traditional medicines (Wagner and Ulrich-Merzenich, 2009), chemical separation and characterization was commenced at a later stage of the project, only after exhaustive biological assessments had been completed. Since a better understanding of the role played by various viral proteins has been achieved (Du et al., 2012a), elucidating the possible modes of virus inhibition demonstrated by the medicinal extracts was the logical next stage of this project.

7.2. MODE OF ACTION STUDIES

Several assays were included in this study to ascertain the mode(s) of antiviral action demonstrated by the plant extracts. All eleven extracts were shown to affect the NA activity of Mem-Bel and PR8 viruses; three extracts (8 from Mussaenda elmeri, 38 from

Tetracera macrophylla and 43 from Albizia corniculata) demonstrated the lowest IC50 range, suggesting the presence of NAI components at higher concentrations or having a larger response at lower concentrations. The antiviral efficacy of extracts studied in time of addition assays further indicated their potential in virus inhibition. Four extracts (8, 41/42 and 43) were shown to inhibit virus-induced hemagglutination against both viruses within specific ranges of concentrations. Since drugs with a different mode of action to those currently available on the market, such as NAIs and adamantanes, may help to tackle the threats posed by drug resistant viruses, the proven HI activities of the extracts are a promising result for further research.

Since four extracts demonstrated activity against both surface glycoproteins of the influenza viruses, there may be more than one active component in the extract. The inhibitory effects demonstrated against viral binding and penetration further supported the HI mode of action of the extracts, as HA is essential for virus adsorption and penetration (Wilks et al., 2012). On the contrary, non-HI extracts (13/30 from

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Trigonopleura malayana , 14 from Santiria apiculata, 31 from Trivalvaria macrophylla, 37 from Baccaurea angulata and 38 from Tetracera macrophylla) that demonstrated activity against NA only were also shown to inhibit virus binding and penetration; NAI activity of these extracts may be responsible for the demonstrated inhibitory activity, since NA is also important for viral entry (Hsieh et al., 2012a, Matrosovich et al., 2004).

The loss of HI activity upon RDE treatment, and the reduction in antiviral potential in in vitro micro-inhibition assay collectively indicate that the HI inhibitory component(s) may contain sialic acid-like chemical structure(s). HI activities of the extracts were not affected by trypsin treatment, indicating that the molecule of interest is unlikely to be proteinaceous. Although extracts with HI activities serves as a potential source for the development of drugs with novel mode of action, one batch of Extract 8 did not show any HI activity. This phenomenon needs to be further examined. The effects of Extract 8, a representative of the medicinal extracts, on the morphology of Mem-Bel and PR8 viruses were studied using TEM; these results are in agreement with specific activities of the extracts on the viral proteins rather than non-specific viral inhibition. The following stage of this project involved antiviral studies of HI extracts against six other influenza viruses, including NAI drug resistant strains.

7.3. ANTIVIRAL ACTIVITIES OF SELECTED MEDICINAL PLANT EXTRACTS

AGAINST TYPES A AND B VIRUSES, INCLUDING NAI RESISTANT STRAINS

The HI extracts were studied for inhibitory activity against six influenza viruses, seasonal A (H1N1), seasonal A (H1N1) (H275Y), pandemic A (H1N1) and pandemic A (H1N1) (H275Y), Type B and Type B (D197E) viruses. The viruses with the H275Y mutation are resistant to Oseltamivir (Samson et al., 2013) and the D197E mutation in Type B virus confers cross resistance to all NA inhibitors, whether commercially available or under trials (McKimm-Breschkin, 2013). Even though all four HI extracts were shown to demonstrate antiviral activity against types A and B viruses, Extract 8 was shown to be the most potent with a relatively low IC50 compared to other extracts. Generally, extracts were more potent against the wild type viruses than the mutant strains (H275Y or D197E), indicating that changes in the amino acid sequence of the virus affect the inhibitory potential of the extracts and a higher concentration of the

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plant extracts may be necessary to inhibit the mutant viruses. Furthermore, the IC50 of extracts against the six viruses were higher than those obtained against Mem-Bel and PR8 viruses, hence the antiviral potential of the extracts is greater against Mem-Bel and PR8 viruses than the other six viruses. Variations in the antiviral potential of extracts 41 and 42, which were collected from different parts of Calophyllum lanigerum, were evident in the antiviral assays. The stem extract (42) was shown to exhibit greater antiviral activities than the whole plant extract (41).

