phytochemical study, anti-toxoplasmosis and antimalarial activities

PHYTOCHEMICAL STUDY,

ANTI-TOXOPLASMOSIS AND ANTIMALARIAL

ACTIVITIES OF TINOSPORA CRISPA MIERS

METHANOL EXTRACT AND ITS BIOACTIVE

FRACTIONS

LEE WEI CAI

UNIVERSITI SAINS MALAYSIA

2015

PHYTOCHEMICAL STUDY,

ANTI-TOXOPLASMOSIS AND ANTIMALARIAL

ACTIVITIES OF TINOSPORA CRISPA MIERS

METHANOL EXTRACT AND ITS BIOACTIVE

FRACTIONS

By

LEE WEI CAI

Thesis submitted in fulfilment of the requirement for the Degree of

Master of Science

February 2015

This thesis is dedicated to.....

My parent

ii

ACKNOWLEDGEMENT

First and foremost, I would like to express my sincere gratitude to my loving parents

who have constantly been giving support and encouragement the entire time. I would

like to convey my deep appreciation to my supervisor, Associate Prof. Dr.

Roziahanim Mahmud, for her continuous support, guidance, knowledge and

invaluable encouragement from the beginning to the final stage of my study.

My sincere thanks to my co-supervisor, Associate Prof. Dr. Sabariah Ismail and field

supervisor, Associate Prof. Dr. Rusliza Basir, for their advice and assistance

throughout my study and to Professor Rahmah Noordin for her kind guidance and

advice for my in vitro studies.

I would like to express my gratitude to the Dean of School of Pharmaceutical

Sciences, Universiti Sains Malaysia for giving me an opportunity and support to

further my MSc study as a full time research student.

I also would like to thank my dear colleagues Ms. Shanmugapriya Perumal, Mr.

Suthagar Pillai Piaru, Miss Lee Yuan-E for their invaluable help, support and

encouragement which made the work possible. I would also like to extend my

gratitude to scientific officers and lab assistants of School of Pharmaceutical

Sciences for their invaluable help in instrumentation and phytochemical analysis.

The financial support of the FRGS research grant (No: 203/PFARMASI/6711189)

from MOHE and USM Short-Term Research Grant (304/PFARMASI/6311013) are

gratefully acknowledged. I would like to acknowledge MOSTI's MyBrain15

(MyMaster) scholarship for providing financial assistance to complete my studies.

Finally, I would like to thank all my family members and friends for their endless

support and understanding throughout my study.