The modes of action studies showed that the four extracts affected viral NA of all the six viruses, and Extract 8 showed a potent NAI effect compared to other extracts. HI activity was demonstrated by extracts 41and 42 against both the wild type and mutant pandemic A, and Type B viruses, while Extract 43 showed similar activity to 41/42 but no HI activity was evidenced against wild type B virus. Since stable coactions of HA and NA are essential for viral replication (Grienke et al., 2012), single amino acid changes in NA may affect the antiviral potential or HI activity of the extract, as NA is also important for viral entry (Hsieh et al., 2012a, Matrosovich et al., 2004, Su et al., 2009b, Su et al., 2009a). As expected, the batch of Extract 8 that did not show any HI effect on the Mem-Bel and PR8 viruses did not display HI effects towards the six virus strains.

HI activity that was originally demonstrated by the extracts was suppressed upon RDE treatment, thereby suggesting that SA mimics may be responsible for the evidenced HI antiviral effects. Since, the antiviral potential of extracts was only reduced and not completely suppressed by RDE treatment (as determined from the in vitro micro- inhibition assays) the extracts may contain anti-influenza component(s) that are non- sialic acid-like in structure. RDE treatment also reduced the original NA inhibitory activity demonstrated by the extracts, and, in some cases, complete loss of the effect was evident. Overall, the main results of this study suggested the probability of synergic effects between sialic acid-like and non sialic acid-like components contained in the plant extracts. Hence, multiple antiviral components of the plant extracts may act against different viral proteins, apart from HA and NA, or interfere with the process of viral replication at different stages.

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7.4. CHEMICAL FRACTIONATION AND ANTIVIRAL ASSESSMENT OF

EXTRACT 8

Extract 8 was selected for further fractionation to study the chemical nature of the extract. Bioactivity-guided fractionations of Extract 8 with acetonitrile (ACN) of varying polarities, by mixing with water, resulted in two active fractions, 40% ACN and 100% ACN, which demonstrated antiviral activity against eight influenza viruses. In a plaque neutralization assay, both fractions and crude extract were shown to inhibit more than 97% of Mem-Bel and PR8 plaques, supporting the results obtained in the in vitro micro-inhibition assays. The batch of Extract 8 used for fractionation did not display HI activity, although other batches of Extract 8 did. NAI activity of the 40% ACN fraction was shown to affect NA of all the viruses included in the study. On the contrary, the 100% ACN fraction demonstrated NAI activity only against PR8 virus, while being inactive against the NAs of other viruses; also, no HI activity was observed against any of the viruses. Hence, there is a possibility that the 100% ACN fraction contains antiviral component(s) that target other proteins or stages of viral replication. Similar to the conclusions from experiments with the crude extract, RDE treatment of the fractions indicated that sialic acid-like and non-sialic acid-like components may act in synergy to achieve optimal antiviral potential, as indicated by experiments conducted with RDE. TEM studies on the extract fractions indicated that the virus structures were intact, supporting the specific activity of the fractions against viral proteins, as indicated by the bioassays, rather than non-specific destruction of virus particles.

GC-MS analyses of the crude extract and its eight fractions were performed to determine their chemical nature. The predominant compound present in the crude extract w as quinic acid, a cyclic polyol known to inhibit influenza virus. The other components present in the fractions, fatty acids, amides and esters, are also known to inhibit influenza. Amides have been recently studied for their anti-influenza activity and were shown to affect the viral endonuclease (Carcelli et al., 2013, Hao et al., 2012). Methyl palmitate, which was predominant in the 100% ACN fraction was shown to display NAI activity in other studies (Kim et al., 2003). Hence, the presence of more than one active compound in Extract 8 is supported by the outcomes of this study. However, the presence of components across more than one fraction indicates that further fractionations, following a variety of protocols, would increase the likelihood of

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Chapter 7 isolating active component(s) without the demonstrated activity being distributed across several fractions.

This study serves as a starting point for the isolation of influenza inhibitory active components from the medicinal plants of Sarawak. Overall, the research work supports the exploration of traditional medicinal plants for antiviral activity against influenza viruses. The novel HI activity of plant extracts against the infectious viruses, including NAI drug resistant strains, may serve as potential sources of anti-influenza compounds that could lead to the development of third generation anti-influenza drugs. Thus, treatment options for influenza infections could be strengthened by conducting various scientific studies on medicinal plants and develop novel drugs with different modes of action against the viruses.

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Appendix I

APPENDICES

APPENDIX I

AI.1. Tropical rainforests of Sarawak

Sarawak is the largest Malaysian state located along the north-western region of the island of Borneo comprising land area of 124, 000 square kilometres. The tropical rainforests of Sarawak are considered to be one among the oldest and richest in the world. Climatic factors and variations in the soil have together contributed an extensive range of forest types. The presence of about 9,500 species of plants has been recorded in the state of Sarawak. Sarawak has a population of 2.2 million made up of 30 ethnic groups. For many generations, the indigenous communities have relied on the forest for their needs. A total of 608 species of medicinal plants used by 14 ethnic communities have been recorded. The medicinal plants differ extensively in habits and forms – from epiphytes, climbers and herbs, to large trees and shrubs. The plants are found in all types of forests, mainly in the primary forests while secondary forests that are made up of different ages and species compositions also contribute significantly; only a small number of plants are cultivated to meet medicinal purposes (Chai, 2006).

AI.2. Mussaenda elmeri (Rubiaceae)

Mussaenda elmeri belongs to the family Rubiaceae and is mostly found in frequently flooded areas in the Climber riparian forest. The plant bores obovate or ovate leaves up to 15 cm long and 7 cm wide that may be hairy downwards. Whitish enlarged sepals that are leaf-like and orange flowers are characteristic features of the plant. Several ethnic groups of Sarawak have been using this plant as a remedial measure for treating ailments. A decoction of the leaves and root is taken to alleviate toothache. The leaves of the plant are warmed over a fire and placed on the forehead to mitigate headaches. Regular consumption of a tea made out of boiling root with water is believed to regulate the blood glucose level in diabetic patients (Chai, 2006). Several plants belonging to the same genus have been shown to possess antimicrobial properties. Chemical analyses of some plants belonging to the same genus have shown the presence of saponins, flavonoids and phenolic compounds (Gopalakrishnan and Vadivel, 2011, Xu et al., 1992 , Zhao et al., 1996a, Zhao et al., 1997, Zhao et al., 1994, Zhao et al., 1996b, Zhao

161

Appendix I et al., 1996c). Antioxidant activities of plants belonging to the genus Mussaenda have also been reported (Li et al., 2011).

AI.3. Calophyllum lanigerum (Clusiaceae)

This species was found in peat soil and heath land near Lundu and Sibu in Sarawak. Canolide-A, a coumarin extracted from this plant has demonstrated activity against the replication of HIV virus and is also known to reduce tuberculosis in HIV patients (Kaur and Kharb, 2011, Salleh et al., 2002). It is believed that Calophyllum lanigerum helps to relieve headache and treats eye related disorders (Yaacob et al., 2009 ) (Musa Yaacob, 2009).

AI.4. Albizia corniculata (Fabaceae)

This plant belongs to the family Fabaceae, also referred to as the family of legumes. Members of this family vary widely in vegetative forms as trees, shrubs, climbers or herbs. Many climbers and herbs are thorny. Trifoliate or compound pinnate leaves with showy flowers are characteristic features of the family Fabaceae (Chai, 2006) . Species belonging to the genus Albizia have been reported to possess activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, Vibrio cholerae, and Candida albicans (Ali et al., 2001, Maji et al., 2010) but activity against influenza viruses have not been reported. Albizia corniculata was traditionally used to treat sore throat. The plant was also used as poultice on ulcers and believed to relieve gum inflammation. Leprosy, skin infection and ring worm treatment are other medicinal uses of this plant.