iii

TABLE OF CONTENT

Page

Acknowledgement ii

Table of Content iii

List of Tables ix

List of Figures xi

List of Abbreviations xv

List of Appendices xvii

Abstrak xxi

Abstract xxiv

CHAPTER 1: LITERATURE REVIEW

1.1 Apicomplexan Parasites 1

1.1.1 Toxoplasma gondii 3

1.1.1.1 Life Cycle and Transmission of Toxoplasma gondii 5

1.1.1.2 Clinical Manifestations and Pathogenesis of Human

Toxoplasmosis 9

1.1.1.3 Treatment of Toxoplasmosis 11

1.1.1.4 Current Challenges in the Treatment of Toxoplasmosis 12

1.1.2 Plasmodium Parasites 13

1.1.2.1 Life Cycle and Transmission of Plasmodium Parasites 15

1.1.2.2 Clinical Manifestations and Pathogenesis of Human

Malaria 18

1.1.2.3 Treatment of Malaria 20

1.1.2.4 Current Challenges in the Treatment of Malaria 24

1.2 Oxidative Stress 25

iv

1.3 Potential of Herbal Medicines 28

1.4 Tinospora crispa Miers 30

1.4.1 Medicinal Use of Tinospora crispa Miers 32

1.4.2 Phytochemical Study of Medicinal Plants 33

1.4.2 Chemical Constituents and Anti-parasitic Biological Activities

of T. crispa Miers 34

1.5 Statement of the Problem and Rationale of the Study 38

1.6 Research Objectives 38

1.7 Expected Outcome of the Study 39

CHAPTER 2: IN VITRO CYTOTOXICITY AND ANTI-PARASITIC

STUDIES AGAINST TOXOPLASMA GONDII OF

CRUDE METHANOL TINOSPORA CRISPA MIERS

STEM EXTRACT AND ALL ITS FRACTIONS

2.1 Introduction 41

2.2 Materials and Methods 42

2.2.1 Chemicals and Solvents 42

2.2.2 Plant Material 42

2.2.3 Preparation of Crude Methanol T. crispa Miers Extract 43

2.2.4 Fractionation and Profiling of Crude Methanol T. crispa Miers

Extract by Column Chromatography and TLC 43

2.2.5 Cell Culture 44

2.2.5.1 Subculturing of Vero Cells 45

2.2.5.2 Cell Counting 46

2.2.6 Determination of Cytotoxicity of Crude Methanol T. crispa

Miers Extract and Its Fractions 47

2.2.7 Determination of Anti-parasitic Activity of Crude Methanol T.

crispa Miers Extract and Its Fractions Against Toxoplasma gondii 48

2.2.7.1 Toxoplasma gondii Parasite 48

2.2.7.2 In vitro Anti-parasitic Assay Against Toxoplasma gondii 49

v

2.2.8 Statistical analysis 49

2.3 Results and Discussion 50

2.3.1 Cytotoxicity of Crude Extract and Fractions of T. crispa Miers 50

2.3.2 Anti-parasitic Activity of Crude Extract and Fractions of T.

crispa Miers against Toxoplasma gondii 52

2.4 Conclusion 53

CHAPTER 3: IN VIVO ANTIMALARIAL STUDIES OF CRUDE

METHANOL TINOSPORA CRISPA MIERS EXTRACT

AND THE BIOACTIVE FRACTION F3 AND F4

3.1 Introduction 54





Extract by Column Chromatography and TLC 56

3.2.4 Animals 56

3.2.5 Measurement of Body Weight and Body Temperature 56

3.2.6 Induction and Maintenance of Murine Malaria Parasite 57

3.2.7 Parasitaemia Measurement 58

3.2.8 Malaria Model Establishment 59

3.2.9 Determination of Antimalarial Activities of Crude Methanol

T. crispa extract, Fraction F3 and F4 60

3.2.10 Statistical Analysis 61


3.3.1 Establishment of Malaria Model 62

3.3.2 Visual Observation on the Physical Signs of Illness of

Malaria-infected mice 62

3.3.3 Post-mortem Examinations on the Internal Organs of Control

and Malaria-infected Mice 67

vi

3.3.4 Effect of Malaria on Body Weight 69

3.3.5 Effect of Malaria on Body Temperature 71

3.3.6 Parasitaemia Level and Parasitaemia Suppression Level in

Malarial Mice 74

3.3.7 Survival of the Malarial Mice 82

3.4 Conclusion 89

CHAPTER 4: PHYTOCHEMICAL IDENTIFICATION OF THE

BIOACTIVE FRACTION F4 OBTAINED FROM

CRUDE METHANOL TINOSPORA CRISPA MIERS

EXTRACT

4.1 Introduction 90


4.2.1 Chemicals and Solvents 93

4.2.2 Instruments 93




Extract by Column Chromatography 94

4.2.6 Qualitative Analysis of Crude Methanol T. crispa Miers Extract

and Its Bioactive Fractions 94

4.2.6.1 Ultra-violet and Visible (UV-Vis) Spectroscopy 94

4.2.6.2 Fourier Transform Infra-red (FT-IR) Spectroscopy 95

4.2.6.3 TLC Profiling of Crude Methanol Extract and 8

Fractions of T. crispa Miers 95

4.2.7 Development of HPLC Method 95

4.2.7.1 Chromatographic Condition 95

4.2.7.2 HPLC Analysis of Fraction F4 96

4.2.8 Development of Preparative LC Method 96

4.2.8.1 Chromatographic Condition 96

vii

4.2.8.2 Preparative LC Analysis of Fraction F4 97

4.2.9 Mass Spectrometry Analysis 97

4.2.10 Determination of Total Phenolic Contents of Crude Methanol

T. crispa Miers Extract and Its Fraction F3 and F4 98

4.2.11 Determination of Total Flavonoid Contents of Crude Methanol

T. crispa Miers Extract and Its Fraction F3 and F4 98


4.3.1 UV-Vis Results 99

4.3.2 FTIR Results 101

4.3.3 TLC Profile Results 104

4.3.4 HPLC Analysis of Fraction F4 107

4.3.5 Preparative LC Result of Fraction F4 109

4.3.6 Mass Spectrometry Result of Purified Peak B Obtained

From Preparative LC 111

4.3.7 Total Phenolic Contents of Crude Methanol T. crispa Miers

Extract and Its Fraction F3 and F4 113

4.3.8 Total Flavonoid Contents of Crude Methanol T. crispa Miers

Extract and Its Fraction F3 and F4 114

4.4 Conclusion 115

CHAPTER 5: ANTIOXIDANT STUDIES OF CRUDE METHANOL

TINOSPORA CRISPA MIERS EXTRACT AND THE

BIOACTIVE FRACTION F3 AND F4

5.1 Introduction 116



5.2.2 Preparation of Crude T. crispa Miers Extract 118

5.2.3 Fractionation and Profiling of Crude Methanol T. crispa

Miers Extract by Column Chromatography and TLC 118

5.2.4 Free Radical Scavenging Activity 118

viii

5.2.5 β-Carotene/Linoleic Acid Bleaching Assay 119

5.2.6 Estimation of Reducing Power 120

5.2.7 Statistical Analysis 121


5.3.1 Antioxidant Activity by Free Radical Scavenging Activity 121

5.3.2 Antioxidant Activity by β-Carotene Bleaching Assay 124

5.3.3 Antioxidant Activity by Reducing Power Assay 127

5.4 Conclusion 130

CHAPTER 6: CONCLUSION AND RECOMMENDATION

6.1 Conclusion 131

6.2 Recommendation for further studies 133

REFERENCES 134

APPENDICES 157

Appendix 1 158

Appendix 2 162

Appendix 3 168

Appendix 4 175

Appendix 5 183

LIST OF PUBLICATIONS 186

ix

LIST OF TABLES

Content Page

Table 1.1 Taxonomic classification of Toxoplasma gondii. 4

Table 1.2 Taxonomic classification of Plasmodium parasites. 15

Table 1.3 Classes of chemical family for antimalarial drugs. 23

Table 1.4 Taxonomic classification of Tinospora crispa Miers. 32

Table 2.1 In vitro cytotoxicity of crude methanol T. crispa Miers

extract, its eight hexane-acetone fractions (F1-F8) and

clindamycin, respectively on Vero cell.

51

Table 2.2 Anti-parasitic activity of crude methanol T. crispa Miers

extract, its eight hexane-acetone fractions (F1-F8) and

clindamycin respectively, against Toxoplasma gondii on

Vero cell.

53

Table 3.1 Physical signs of illness observed for different groups of

P. berghei-infected mice treated with 10 mg/kg animal

weight, 50 mg/kg animal weight and 100 mg/kg animal

weight of crude methanol T. crispa Miers extract,

fraction F3, fraction F4, chloroquine (20 mg/kg animal

weight) and untreated malarial-infected mice (negative

control), respectively.

65

Table 3.2 Post-mortem examinations on major organs in untreated

malaria-infected mice.

68

Table 5.1 Median inhibition concentration (IC50) values of free

radical scavenging activity of crude methanol T. crispa

Miers stem extract, fraction F3, fraction F4 and BHT

(positive control), respectively.

123

Table 5.2 Beta-carotene/linoleic acid bleaching activity of crude

methanol T. crispa Miers stem extract, fraction F3,

fraction F4, BHT (positive control) and negative control

(DMSO).

125

Table 5.3 Reducing power activity [ferric (III) ions (Fe3+

) to ferrous

(II) ions (Fe2+

)] of crude methanol T. crispa Miers stem

extract, fraction F3 and fraction F4.

128

x

Table 5.4 Reducing power activity [ferric (III) ions (Fe3+

) to ferrous

(II) ions (Fe2+

)] of ascorbic acid (positive control).

128

xi

LIST OF FIGURES

Content Page

Figure 1.1 The morphology of apicomplexan parasites. 3

Figure 1.2 Ultra structure comparisons of a T. gondii tachyzoite and

a P. falciparum merozoite.

5

Figure 1.3 Life cycle, transmission route and clinical manifestations

of T. gondii.

8

Figure 1.4 Life cycle of Plasmodium parasites in the human body

and the anopheline mosquito.

17

Figure 1.5 Gametocyte and trophozoite (ring form) of P. falciparum

in a thin blood smear.

17

Figure 1.6 Tinospora crispa Miers plant with blunt tubercles on the

stem.

31

Figure 1.7 Collected Tinospora crispa Miers plant. 31

Figure 1.8 Chemical structure of compounds identified from

methanol-water extract of T. crispa Miers stem.

36

Figure 1.9 Flow chart of research strategy. 40

Figure 3.1 Daily body weight change (g) of different groups of P.

berghei-infected mice treated with different doses of

crude methanol stem extract of T. crispa Miers, fraction

F3, fraction F4, chloroquine and untreated malarial-

infected mice (negative control), respectively.

70

Figure 3.2 Daily body temperature change (ºC) of different groups

of P. berghei-infected mice treated with different doses

of crude methanol stem extract of T. crispa Miers,

fraction F3, fraction F4, chloroquin and untreated

malarial-infected mice (negative control), respectively.

73

Figure 3.3 Daily parasitaemia level (%) of different groups of P.

berghei-infected mice treated with different doses of

crude methanol stem extract of T. crispa Miers, fraction

F3, fraction F4, chloroquin and untreated malarial-

infected mice (negative control), respectively.

78

xii

Figure 3.4 Daily parasitaemia suppression level (%) of different

groups of P. berghei-infected mice treated with different

doses of crude methanol stem extract of T. crispa Miers,

fraction F3, fraction F4, chloroquin and untreated


79

Figure 3.5 Leishman’s stained-blood film of untreated malarial-

infected (negative control) mouse tail blood on Day 6

post-inoculation observed under 1000 x magnification

with oil immersion field.

80

Figure 3.6 Leishman’s stained-blood film of chloroquine-treated

(positive control) mouse tail blood on Day 6 post-

inoculation observed under 1000 x magnification with oil

immersion field.

80

Figure 3.7 Leishman’s stained-blood film of crude T. crispa Miers

100 mg/kg animal weight treated-mouse tail blood on

Day 6 post-inoculation observed under 1000 x

magnification with oil immersion field.

81

Figure 3.8 Leishman’s stained-blood film of F3 100 mg/kg animal

weight treated-mouse tail blood on Day 6 post-


immersion field.

81

Figure 3.9 Leishman’s stained-blood film of F4 100 mg/kg animal

weight treated-mouse tail blood on Day 6 post-


immersion field.

82

Figure 3.10 Survival curve of different groups of P. berghei-infected

mice treated with different doses of crude methanol stem

extract of T. crispa Miers, chloroquine and untreated


86


mice treated with different doses of fraction F3,

chloroquine and untreated malarial-infected mice

(negative control), respectively.

87


mice treated with different doses of fraction F4,

chloroquine and untreated malarial-infected mice

(negative control), respectively.

88

xiii

Figure 4.1 UV-Vis spectra of crude methanol T. crispa Miers

extract.

100

Figure 4.2 UV-Vis spectra of fraction F3 of crude methanol T.

crispa Miers extract.

100

Figure 4.3 UV-Vis spectra of fraction F4 of crude methanol T.

crispa Miers extract.

101

Figure 4.4 FT-IR spectra of crude methanol T. crispa Miers extract. 102

Figure 4.5 FT-IR spectra of fraction F3 of crude methanol T. crispa

Miers extract.

103

Figure 4.6 FT-IR spectra of fraction F4 of crude methanol T. crispa

Miers extract.

103

Figure 4.7 Thin layer chromatography (TLC) analysis of crude

methanol T. crispa Miers extract and all eight fractions

(F1-F8) viewed under short UV wavelength (254 nm).

105

Figure 4.8 Thin layer chromatography (TLC) analysis of crude

methanol T. crispa Miers extract and all eight fractions

(F1-F8) viewed under long UV wavelength (360 nm).

106

Figure 4.9

HPLC chromatogram of fraction F4 of crude methanol T.

crispa Miers extract detected at 260 nm.

108

Figure 4.10 HPLC chromatogram of crude methanol T. crispa Miers

extract detected at 260 nm.