AI.5. Trigonopleura malayana (Euphorbiaceae)

This plant belongs to the family Euphorbiaceae. Trigonopleura malayana is a medium- sized tree with spirally arranged leaves. Young parts of the plant are covered in soft short hairs. A tea made out of its leaves is a remedy for cough. Skin diseases like ringworm and diseases of the scalp are treated by rubbing a paste of the leaves onto the infected area (Chai, 2006) . The resin obtained from Trigonopleura malayana is locally known as ‘Gambir Sarawak’ and has been used traditionally to relieve pain associated with toothache, muscle ache, insect bites and minor injuries. Aqueous extract of the resin has been shown to demonstrate antinociceptive and anti-inflammatory activities

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Appendix I

(Sulaiman et al., 2008). Leaves of the plant were obtained from two sources of the same species in the same location but collected at different times and tested for anti-influenza activity.

AI.6. Santiria apiculata (Burseraceae)

The plant belongs to the family Burseraceae which comprises trees in a wide range of forest types. Fresh parts produce a colourless or whitish resinous exudate. The presence of greenish flowers and leaves that are compound pinnate with a terminal leaflet, spiral or alternate are features of this family. Santiria apiculata is medium-sized and can grow up to 25 m tall. There are several varieties that differ in shapes and sizes of leaflets.

Traditionally, Santiria apiculata was used to alleviate cold feet syndromes in mothers after childbirth, reduce body temperature, and ease symptoms of flu and headaches (Chai, 2006) .

AI.7. Anisophyllea disticha (Anisophylleaceae)

Anisophyllea disticha is a tropical evergreen shrub that has a slender main stem which grows up to 7.5 m tall. A distinctive feature is the presence of two kinds of leaves that are arranged in four rows in almost the same plane as a branch. Yellowish or pinkish- white flowers are also present (Research, 2013). Anisophyllea disticha was traditionally used to treat dysentery and diarrhoea. The decoction of the leaves and stem is used to bathe jaundice patients as it is believed to cure the disease. Also, the decoction is used as a tonic, treats stomach aches and insect bites (Salleh et al., 2002) . Extracts from the trees stems were tested for anti-influenza effect.

AI.8. Trivalvaria macrophylla (Annonaceae)

Trivalvaria macrophylla belongs to the family Annonaceae which is known for a range of non-timber products such as kernels, edible fruits and medicines. Members of Annonaceae have been used traditionally as remedies against malaria, yellow fever, diarrhoea, dysentery, cough and poisons. Internal application through the oral route by inhaling vapours from decoctions, concoctions or infusions is the general mode of treatment for the above mentioned ailments whereas external administration involving steam baths and direct application of crushed plant part mixed with water or oil on the body are modes of application that are used to treat swellings, fractures, abscesses, skin

163

Appendix I diseases and wounds (Focho et al., 2010). Trivalvaria macrophylla was used by ethnic groups in Sarawak to relieve flu, headache and stomach aches. The plant extract obtained from the roots of Trivalvaria macrophylla were tested for activity against influenza viruses in this study.

AI.9. Baccaurea angulata (Euphorbiaceae)

Baccaurea angulata belongs to the family Euphorbiaceae. Members belonging to Euphorbiaceae include trees, shrubs and herbs. Several species of Baccaurea genus are known to treat eye diseases, stomach ache, pain, skin diseases, drunkenness, menstrual disease and complications of women after child-birth. Baccaurea angulata was traditionally used to treat conjunctivitis (Chai, 2006, Salleh et al., 2002). Fruits of Baccaurea angulata have been reported to demonstrate antioxidant activity due to the presence of phenolic and flavanoid content (Jauhari et al., 2013). Plant extracts obtained from the stems of Baccaurea angulata were tested for anti-influenza activity.

AI.10. Tetracera macrophylla (Dilleniaceae)

Tetracera macrophylla belongs to the family, Dilleniaceae. The plant is a woody climber with hairy, broadly obovate leaves with toothed margins and several lateral veins. Consumption of water from the stem is a remedial measure for cough and diarrhoea. A decoction of the root is taken as a treatment against painful urination and blood in the urine (Chai, 2006). Hepatoprotective, antioxidant nature and anti- tuberculosis potential of plants belonging to Tetracera have been published (Kukongviriyapan et al., 2003, Mbatchi et al., 2006).

164

Appendix II

APPENDIX II

Inhibitory effects of anti-influenza extracts on the penetration of H3N1 and H1N1 strains at 60 min

Antiviral activity (%) of extract (concentration in µg/mL) against the penetration of Extract H3N1 strain H1N1 strain 50 12.5 50 12.5 8 94.0±1.8 52.6±4.9 91.0±0.6 78.5±2.9 13/30 81.6±16.2 47.0±5.8 73.2±3.3 53.9±5.9 14 56.7±1.9 - 60.3±0.3 50.1±1.2 29 - - - - 31 72.4±3.7 51.1±1.6 90.4±0.5 77.2±4.1 37 - - 58.9±1.2 - 38 73.9±1.6 53.9±5.2 91.6±0.3 80.2±2.1 41 68.5±7.9 - 84.5±0.9 65.2±3.7 42 70.1±4.7 - 77.1±1.0 55.3±4.1 43 82.2±0.6 - 85.7±0.3 64.9±3.7 Zanamivir 68.1±2.8 - - - Oseltamivir - - - -

The activities of extracts against the penetration of influenza viruses (200 TCID50) at 50 and 12.5µg/mL are shown along with standard errors.

165

Appendix III

APPENDIX III

(a) (b)

HI against H3N1 Trypsin Extract Treatment on HI extracts

Virus control Cell

Control Effect of trypsin treatment on the antiviral activity of plant extracts

This experiment is essentially the same as explained in Figure 4.9, except that H1N1 virus was employed in the study. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

166

Appendix IV

APPENDIX IV

NAI activity of neuraminidase inhibitors

Viruses IC 50 range (nM)* Oseltamivir Zanamivir

Seasonal A (H1N1) 0.4-10 0.3-1

Seasonal A (H1N1) (H275Y) 257-3455 0.3-2

Pandemic A (H1N1) 0.2-10 0.2-1

Pandemic A (H1N1) 132-2179 0.2-2 (H275Y)

Type B 8-128 0.5-12

Type B (D197E) 79-966 3-290

The NAI activity of the controls, Zanamivir and Oseltamivir were measured at concentration ranging between 0.01 to 10,000 nM as recommended by the manufacturer. The optimum virus dilution for the NAI assay was selected by titration of virus stock in an NA activity assay; 1:4 dilutions of the virus strains was selected in the NA activity assay to perform NAI assay.

* The IC50 range was based on results determined by seven different laboratories using a range of fluorescence-based protocols (ISIRV, 2012).

167

Appendix V

APPENDIX V

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match Crude extract

5.230 Glycerin C3H8O3 92 96 5.06 6.350 Mono-methyl hydrogen succinate C5H8O4 132 95 1.34 6.780 Glycerol C3H8O3 92 79 0.66 7.330 5-(Hydroxymethyl)furfural C6H6O3 126 87 1.6 8.390 5-oxo-pyrrolidine-2-carboxylic acid methylester C6H9NO3 143 94 1.6 8.725 Lactone G C5H8O4 132 83 1.83 8.920 2-amino-9-(3,4-dihydroxy-5-hydroxymethyl- C10H13N5O5 283 82 8.49 tetrahydro-furan-2-yl)-3,9-dihydro-purin-6-one 9.060 2-hydroxy-4-methylbenzaldehyde C8H8O2 136 75 0.75 9.240 Veratroylzygadenine C3H51NO10 657 78 1.02 9.780 Diethyl phthalate C12H14O4 222 93 1.42 10.070 (1R,3R,4R,5R)-(-)-Quinic acid C7H12O6 192 82 24.11 10.225 Mome inositol (cyclohexane polyol) C7H14O6 194 90 6.09 10.540 n-Decyl methylphosphonofluoridate C11H24FO2P 238 77 0.83 10.935 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-6- C11H16O3 196 84 0.77 hydroxy-4,4,7a-trimethyl-, (6S,7aR)- 11.610 Methyl palmitate C17H34O2 270 86 0.86 11.795 Palmitic acid C16H32O2 256 87 1.53 12.86 Octadecanoic acid C18H36O2 284 82 0.66 12.995 Hexadecanamide C16H33NO 255 82 2.19 13.120 Phenol, 4, 4’-(1-methylethylidene)bis- C15H16O2 228 91 5.94