109

Figure 4.11 Preparative liquid chromatography (LC) chromatogram

of fraction F4 of crude methanol T. crispa Miers extract

detected at 260 nm.

110

Figure 4.12 Real time chromatogram of fraction F4 of crude

methanol T. crispa Miers extract detected at 260 nm on

preparative LC.

111

Figure 4.13 Mass spectrum and the proposed fragment ions of the

peak B isolated from fraction F4.

112

Figure 4.14 Fragmentation pattern 13(S)-HPODE. 113

Figure 5.1 DPPH free radical scavenging activity of crude methanol

stem extract of T. crispa Miers, fraction F3, fraction F4

and tert-butylated hydroxytoulene (BHT) (n=3).

123

xiv

Figure 5.2 Beta carotene-linoleic acid bleaching activity of crude

methanol stem extract of T. crispa Miers, fraction F3,

fraction F4, tert-butylated hydroxytoulene (BHT) and

negative control (n=3).

126

Figure 5.3 Reducing power activity [ferric (III) ions (Fe3+

) to

ferrous (II) ions (Fe2+

)] of crude methanol stem extract of

T. crispa Miers, fraction F3, fraction F4 and ascorbic

acid (n=3).

128

xv

LIST OF ABBREVIATIONS

µg/mL microgram per millilitre

ACN Acetonitrile

AIDS Acquired immunodeficiency syndrome

AlCI3.H2O Aluminium chloride

ALP Alkaline phosphatase

ANOVA Analysis of variance

ATCC American Type & Culture Collection

ATR Attenuated Total Reflectance

BHT tert-butylated hydroxytoulene

CAT Catechin equivalents

CDC Centers for Disease Control and Prevention

CGM Complete Growth Medium

CNS Central nervous system

CO2 Carbon dioxide

DAD Photodiode array detector

dd H2O Doubly-distilled water

DMEM Dulbecco's Modified Eagle Medium

DMSO Dimethyl sulfoxide

DPPH 2,2-diphenyl-1-picrylhydrazyl

EC50 Median effective concentration

ESI Electrospray ionization

FBS Foetal bovine serum

FeCl3 Ferric chloride

FTIR Fourier Transform Infra-Red

g Gram

GAE Gallic acid equivalent

GCMS Gas chromatography-mass spectrometry

GSH Glutathione

GSH-Px Glutathione peroxidase

h Hour

HIV Human immunodeficiency virus

HPLC High performance liquid chromatography

IC50 Inhibitory concentration of 50 %

IL-1β Interleukin-1 beta

IL-6 Interleukin-6

i.p. Intraperitoneal

K3Fe(CN)6 Potassium ferricyanide

LCMS Liquid chromatography-mass spectrometry

LDH Lactate dehydrogenase

mg Milligram

mg/mL Milligram per millilitre

min Minutes

mL Millitre

MIP-1α Macrophage inflammatory protein-1 alpha

xvi

MIP-1β Macrophage inflammatory protein-1 beta

MS Mass spectrometry

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

(4-sulfophenyl)-2H-tetrazolium

MTT 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide

NaCl Sodium chloride

Na2CO3 Sodium carbonate

NaHCO3 Sodium bicarbonate

NaOH Sodium hydroxide

NaNO2 Sodium nitrate

nm Nanometer

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PGE2 Prostaglandin E2

PRBCs Parasitized red blood cells

Prep LC Preparative liquid chromatography

PS Penicillin-streptomycin solution

psi Pound per square inch

Rf Retention factor

RBCs Red blood cells

RNS Reactive nitrogen species

ROS Reactive oxygen species

RPM Revolution per minute

SEM Standard error of the means

SI Selectivity index

SOD Superoxide dismutase

TFC Total flavonoids content

TIC Total ion counting

TLC Thin layer chromatography

TNF-α Tumour necrosis factor-alpha

TPC Total phenolic content

UV-vis Ultra violet-visible

WHO World Health Organization

5-HT 5-hydroxytryptamine

xvii

LIST OF APPENDICES

Page

Appendix 1 158

Appendix 1.1 Preparation of DMEM Media 159

Appendix 1.2 Cytotoxicity of crude methanol T. crispa Miers extract

and all the eight fractions (F1-F8) on Vero cells.

159

Appendix 1.3 Cytotoxicity activity of crude methanol stem extract of

T. crispa Miers and all its eight fractions (F1-F8)

respectively, on Vero cells.

160

Appendix 1.4 Cytotoxicity of clindamycin on Vero cells. 160

Appendix 1.5 Cytotoxicity activity of clindamycin on Vero cells. 160

Appendix 1.6 In vitro anti-toxoplasmosis activity of crude methanol

T. crispa Miers extract, the eight fractions (F1-F8) and

clindamycin on Vero cells.

161

Appendix 1.7 Anti-toxoplasmosis activities of crude methanol stem

extract of T. crispa Miers, the eight fractions (F1-F8)

and clindamycin on Vero cell line (n=5).

161

Appendix 2 162

Appendix 2.1 Daily mean body weight of different groups of P.

berghei-infected mice treated with 10 mg/kg animal



fraction F3 and fraction F4, chloroquine (20 mg/kg

animal weight), untreated malarial-infected mice

(negative control) and uninfected control mice,

respectively.

163

Appendix 2.2 Daily mean body temperature of different groups of P.





weight), untreated malarial-infected mice (negative

control) and uninfected control mice, respectively.

164

xviii

Appendix 2.3 Daily mean percentage of parasitaemia of different groups

of P. berghei-infected mice treated with 10 mg/kg

animal weight, 50 mg/kg animal weight and 100 mg/kg

animal weight of crude methanol T. crispa Miers

extract, fraction F3, fraction F4, chloroquine (20 mg/kg

animal weight), untreated malarial-infected mice

(negative control) and uninfected control mice,

respectively.

165

Appendix 2.4 Daily mean percentage of parasitaemia suppression of

different groups of P. berghei-infected mice treated

with 10 mg/kg animal weight, 50 mg/kg animal weight

and 100 mg/kg animal weight of crude methanol T.

crispa Miers extract, fraction F3, fraction F4 and

chloroquine (20 mg/kg animal weight), respectively.

166

Appendix 2.5 Daily survival percentage of different groups of P.





weight), untreated malarial-infected mice (negative

control) and uninfected control mice, respectively.

167

Appendix 3 168

Appendix 3.1 Crude methanol extract yielded and percentage of yield

for each round of maceration of T. crispa Miers stem

powder.

169

Appendix 3.2 Yield of each fraction of crude methanol T. crispa

Miers extract obtained from each round of fractionation

through column chromatography.

169

Appendix 3.3 HPLC chromatogram of peak A purified from

preparative LC. The chromatogram was detected at 260

nm.

170

Appendix 3.4 HPLC chromatogram of peak B purified from


nm.

170

Appendix 3.5 HPLC chromatogram of peak C purified from


nm.

171

xix

Appendix 3.6 HPLC chromatogram of peak D purified from


nm.

171

Appendix 3.7 Mass spectrum of peak D purified from preparative LC. 172

Appendix 3.8 Calibration curve of absorbance versus concentration of

gallic acid for total phenolic content (TPC) study.

173

Appendix 3.9 Raw data for total phenolic contents (TPC) study. 173

Appendix 3.10 Calibration curve of absorbance versus concentration of

catechin for total flavonoids content (TFC) study.

174

Appendix 3.11 Raw data for total flavonoid contents (TFC) study. 174

Appendix 4 175

Appendix 4.1 Calibration curve of crude methanol T. crispa Miers

extract on DPPH free radical scavenging activity.

176

Appendix 4.2 Calibration curve of fraction F3 on DPPH free radical

scavenging activity.

176

Appendix 4.3 Calibration curve of fraction F4 on DPPH free radical


177

Appendix 4.4 Calibration curve of BHT on DPPH free radical


177

Appendix 4.5 Raw data for beta carotene bleaching assay. 178

Appendix 4.6 Inhibition of beta carotene oxidation of linoleic acid by

crude methanol T. crispa Miers extract, fraction F3,

fraction F4, BHT and control (DMSO).

179

Appendix 4.7 Calibration curve of crude methanol T. crispa Miers

extract on reducing power activity.

179

Appendix 4.8 Calibration curve of fraction F3 on reducing power

activity.

180

Appendix 4.9 Calibration curve of fraction F4 on reducing power

activity.

180

Appendix 4.10 Calibration curve of ascorbic acid on reducing power

activity.