168

Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 100% Water

6.150 1,3,5-Triazine-2,4,6-triamine C3H6N6 126 86 2.69 6.410 Butanedioic acid, monomethyl ester C5H8O4 132 96 5.0 6.710 2,3-dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4- C6H8O4 144 90 2.23 one 7.285 2-(Isopropylthio)pentane C8H18S 146 81 1.7 7.340 2-Furancarboxaldehyde, 5-(hydroxymethyl)- C6H6O3 126 95 4.91 7.550 1,2,3-propanetriol, 1-acetate C5H10O4 134 85 3.91 8.165 Ethyl-5,5-diethoxy valerate C11H22O4 218 77 0.58 8.235 Propyl isovalerate C8H16O2 144 77 1.55 8.445 DL-Proline,5-oxo-,methyl ester C6H9NO3 143 95 4.36 8.840 Lactone G C5H8O4 132 77 0.98 9.060 2-hydroxy-4-methylbenzaldehyde C8H8O2 136 80 0.77 10.200 (1R,3R,4R,5R)-(-)-Quinic acid C7H12O6 192 80 30 10.975 Mome inositol C7H14O6 194 88 2.58 11.805 1,2-benzenedicarboxylic acid, butyl 8- C22H34O4 362 81 0.70 methylnonyl ester 13.000 Hexadecanamide C16H33NO 255 95 6.15 14.340 Cis-octadecenamide C18H35NO 281 94 5.54 14.555 N-tetradecanoic acid amide C14H29NO 227 91 6.72

169

Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 10% ACN

4.475 α-Hydroxyisobutyric acid C4H8O3 104 83 0.73 6.360 Butanedioic acid, monomethyl ester C5H8O4 132 95 1.45 6.395 Maltol C6H6O3 126 91 0.61 6.930 Benzoic acid C7H6O2 122 96 3.47 7.040 Pentanedioic acid, monomethyl ester C6H10O4 146 88 0.82 7.540 Benzeneacetic acid C8H8O2 136 96 1.10 8.080 2-hydroxybenzoic acid (salicylic acid) C7H603 138 93 1.4 8.315 1-hydroxylinalool C10H18O2 170 83 0.69 8.400 DL-Proline, 5-oxo-, methyl ester C6H9NO3 143 95 1.1 8.605 4-hydroxy-3-methoxybenzaldehyde (vanillin) C8H8O3 152 96 1.86 9.245 Angeloylzygadenine C32H49NO8 575 79 6.22 9.275 2,6-di-tert-butyl-4-methylphenol C15H240 220 89 0.56 9.455 Homovanillyl alcohol C9H12O3 168 90 3.95 9.640 Vanillic acid C8H8O4 168 91 1.32

170

Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 10% ACN

10.060 D-(-)-Quinic acid C7H12O6 192 86 10.91 10.185 Mome inositol C7H14O6 194 75 4.22 10.445 Acetylphenylalanine methyl ester C12H15NO3 221 87 0.91 10.940 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-6- C11H16O3 196 85 6.81 hydroxy-4,4,7a-trimethyl-, (6S,7aR)- 10.995 Pluchidiol C13H20O2 208 84 3.48 11.080 Ethylene glycol monohexadecyl ether C18H38O2 286 80 0.7 11.130 Ethyl 4-hydroxy-3-methoxybenzoate C10H12O4 196 83 1.39 11.805 Dibutyl phthalate C16H22O4 278 86 0.95 11.930 7-hydroxy-5-methoxycoumarin C10H8O4 192 90 1.12 12.440 9-Tetradecenal, (Z)- C14H26O 210 80 0.6 12.995 Hexadecanamide C16H33NO 255 95 17.86 14.335 Cis-9-octadecenamide C18H35NO 281 94 3.33

171

Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 25% ACN

2.530 Methyl-D3 1-Dideuterio-2-propenyl ether C4H3D5O 77 83 0.58 4.525 Methoxy, phenyl- ,oxime C8H9NO2 151 75 0.53 8.790 Cinnamic acid C9H8O2 148 86 1.50 9.125 D-(-)-Quinic acid C7H12O6 192 79 3.16 9.200 Octadecanoic acid C18H36O2 284 75 1.15 9.270 2,6-Di-tert-butyl-4-methylphenol C15H24O 220 79 1.84 10.040 1,3,4,5-tetrahydroxy-cyclohexanecarboxylicacid C7H12O6 192 94 7.12 10.400 1,6,6-trimethyl-7-(3-oxo-but-1-enyl)-3,8-dioxa- C13H16O4 236 81 1.19 tricyclo[5.1.0.0 2,4]octan-5-one 10.445 Acetyl-l-phenylalanine methyl ester C12H15NO3 221 81 0.59 10.890 6,7-dehydro-7,8-dihydro-3-oxo-.alpha.-ionol C13H20O2 208 86 3.17 10.940 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-6- C11H16O3 196 85 4.47 hydroxy-4,4,7a-trimethyl-, (6S,7aR)- 11.805 1,2-benzenedicarboxylic acid, dibutyl ester C16H22O4 278 88 1.95 11.930 7-Hydroxy-5-methoxycoumarin C10H8O4 192 79 2.68 12.440 (3E)-3-Hexadecenenitrile C16H29N 235 81 0.69 12.995 Hexadecanamide C16H33NO 255 95 59.66 14.340 Cis-9-octadecenamide C18H35NO 281 94 10.65

172

Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 40% ACN

3.815 1,2-Ethanediol, monoacetate C4H8O3 104 90 0.79 8.540 Tetradecane C14H3O 198 89 0.91 9.275 2,6-Di-tert-butyl-4-methylphenol C15H24O 220 90 0.74 9.540 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a- C11H16O2 180 87 2.26 trimethyl- 9.815 Heneicosane C21H44 296 95 6.7 10.035 1,3,4,5-tetrahydroxy-cyclohexanecarboxylic acid C7H12O6 192 89 6.69 10.155 3-Oxo-, alpha-ionol C13H20O2 208 80 1.11 10.080 Nerolidol-epoxyacetate C17H28O4 296 76 2.12 10.195 2-Hexyl-1-decanol C16H34O 242 78 0.73 10.505 Nonylphenol C15H24O 220 81 2.89 10.820 3,5-Di-tert-butyl-4-hydroxybenzaldehyde C15H22O2 234 81 1.18 11.080 2-Icosyloxyethanol C22H46O2 342 77 1.07 11.335 1-Octadecanol C18H380 270 86 1.08 11.505 Heptadecanenitrile C17H33N 251 82 0.86 11.615 Ethylene glycol monohexadecyl ether C18H38O2 286 80 0.85 11.805 1,2-Benzenedicarboxylicacid, 1-butyl 2-(8- C22H34O4 362 85 4.75 methylnonyl) ester 12.385 1-Eicosanol C20H420 298 88 0.72 12.440 9-tetradecenal C14H26O 210 82 1.89

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Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 40% ACN

12.510 Octadecanoic acid, 2-oxo-, methyl ester C19H36O3 312 77 1.23 12.865 Octadecanoic acid (Stearic acid) C18H36O2 284 90 1.55 12.995 Hexadecanamide C16H33NO 255 95 20.07 13.090 Octacosane C28H58 394 83 0.74 14.340 Cis-9-octadecenamide C18H35NO 281 91 10.00 14.555 N-Tetradecanoic acid amide C14H29NO 227 91 18.96 55% ACN

3.840 1,2-Ethanediol, monoacetate C4H8O3 104 87 2.49 4.530 Methyl N-hydroxybenzenecarboximidoate C8H9NO2 151 79 2.03 9.275 2,6-Di-tert-butyl-4-methylphenol C15H24O 220 85 1.62 10.035 1,3,4,5-tetrahydroxy-cyclohexanecarboxylicacid C7H12O6 192 92 5.76 11.805 1,2-benzenedicarboxylic acid, dibutyl ester C16H22O4 278 89 2.68 12.440 9-Octadecenal C18H34O 238 80 2.12 12.995 Hexadecanamide C16H33NO 255 94 11.70 14.335 9-octadecenamide C18H35NO 281 91 4.56 14.550 N-tetradecanoic acid amide C14H29NO 227 91 11.69

174

Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 70% ACN

4.530 Methyl N-hydroxybenzenecarboximidoate C8H9NO2 151 78 0.26 6.315 Nonanal C9H180 142 87 1.67 9.325 1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid C7H12O6 192 77 6.72 12.995 Hexadecanamide C16H33N0 255 92 8.75 14.550 Tetradecanamide C14H29NO 227 90 7.52 85% ACN