181

xx

Appendix 4.11 Raw data of crude methanol T. crispa Miers extract,

fraction F3 and fraction F4 on reducing power assay.

182

Appendix 4.12 Raw data of ascorbic acid on reducing power assay. 182

Appendix E 183

Appendix 5.1 Approval Letter from Animal Care and Use Committee,

Universiti Putra Malaysia.

184

Appendix 5.2 Certificate of Acknowledgement of presentation at the

School of Pharmaceutical Sciences, USM.

185

xxi

KAJIAN FITOKIMIA, AKTIVITI ANTI-TOKSOPLASMOSIS DAN

AKTIVITI ANTIMALARIA EKSTRAK METANOL TINOSPORA CRISPA

MIERS DAN FRAKSI BIOAKTIFNYA

ABSTRAK

Toksoplasmosis dan malaria merupakan dua penyakit serius yang mengakibatkan

bebanan terhadap kesihatan manusia. Pada masa kini, tiada banyak kemajuan yang

dicapai bagi pembangunan agen-agen kimoterapeutik untuk rawatan toksoplasmosis

dan malaria. Agen-agen kimoterapeutik semasa telah digunakan selama beberapa

dekad dan parasit telah mencapai rintangan terhadap ubat-ubatan semasa. Secara

tradisi, Tinospora crispa Miers telah digunakan dengan meluas untuk merawat

malaria, demam dan penyakit-penyakit parasit lain yang berkaitan. Dalam kajian ini,

ekstrak mentah metanol dan fraksi-fraksi bioaktif Tinospora crispa Miers dikaji

untuk aktiviti-aktiviti anti-parasitik terhadap Toxoplasma gondii dan Plasmodium

berghei, kajian fitokimia dan aktiviti anti-oksida. Ekstrak mentah telah diekstrak

daripada batang Tinospora crispa Miers menggunakan kaedah rendaman dan

dipencilkan kepada kumpulan komponen yang kecil dengan menggunakan teknik

kromatografi lajur dengan pengelusi sistem pelarut heksana-aseton. Aktiviti

sitotoksik ekstrak mentah dan kesemua fraksi yang mempunyai kekutuban berbeza

(F1 hingga F8 masing-masing) telah ditentukan dengan in vitro assai MTT ke atas sel

Vero. Kajian ini mendapati bahawa ekstrak mentah dan kesemua fraksi menunjukkan

tiada aktiviti sitotoksik ke atas sel Vero. Ekstrak mentah dan kesemua fraksi telah

dikaji selanjutnya untuk aktiviti anti-parasitik terhadap Toxoplasma gondii. Ekstrak

mentah menunjukkan aktiviti perencatan terhadap Toxoplasma gondii dengan nilai

EC50 7.83 ± 0.12 µg/mL. Daripada kesemua fraksi, fraksi F3 dan fraksi F4

xxii

menunjukkan aktiviti paling bioaktif terhadap T. gondii dengan nilai EC50 6.77 ±

0.03 µg/mL dan 6.04 ± 0.02 µg/mL, masing-masing. Siasatan lanjut untuk aktiviti

antimalaria terhadap Plasmodium berghei dalam model tikus secara in vivo

dijalankan dengan ekstrak mentah dan dua fraksi poten F3 dan F4 yang telah

ditentukan daripada kajian in vitro. Tikus dijangkiti dengan malaria yang menerima

dos rawatan 100 mg/kg berat haiwan ekstrak mentah, fraksi F3 dan fraksi F4 masing-

masing, menunjukkan tahap perencatan parasitemia yang signifikan berbanding

dengan kawalan positif klorokuin. Tahap perencatan parasitemia bagi dos 100 mg/kg

berat haiwan fraksi F4 adalah lebih tinggi daripada ekstrak mentah dan fraksi F3

pada hari ke-6 selepas jangkitan, iaitu pada 54.32 ± 0.24 %, 42.85 ± 0.58 % dan

41.01 ± 0.46 % masing-masing, berbanding dengan kawalan positif klorokuin yang

ditentukan pada 97.17 ± 0.03 %. Tambahan pula, tikus dijangkiti malaria yang

menerima dos rawatan 100 mg/kg berat haiwan fraksi F4 menunjukkan kadar

kehidupan harian yang lebih tinggi berbanding dengan tikus yang menerima rawatan

dos rawatan 100 mg/kg berat haiwan ekstrak mentah dan fraksi F3. Komposisi kimia

fraksi F4 yang bioaktif telah ditentukan dengan menggunakan kajian spektroskopi

dan kromatografi (TLC, FT-IR, Uv-vis, HPLC, prep-LC dan MS). Komposisi kimia

daripada fraksi F4 yang berpotensi telah ditentukan secara kualitatif menggunakan

ESI-MS. 13(S)-HPODE telah didapati wujud dalam fraksi F4. Dalam aktiviti anti-

oksida, ekstrak mentah menunjukkan aktiviti yang lebih tinggi berbanding dengan

fraksi F3 dan fraksi F4 dalam asai DPPH, asai pelunturan beta-karotena/asid linolik

dan asai kuasa penurunan. Namun, jumlah kandungan fenolik dalam ekstrak mentah

adalah 1.6 kali lebih rendah berbanding dengan fraksi F4. Tambahan pula, jumlah

kandungan flavanoid dalam ekstrak mentah adalah 14.8 kali lebih rendah berbanding

dengan fraksi F4. Sebagai kesimpulannya, kajian ini telah mendapati ketinggian

xxiii

potensi ekstrak Tinospora crispa Miers dalam aktiviti-aktiviti anti-toksoplasmosis

dan antimalaria, di mana Fraksi F4 yang bioaktif telah menyerlahkan aktiviti

farmakologi signifikan dalam assai anti-T. gondii in vitro dan model tikus P. berghei

secara in vivo. Kajian ini membuka ruang kajian selanjutnya yang akan

memberangsangkan pembangunan praklinikal agen anti-parasitik daripada sumber

tumbuhan ubatan tempatan.

xxiv

PHYTOCHEMICAL STUDY, ANTI-TOXOPLASMOSIS AND

ANTIMALARIAL ACTIVITIES OF TINOSPORA CRISPA MIERS

METHANOL EXTRACT AND ITS BIOACTIVE FRACTIONS

ABSTRACT

Toxoplasmosis and malaria are two serious illnesses resulting in huge toll on human

health. To date, there is not much advancement in the chemotherapeutic agents used

to treat toxoplasmosis and malaria. The current chemotherapeutic agents have been

used for decades and the associated parasites have developed resistance towards the

current mainstay drugs. Traditionally, Tinospora crispa Miers are widely used to

treat malaria, fever and some other parasite-related illnesses. In this work, the crude

methanol extract and bioactive fractions of Tinospora crispa Miers were studied for

anti-parasitic activities against Toxoplasma gondii and Plasmodium berghei,

phytochemical content and antioxidant activities. The crude extract was extracted

from stems of Tinospora crispa Miers using maceration method and further

fractionated into smaller group of components by elution with the hexane-acetone

solvent system on column chromatographic technique. The cytotoxic activity of the

crude extract and all the differing polarity fractions (F1 to F8 respectively) on Vero

cell line was determined by in vitro MTT assay. This study found that the crude

extract and all the fractions showed no cytotoxic activity on Vero cells. The crude

extract and all fractions were then further investigated for anti-parasitic activity

against Toxoplasma gondii. The crude extract showed inhibitory activity against T.

gondii with EC50 value of 7.83 ± 0.12 µg/mL. Of all the fractions, fractions F3 and

F4 showed the most bioactive activity against T. gondii with EC50 values of 6.77 ±

0.03 µg/mL and 6.04 ± 0.02 µg/mL, respectively. Further investigation for

xxv

antimalarial activity against Plasmodium berghei in the in vivo mice model was then

carried out with crude extract and its two potent fractions F3 and F4 determined from

the in vitro study. Malarial-infected mice receiving treatment of dose at 100 mg/kg

animal weight of crude extract, fraction F3 and fraction F4 respectively, showed

significant parasitaemia suppression level as compared to positive control

chloroquine. The parasitaemia suppression levels of 100 mg/kg animal weight dose

of Fraction F4 was found to be higher compared to the crude extract and fraction F3

on Day 6 post-inoculation, which were 54.32 ± 0.24 %, 42.85 ± 0.58 % and 41.01 ±

0.46 %, respectively in comparison with the positive control chloroquine at 97.17 ±

0.03 %. In addition, malarial-infected mice receiving 100 mg/kg animal weight dose

of fraction F4 treatment showed higher daily survival rate compared to the mice

receiving same doses of crude extract and fraction F3. The chemical composition of

bioactive fraction F4 was determined by various spectroscopic and chromatographic

studies (TLC, FT-IR, UV-vis, HPLC, prep-LC and MS). The chemical composition

of bioactive fraction F4 was determined qualitatively on ESI-MS. The 13(S)-HPODE

was found to be present in fraction F4. In the antioxidant activities, the crude extract

showed better activities than fractions F3 and F4 in DPPH assay, beta

carotene/linoleic acid bleaching assay and reducing power assay. However, the total

phenolic content of the crude extract was 1.6 times lower compared to fraction F4,

while the total flavonoids content of the crude extract was 14.8 times lower than

fraction F4. In conclusion, this study have uncovered potent anti-toxoplasmosis and

antimalarial activities of Tinospora crispa Miers extract, where its bioactive fraction

F4 exhibited significant pharmacological activities in the in vitro anti-T. gondii assay

and in vivo P. berghei mice model. This work opens the door to further promising

pre-clinical development of anti-parasitic agent(s) of local medicinal plant origin.

1

CHAPTER 1: LITERATURE REVIEW

1.1 Apicomplexan Parasites

A parasite refers to an organism that lives on or in a host, and gets its food from or at

the expense of its host (CDC, 2014). Pathogenic parasites can cause serious diseases

in humans and many economically important animal species. One of the important

classifications of the parasite is the apicomplexan parasites that consist of pathogenic

single-cell intracellular parasites. Some important apicomplexan parasites that cause

diseases in human are Plasmodium species that is well known for the deadly malaria,

Cryptosporidium species for causing cryptosporidiosis and Toxoplasma species for

causing toxoplasmosis. Two apicomplexan parasites (Plasmodium and Toxoplasma)

that cause severe diseases in human population were the main highlights in the study.

All apicomplexan are parasitic and share many specific features related to parasitism

(Figure 1.1). The most important feature is the presence of the apicoplast (McFadden

et al., 1996). Apicoplast is a non-photosynthetic organelle of algal origin (Tawk et al.,

2011). However, an exception to this rule is the Cryptosporidium parasites that loss

the organelle during evolution (Zhu et al., 2000). Several studies have proven that

the apicoplast fulfils critical functions for parasites invasion and survival. Apicoplast

is the site of several plastid-derived biochemical pathways such as biosynthesis of

fatty acids, isoprenoids, haem and iron–sulfur clusters (Ralph et al., 2004). Ralph et

al. (2004) also stated that the metabolic pathways of the apicoplast contribute to lipid

production and the modification of lipid-bound proteins. These findings proved that

the apicoplast plays a crucial role in the successful establishment of parasite-host

interaction, such as in the formation of parasitophorus vacuole during parasites

2

invasion. Moreover, apicoplast is needed for survival in both erythrocytic (Goodman

et al., 2007) and liver stages (Vaughan et al., 2009; Yu et al., 2008) of P. falciparum.

In fact, the apicoplast forms close associations with mitochondria and endoplasmic

reticulum (ER) during development of Plasmodium species (van Dooren et al., 2005),

while in Toxoplasma gondii, apicoplast is tightly associated with ER (Tomova et al.,

2009).

Apart from the apicoplast, the apicomplexan parasites also have a characteristically

polarized cell structure, complex cytoskeletal and secretory organelle at the apical

end of the cell (Dubey et al., 1998). The cytoskeleton and organelles mediate the

parasites' locomotion and cellular invasion (Templeton et al., 2004). Apicomplexan

parasites also contain the rhoptries and micronemes that are deployed during the

invasion process and the delivery of parasites protein into the host cells

(Anantharaman et al., 2007).

Each apicomplexan lineage has evolved unique spectrum of protein that modify the

host in diverse ways. The mechanisms of invasion into the hosts are largely

conserved among the apicomplexan (Kim and Weiss, 2004). However, the host range,

cell specificity and life cycle stages within species are quite different among the

apicomplexan parasites.

3

Figure 1.1: The morphology of apicomplexan parasites. Apicomplexan parasites are

highly polarized cells containing a collection of organelles that are specific to the

phylum. The figure was adapted from Morrissette and Sibley (2002).

1.1.1 Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite which is a

significant human and veterinary pathogen (Kim and Weiss, 2004). Figure 1.2

illustrates the ultra structure of a T. gondii tachyzoite. T. gondii is capable to infect

any nucleated mammalian or avian cell including humans (Dubey, 1998). This makes

T. gondii one of the most successful parasitic organisms worldwide and can be found

in most regions of the world (Innes, 2010). Felidae (cat) species are the definitive

host of T. gondii for its reproductive cycle while the other warm-blooded organisms

such as humans serve as intermediate hosts for its propagation.

The apicomplexan parasite Toxoplasma gondii causes a flu-like disease known as

toxoplasmosis. The symptoms of toxoplasmosis include fever, headache, sore throat,

cough and dizziness. Toxoplasmosis can also cause significant morbidity and

mortality in the fetus and immunocompromised individuals if not treated (Montoya

and Liesenfeld, 2004). According to Flegr et al. (2014), infection with T. gondii is

4

the most prevalent one in humans, which estimated to affect about 30-50 % of the

world population. On top of that, there was a T. gondii seroprevalence of 55.7 %

among rural Malays in a study from Malaysia (Petersen, 2007). In addition,

estimation of toxoplasmosis disease burden ranks this disease at the same level as

salmonellosis or campylobacteriosis in the food borne disease category (Kijlstra and

Jongert, 2008).

Numerous researchers supported the use of T. gondii parasite as the model organism

to study the apicomplexan parasites. The advantages of using T. gondii as model

organism lays in its various biotechnology establishments that includes the well-

established methodology for classic and reverse genetics of T. gondii (Roos et al.,

1999), identification of numerous T. gondii molecular markers and the relative ease

with which the parasite can be studies using microscopic techniques (Kim and Weiss,

2004). All these factors make T. gondii the best model system for cell biology

investigation of apicomplexan parasites. The studies of mechanisms of drug

resistance, the biology of the apicoplast, and the process of host cell invasion have all

been achieved in T. gondii (Kim and Weiss, 2004). In fact, T. gondii is the most

experimentally tractable among the apicomplexan parasites (Roos et al., 1999).

Table 1.1: Taxonomic classification of Toxoplasma gondii (Adapted from NCBI,

2015a).

Taxonomic Classification

Kingdom Alveolata

Phylum Apicomplexa

Class Conoidasida

Order Eucoccidiorida

Family Sarcosystidae

Genus Toxoplasma

Species T. gondii

5

Figure 1.2: Ultra structure comparisons of a T. gondii tachyzoite and a P. falciparum

merozoite (adapted from Baum et al., 2006).

1.1.1.1 Life Cycle and Transmission of Toxoplasma gondii

The life cycle and transmission routes of T. gondii are illustrated in Figure 1.3. The

life cycle of T. gondii is facultatively heteroxenous. This means that T. gondii has

more than one host in its life cycle. There are three infectious stages of T. gondii

during their life cycle and they are tachyzoites, bradyzoites contained in tissue cysts

and sporozoites contained in sporulated oocysts. All the three stages are infectious

for both intermediate and definitive hosts (Tenter et al., 2000).

The sexual reproduction of T. gondii only occurs in the felid's intestinal tract. The

cats become infected after consuming intermediate hosts contaminated with tissue

6

cysts or oocysts-contaminated water (Montoya and Liesenfeld, 2004). Next, the

parasites invade the cat enterocytes and undergo division and differentiation into

microgametocytes and macrogametocytes. The gametocytes fuse to form zygotes or

oocysts that are shed into environment together with cat feces. The oocysts produce

highly infectious sporozoites that are very resistant to environmental damage. The

sporozoites may persist for years in warm and humid condition (Montoya and

Remington, 2000). The oocysts spread and contaminate the water, soil, fruits and

vegetables. Animals (intermediate hosts) will be infected following the consumption

of contaminated water, soil, fruits or vegetables (Tenter et al., 2000).