10.035 1,3,4,5- tetrahydroxy-cyclohexanecarboxylic acid C7H12O6 192 91 1.95 11.805 1,2-Benzenedicarboxylic acid, dibutyl ester C16H22O4 278 88 1.99 12.440 9-Tetradecenal, (Z)- C14H26O 210 80 1.50 12.995 Hexadecanamide C16H33N0 255 95 33.30 14.335 9-Octadecenamide C18H35NO 281 93 6.33 10.035 1,3,4,5- tetrahydroxy-cyclohexanecarboxylic acid C7H12O6 192 91 1.95

175

Appendix V

APPENDIX V continued

Retention Compound Molecular Molecular Percentage Area (%) time formula weight match 100% ACN

6.315 Nonanal C9H180 142 93 0.65 7.585 Trans-2-decenal C10H180 154 90 0.79 10.035 1,3,4,5- tetrahydroxy-cyclohexanecarboxylic acid C7H12O6 192 90 0.93 11.190 2-Pentadececanone, 6, 10, 14-trimethyl- C18H36O 268 95 1.74 11.610 Methyl palmitate C17H34O2 270 95 20.96 11.805 Pentadecanoic acid (stearic acid) C15H30O2 242 88 14.68 12.115 Hexadecanoic acid, 14-methyl-, methyl ester C18H3602 284 88 0.67 12.435 5-tetradecyloxolan-2-one;4-Hydroxyoctadecanoic C18H34O2 282 79 2.3 acid γ-lacton 12.515 9-Octadecanoic acid, methyl ester C19H36O2 296 90 2.3 12.645 Octadecanoic acid methyl ester C19H38O2 298 94 2.73 12.865 Octadecanoic acid C18H36O2 284 89 3.05 12.995 Hexadecanamide C16H33NO 255 94 10.63 14.240 14-Beta-H-Pregna C21H36 288 79 2.24 14.330 9-Octadecenamide C18H35NO 281 94 4.92 14.555 N-teradecanoic acid amide C14H29NO 290 91 8.61 16.135 1,2-Benzenedicarboxylic acid C24H38O4 390 89 0.91

176

Appendix VI

APPENDIX VI

Chromatogram of 100% ACN fraction of Extract 8

Crude Extract 8 and its eight fractions obtained through SPE were analysed using a gas chromatograph coupled to a mass spectrometer as detector (GCMS-QP2010 Ultra, Shimadzu), equipped with an Rxi- 5SIL-MS column. The chromatogram for the 100% ACN fraction is shown in the figure. Helium was used as the carrier gas with total flow, column flow and purge flow set to 7.5 mL/min, 1.50 mL/min and 3.0 mL/min respectively; 2 µL samples were injected. The injector temperature was set to 200ºC and split injection mode (1:2) was selected at pressure 88.9 kPa. The column oven temperature was programmed as follows; held at 50ºC for the first two minutes and ramping at a rate of 20ºC/min to a final temperature of 250ºC , then held for eight minutes. The total GC run time was 20 minutes. The chromatogram obtained after the run was used to generate a qualitative table of the 50 most abundant compounds using peak integrate option. A similarity search was then performed to determine the possible structural units. The compounds were identified by comparison with the NIST and Wiley 8.0 MS libraries. Only peaks with a match greater than 75% and area % greater than 0.5% were reported. Only the major peaks are labelled in the figure shown, zooming in, using the software was required to visualize several small peaks.

177

Appendix VII

APPENDIX VII

(A) Inhibitory activities of extract 8 fractions against Mem-Bel (H3N1) strain

Cells at 80% confluence were treated with two-fold serial dilutions of plant extract (50-400 µg/mL) and

100 TCID50 of Mem-Bel virus. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

178

Appendix VII

APPENDIX VII continued

(B) Inhibitory activities of extract 8 fractions against PR8 (H1N1) strain

Cells at 80% confluence were treated with two-fold serial dilutions of plant extract (50-400 µg/mL) and

100 TCID50 of PR8 virus. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p < 0.05 (one way ANOVA).

179