The asexual reproduction of T. gondii occurs in intermediate hosts (Montoya and

Liesenfeld, 2004). Shortly after ingestion, the oocysts are rapidly transformed into

tachyzoites. Tachyzoites enter all nucleated cells and replicate rapidly within the

cells. Replication of the tachyzoites inside the cells eventually leading to cell death.

Once the host cells are disrupted, the tachyzoites are released and disseminated via

the bloodstream and rapidly invades many tissues such as central nervous system

(CNS), skeletal muscle, heart, eye, placenta and fetus (Montoya and Liesenfeld,

2004). The tachyzoites cause massive tissue destruction, stimulate host immune

system and cause clinical manifestations of the disease (Montoya and Remington,

2000).

Under the pressure of the immune response, tachyzoites can transform into

bradyzoites to form cysts that reside and persist in tissues and muscles, especially in

brain, skeletal and heart muscles for the life of the host (Montoya and Liesenfeld,

2004). The bradyzoites can be released and transformed back into active tachyzoites

7

that cause recrudescence infection. The cysts are the infective stages for both

intermediate and definitive hosts (Montoya and Remington, 2000).

Transmission of T. gondii in human can occur via several routes. The main route is

through oral consumption (Montoya and Liesenfeld, 2004). This happens when

eating raw or undercooked cysts-contaminated meat, foods or water contaminated

with cat feces or environmental samples such as fecal-contaminated soil, changing

the litter box of pet cat, unwashed fruits or vegetables and unfiltered water. There

was high seroprevalence of T. gondii in countries and regions where the humans

were common to eat undercooked or raw meat (Dubey and Beattie, 1988). Second,

humans can acquire toxoplasmosis during blood transfusion or organ transplantation

(Montoya and Liesenfeld, 2004). However, the risk of transmission through this

route is very low (Dubey and Beattie, 1988). Lastly, the fetus can get infected with T.

gondii through transplacental infection from the pregnant mother (Montoya and

Remington, 2000).

8

Figure 1.3: Life cycle, transmission route and clinical manifestations of T. gondii

(Adapted and modified from Montoya and Liesenfeld, 2004).

9

1.1.1.2 Clinical Manifestations and Pathogenesis of Human Toxoplasmosis

The clinical manifestations and pathogenesis of toxoplasmosis in humans mainly

depend on the patient's immune status and the clinical setting such as

immunocompetent, immunocompromised, congenital toxoplasmosis, or ocular

disease (Montoya and Liesenfeld, 2004).

(i) Immunocompetent people

The primary infection usually causes a self-limited and non-specific flu-like illness.

The general symptoms of toxoplasmosis are fever, lymphadenopathy, headache, sore

throat, cough, myalgia and dizziness. However, infection can remain for life and

reactivation of T. gondii infection can occur when the immune system is

compromised. Though very rarely, myocarditis, polymyositis, pneumonitis, hepatitis

or encephalitis can occur as a result of T. gondii infection (Montoya and Liesenfeld,

2004).

(ii) Immunocompromised and immunosuppressed patients

Toxoplasmosis can be life-threatening (Liesenfeld et al., 1999) and always happens

as a result of reactivation of chronic infection in immunocompromised individuals

(Porter and Sande, 1992). The most common clinical manifestation of toxoplasmosis

in acquired immunodeficiency syndrome (AIDS) patient is toxoplasmic encephalitis.

In fact, toxoplasmic encephalitis is the most common cause of death among

toxoplasmosis patients with AIDS (Dubey, 2004). Other clinical manifestations

include mental status changes, seizures, focal motor deficits, cranial nerve

disturbances, sensory abnormalities, cerebellar signs, movement disorders, and

neuropsychiatric findings (Montoya and Liesenfeld, 2004). Severe manifestations of

toxoplasmosis may also include chorioretinitis, pneumonitis, or multiorgan

10

involvement with acute respiratory failure and haemodynamic abnormalities similar

to septic shock (Liesenfeld et al., 1999). Severe symptoms of toxoplasmosis can also

be observed in immunosuppressed patients with malignancies and after transfusions

or transplant with immunosuppressive therapy (Dubey and Jones, 2008).

(iii) Congenital toxoplasmosis

Infection with T. gondii during pregnancy causes serious consequences. The severity

of the infection depends on the stage of pregnancy at the time of infection (Montoya

and Liesenfeld, 2004). The tachyzoites of T. gondii can cross the placenta to infect

the fetus, thus causing congenital infection. Chances of congenital infection are 10-

25 % in untreated women in the first trimester of pregnancy (Emelia, 2009). The

incidence of fetal infection ranges between 30-54 % and 60-65 %, respectively, if

infection occurs during second and third trimester of pregnancy (Lynfield and

Guerina, 1997). Consequences to fetus are more severe if the infection occurs in

early stages of pregnancy (Remington and Desmonts, 1990). Some of the

consequences that might occur include miscarriage, intra-uterine growth retardation

and premature birth. Moreover, neonatal clinical manifestations of congenital

toxoplasmosis vary widely. These may include hydrocephalus, microcephaly,

intracranial calcifications, chorioretinitis, strabismus, blindness, epilepsy,

psychomotor or mental retardation, petechia due to thrombocytopenia, and anaemia

(McAuley et al., 1994; Swisher et al., 1994).

11

(iv) Ocular disease

Toxoplasmic chorioretinitis refers to severe eye infection and it results from

congenital or postnatally acquired infection of acute infection or reactivation

(Montoya and Remington, 1996; Holland, 1999). The symptoms include white focal

lesions in retina with overlying and intense vitreal inflammatory reaction. The retina

lesion will slowly resolve and leaving a focus of retinochoroidal scarring (Koch et al.,

1943). Besides, mild ocular pain, blurred vision and new onset of floating spots,

segmental periarteritis, retinal and vitreous haemorrhage, choroidal or retinal

neovascularization are among other symptoms of active toxoplasmic choretinitis

(Emelia, 2009).

1.1.1.3 Treatment of Toxoplasmosis

There is not much advancement in the search for new anti-toxoplasmosis drugs. The

current mainstay anti-toxoplasmosis agents have been used for decades. The classic

clinical drug combinations to treat toxoplasmosis are combination of pyrimethamine,

sulfadiazine and folinic acid (Montoya and Remington, 2000).

The immunosuppressed patients such as recipients of organs transplant need to be

tested for anti-Toxoplasma antibodies, and highly effective prophylaxis

trimethoprim/sulfamethoxazole combinations are given (Montoya and Liesenfeld,

2004). Clindamycin is given to the patients with sulfonamides intolerance.

For women with suspected or confirmed acute T. gondii infection during pregnancy,

spiramycin treatment is recommended for the first and early second trimester while

pyrimethamine/sulfadiazine is recommended for late second and third trimester

12

(Gilbert et al., 2001; Gras et al., 2001). Suspected or confirmed maternal infection

acquired during gestation must be confirmed by prenatal diagnosis by PCR of

amniotic fluid (Rormand et al., 2001). Prenatal treatment with

pyrimethamine/sulfadiazine in women suspected or confirmed with fetal infection

reduces the sequelae of the disease in newborn (Foulon et al., 1999).

Toxoplasmic infection can cause severe eyes infection and the treatment should start

when severe inflammatory responses, proximity of retinal lesions to the fovea or

optic disk, or both are found (Holland, 1999). The combination of drugs used is

pyrimethamine, sulfadiazine and prednisone. Besides, clindamycin or

trimethoprim/sulfamethoxazole for a minimum of 3 weeks can also be used to treat

the disease (Luft et al., 1986). Spiramycin is not recommended to treat toxoplasmic

chorioretinitis due to high recurrence rate (Ghosh et al., 1965).

1.1.1.4 Current Challenges in the Treatment of Toxoplasmosis

The current standard therapies for toxoplasmosis involve the combinations of

pyrimethamine with sulfadiazine, clindamycin, azithromycin, or atavaquone

(Djurkovic-Djakovic et al., 1999; Chirgwin et al., 2002). However, these drugs do

not completely eliminate the intracellular parasite and are often associated with

severe side effects (Montoya and Liesenfeld, 2004). The combination of

pyrimethamine and sulfadiazine is not recommended for the treatment of acute

toxoplasmosis in pregnancy and AIDS patients, as they can cause bone marrow

suppression, liver toxicity, nausea, vomit, seizures and other symptoms (Haverkos,

1987). Besides, clindamycin can cause adverse effects such as skin rashes and

gastrointestinal problems such as diarrhoea, stomach upset and bleeding (Djurkovic-

13

Djakovic et al., 1999). However, continuous drug therapy is essential for patients to

ensure prevention of severe complications.

An effective vaccine against human T. gondii infection is a very desirable measure to

control the disease. However, the development of effective vaccine is still in early

stage of research. Alternative drugs with lesser side effects are needed to combat the

disease. The knowledge of the parasite’s lifecycle, transmission routes, risk groups

and host immune responses has helped in the development of strategies to control the

disease, reduce transmission of the parasite and limit environmental contamination.

1.1.2 Plasmodium parasites

The apicomplexan Plasmodium parasites are the causative agents of the well-known

malaria disease in humans. Malaria is the most important parasitic disease in human.

According to World Malaria Report 2013 by World Health Organization (WHO),

malaria is endemic in 104 countries throughout the world and 3.4 billion people are

at risk of malaria. An estimated 207 million people suffered from malaria in 2012

and caused about 627 000 death cases. A total of about 90 % of cases and deaths

occurred in Sub-Saharan Africa and about 77 % of death cases were in children

under age of 5. Malaria causes a huge toll on human health and imposes a heavy

social and economic burden in the developing countries, especially in Sub-Saharan

Africa and South Asia. In Malaysia, cases of malaria are restricted in distribution.

Between 2000 and 2012, Malaysia successfully achieved more than 75 % decrease in

microscopically confirmed malaria cases and more than 50 % decrease in reported

malaria mortality rate (WHO, 2013).

14

The malaria-causing Plasmodium parasites are transmitted by Anopheles mosquitoes.

About 30 species out of 400 different species of the Anopheles mosquitoes are the

important vectors in transmitting the parasites (White et al., 2014). The ultra

structure of a P. falciparum merozoite is illustrated in Figure 1.2 under Section 1.1.1.

There are five species of the Plasmodium parasites that cause malarial infections in

human. Majority of the cases are caused by Plasmodium falciparum or Plasmodium

vivax, but infection with Plasmodium ovale and Plasmodium malariae can lead to

malaria as well. Recently, there were cases of malaria caused by monkey malaria

Plasmodium knowlesi being reported in parts of Southeast Asia (Kantele and

Jokiranta, 2011). P. falciparum causes the most serious cases of malaria and is

responsible for almost all malaria death cases. P. falciparum predominates in Africa

while P. vivax has a wider distribution due to its ability to develop in Anopheles

mosquito at lower temperatures, and to survive at higher altitudes and in cooler

climates. Besides, P. vivax also has a long dormant liver stage that enables the

parasites to persist in human (White et al., 2014).

Malaria remains one of the most important diseases in humans in terms of both

mortality and morbidity, with Plasmodium falciparum being the most important

infecting agent (White et al., 2014). The increasing global spread of drug resistance

to most of the available and affordable antimalarial drugs is a major concern and

requires innovative strategies to combat (Nguyen-Pouplin et al., 2007).

15

Table 1.2: Taxonomic classification of Plasmodium parasites (Adapted from NCBI,

2015b)

Taxonomic Classification

Kingdom Alveolata

Phylum Apicomplexa

Class Aconoidasida

Order Haemosporida

Family Plasmodiidae

Genus Plasmodium

Species P. falciparum, P. ovale, P. vivax. P. malariae, P. knowlesi

1.1.2.1 Life Cycle and Transmission of Plasmodium Parasites

The life cycle of the Plasmodium parasites involve three distinct stages (Figure 1.4).

The liver and blood stages occur in host, whereas the sexual stage occurs in the

mosquito gut (White et al., 2014).

The malaria-infected female anopheline mosquito inoculates motile sporozoites into

the human host during a blood meal. The sporozoites then rapidly infect and invade

into hepatocytes. Inside liver cells, the sporozoites mature into schizonts and

multiply to become merozoites. A successful sporozoite invasion can produce about

10000 to 30000 merozoites in 5.5-8 days inside a single hepatocyte. In P. vivax and

P. ovale, infection of sporozoites into liver cells can produce hypnozoites in the

dormant stage that can persist in the liver, which will then causing relapses weeks or

years later. Next, the merozoites are released to invade the red blood cells. The

parasites undergo asexual multiplication in the erythrocytes after the initial liver

replication. An asexual cycle in the blood takes roughly 48 h for P. falciparum, P.

vivax, and P. ovale, 72 h for P. malariae, and 24 h only for P. knowlesi. Inside the

erythrocytes, the merozoites develop into ring stage trophozoite (Figure 1.5) and then

mature into daughter merozoites. The erythrocytes rupture and release daughter

16

merozoites which rapidly invade new erythrocytes and repeat the cycle (White et al.,

2014).

The malaria disease is caused by the effects of erythrocytes parasitization and

destruction, as well as the host immune reaction to fight against the parasites. Inside

the red blood cells, the merozoites consume the cell content, altering the cell

membrane to facilitate nutrients transportation and metabolise the toxic haem into

harmless haemozoin (malaria pigment). Besides, some merozoites differentiate into

gametocytes, which are then ingested by Anopheles mosquito during blood meal and

initiate the sexual reproduction cycle of the parasites (Fujioka and Aikawa, 2002).

The sexual reproduction of the Plasmodium parasites occurs only in mosquitoes.

Upon ingestion of the gametocytes, the microgametocytes fuse with

macrogametocytes inside the mosquito's stomach to produce zygotes. The zygotes

are then developed into ookinetes that invade the midgut wall of mosquitoes.

Ookinetes develop into oocysts that rupture and release the sporozoites. The

sporozoites migrate to the mosquito's salivary gland and inoculate into new host

when the mosquito taken a blood meal. Inoculation of sporozoites into new human

host initiates the new malaria life cycle (Fujioka and Aikawa, 2002).

17

Figure 1.4: Life cycle of Plasmodium parasites in the human body and the

anopheline mosquito (adapted from White et al., 2014).

Figure1.5: Gametocyte and trophozoite (ring form) of P. falciparum in a thin blood

smear [Adapted from Centers for Disease Control and Prevention (CDC) USA,

2014].

18

1.1.2.2 Clinical Manifestations and Pathogenesis of Human Malaria

Malaria causes severe clinical manifestations in human. The initial symptoms of

malaria are nonspecific and they include absence of wellbeing, headache, fatigue,

anaemia, muscle aches, abdominal discomfort and followed by irregular fever. Other

symptoms such as vomiting, nausea and hypotension also occur frequently. Malaria

caused by falciparum parasites is also associated with generalised seizures and might

be followed by coma that is caused by cerebral malaria. For uncomplicated malaria

infections, most patients have fewer physical symptoms. Since malaria parasites

infect the liver, hepatomegaly may often be found in young children while mild

jaundice is more likely to occur in adults. Recurrent malaria infection causes chronic

anaemia and splenomegaly in young children (White et al., 2014).

Infection with P. falciparum can cause the severe malaria type. In falciparum

malaria, the infected erythrocytes tend to adhere to vessel walls and also to each

other. The erythrocytes adherence causes sequestration of red blood cells containing

mature parasites into vital organs particularly the brain (White et al., 2014). Inside

the brain, the sequestered parasites will interfere with microcirculatory flow and the

metabolism and normal functioning of vascular endothelium (Pongponratn et al.,

2003). The erythrocytes in malaria caused by other parasites species are not

sequestered substantially. Severe falciparum malaria is mainly attributed by

extensive parasitised erythrocytes sequestration and consequently dysfunction of

vital organs. However, age is the main factor determining the manifestation of severe

falciparum malaria (Dondorp et al., 2008). In children, severe anaemia and

hypoglycaemia occur more frequently whereas acute pulmonary oedema, acute

kidney injury, and jaundice are more common in adults. Besides, acidosis and

19

cerebral malaria following by subsequent coma can occur in all age. The mortality

rate increases as the proportion of infected red blood cells exceeds 2 %. However,

uncomplicated falciparum malaria has a low mortality rate of about 0.1 % when

properly treated with effective antimalarial drugs (Dondorp et al., 2008).

Severe malaria can also cause other clinical complications such as invasive bacterial

infections by enteric bacteria in children (Bronzan et al., 2007). Malaria can

accelerate the progression and transmission of HIV infection (Kublin et al., 2005).

The mortality rate of clinical and severe malaria is increased in adults with HIV

infection and compromised immune status (Whitworth et al., 2000; Chalwe et al.,

2009). Moreover, the concomitant HIV infection increases the severity and mortality

of severe falciparum malaria in children (Berkley et al., 2000; Hendriksen et al.,

2012).

Falciparum malaria can cause serious complications in pregnant women. The risk of

low birth weight (< 2.5 kg) increases when the mother get infected. The lower birth

weight is associated with increased infant mortality (Desai et al., 2007). Other

malaria complications such as high parasitaemia, severe anaemia, hypoglycaemia

and acute pulmonary oedema are more frequent in pregnant women compared to

non-pregnant women (McGready et al., 2012). Fetal distress, premature labour and

stillbirth often occur in pregnant women with severe malaria (Desai et al., 2007).

There is a higher risk of infant death if maternal malaria occurs during late

pregnancy (Bardaji et al., 2011). Besides, maternal death caused by haemorrhage

during childbirth is correlated with the malaria-induced anaemia (Rijken et al.,

2012).

20

1.1.2.3 Treatment of Malaria

Most of the antimalarial drugs target the asexual erythrocytic stage of the parasite.

During parasitic growth, the parasites metabolize haemoglobin. The haemoglobin

degradation produces free haem that is able to react with oxygen, thus producing

reactive oxygen species as toxic by-products. A major pathway of haem

detoxification is through polymerization to malaria pigment haemozoin (Pagola et

al., 2000). Most of the antimalarial drugs act to disturb the polymerization of haem,

thus killing the parasites with its own metabolic waste (Egan and Marques, 1999).

The main chemical classes of antimalarial drugs are as listed in Table 1.3. Some

antibiotics are also used to treat malaria in combination with other drugs.

The only class of drugs that is active against the gametocytes stage is of 8-

aminoquinolines (Robert et al., 2001) (Table 1.3). Primaquine belongs to 8-

aminoquinolines class and it is widely used against the hypnozoites (liver dormants)

responsible for causing relapse of P. vivax and P. ovale. Primaquine interferes with

the mitochondrial function of Plasmodium parasites (Robert et al., 2001).

The classes of sulfonamides and sulfones, biguanides, diaminopyrimidine and

naphthoquinone act as nucleic acid inhibitors (Table 1.3). Sulfonamides and

sulfones, biguanides and diaminopyrimidine belong to the folate antagonist class

(Olliaro, 2001), though the Plasmodium parasites already developed resistance

against this class of drugs. The inhibition of the enzymes in the folate pathway

results in decreased synthesis of parasites pyrimidine, thus reduces the formation of

methionine, serine and DNA necessary for parasites development. The inhibitory

activity is exerted at all growing stages of the erythrocytic cycle and on young

21

gametocytes (Olliaro, 2001). On the other hand, atovaquone (from class

naphthoquinone) inhibits the respiratory chain of malarial mitochondria at the

cytochrome bc1 complex by mimicking the natural substrate, ubiquinone. This, in

turn, inhibits the mitochondrial electron transfer from ubiquinol to cytochrome c (Fry

& Pudney, 1992). However, the parasites develop resistance to atovaquone quickly

when atovaquone is prescribed alone. Therefore, atovaquone is usually prescribed

with proguanil in fixed combination to treat malaria (Robert et al., 2001).

On the other hand, blood schizonticides refer to drugs that act on erythrocytic stages

of parasites. The primary target is the parasite food vacuole (Olliaro, 2001). These

classes of drugs include the quinoline containing drugs such as 4-Aminoquinolines

and aryl-amino alcohols, as well as the artemisinin-type drugs (Table 1.3). The

quinoline drugs accumulate in the acidic food vacuole within the parasites, and

subsequently alter the haem detoxification processes that eventually leading to

parasites death (Olliaro, 2001).

The artemisinin-type compounds include the natural extract artemisinin and its semi-

synthetic derivatives. They achieve higher parasitaemia reduction rates than any

other drugs (White, 1997), and are being used for the treatment of uncomplicated and

severe forms of malaria. The artemisinin-type compounds mainly act through the

generation of alkylating species by reaction of homolytic cleavage of endoperoxide

bridge with haem (Robert et al., 2001). The generated alkylating radicals then disrupt

the vital biochemical processes of the parasite via alkylation of biomolecules and

thus causing parasites death (Robert et al., 2001).

22

Artemisinin-based combination therapies (ACTs) are the current first-line therapy for

falciparum malaria in all endemic areas (WHO, 2013). It is also highly efficacious

against the other human malarias. Artemisinin combination treatment acts rapidly

and reliably effective. Artemisinin combination therapy has few adverse effects and

has very high curative level (International Artemisinin Study Group, 2004). In ACTs,

the artemisinin derivatives are combined with a partner drug of different mechanisms

of action. The fast-acting artemisinin compound serves the purpose to reduce the

main parasite load during the first three days of treatment, while the partner drug

eliminates the remaining parasites (WHO, 2013). The artemisinin derivatives such as

artesunate is also the first treatment of choice for all types of severe malaria (severe

falciparum, vivax and knowlesi malaria) as artesunate has no important local or

systemic adverse effects (Barber et al., 2013). Chloroquine is still used in regions

where it still remains efficacious.

Atovaquone–proguanil, doxycycline, primaquine, or mefloquine are prescribed to

travellers travel to resistant P. falciparum endemic areas as chemoprophylaxis

purpose. On the other hand, sulfadoxine–pyrimethamine was given as intermittent

preventive treatment to pregnant women in Africa. A minimum of three doses of

sulfadoxine–pyrimethamine are recommended to provide continuous preventive

effects against malaria (White et al., 2014). Besides, mefloquine is recommended for

chemoprophylaxis in pregnant women travelling to areas with drug-resistant malaria.

WHO (2012) recommended monthly amodiaquine and sulfadoxine–pyrimethamine

(maximum four doses) to all children aged 3–59 months in Africa during the yearly

malaria transmission season.

23

Table 1.3: Classes of chemical family for antimalarial drugs. The table was adapted

and modified from WHO (2010).

Chemical Family Common structure Drugs

4-Aminoquinolines

Chloroquine, amodiaquine,

piperaquine

Aryl-amino

alcohols

Quinine, quinidine,

mefloquine, halofantrine,

lumefantrine

Sulfonamides and

sulfones Sulfonamides

Sulfones

Sulfadoxine, sulfalene,

dapsone

Biguanides

Proguanil, chlorproguanil

Diaminopyrimidine

Pyrimethamine, cycloguanil

8-Aminoquinoline Primaquine

Sesquiterpene

lactones

Artemisinin, arteether,

artemether, artesunate,

dihydroartemisinin

Naphthoquinone Atovaquone

Antibiotics

Azythromycin

Clindamycin

Doxycycline

Tetracycline

Azythromycin, clindamycin,

doxycycline, tetracycline

24

1.1.2.4 Current Challenges in the Treatment of Malaria

The current antimalarial drugs such as chloroquine and artemisinin have been used

for decades and there is not much advancement in the search for new effective

antimalarial molecules. Even the newest antimalarial drugs, the artemisinin

derivative, were introduced into market in 1990s (Faurant, 2011).

The use of chloroquine to prevent and treat falciparum malaria has led to the wide

spread appearance of chloroquine-resistance strain of Plasmodium falciparum across

the world (Rahman et al., 1999). The resistance of malaria parasites has been

increasingly extended to other current antimalarial drugs. This is because many of

antimalarial drugs in current usage are chemically related. Thus, development of

resistance to one drug can facilitate the development of resistance to others. In fact,

cross-resistance towards chloroquine and amodiaquine (4-aminoquinolines) has been

reported (Bloland, 2001). Besides, resistance to mefloquine may lead to resistance to

halofantrine and quinine.

In such situation, World Health Organization (WHO) has recommended artemisinin-

based combination therapies in all endemic countries. Moreover, WHO had also

banned the use of artemisinin in monotherapy and recommended the artemisinin

combination therapy instead in order to delay the development of drug resistance and

to improve cure rates (WHO, 2006; Robert et al., 2001). Recently, there have been

signs that the efficacy of artemisinin-based combination therapy and artesunate

monotherapy have declined in western Cambodia. The resistance to artemisinin has

been reported around Thai-Cambodia border (Muller et al., 2009) and Thailand-

Myanmar border (Phyo et al., 2012). The spread of artemisinin resistance parasites to

phytochemical study, anti-toxoplasmosis and antimalarial activities

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